Histone H3


3' end processing of Drosophila melanogaster histone pre-mRNAs: requirement for phosphorylated Drosophila stem-loop binding protein and coevolution of the histone pre-mRNA processing system

Synthetic pre-mRNAs containing the processing signals encoded by Drosophila histone genes undergo efficient and faithful endonucleolytic cleavage in nuclear extracts prepared from Drosophila cultured cells and 0- to 13-h-old embryos. Biochemical requirements for the in vitro cleavage are similar to those previously described for the 3' end processing of mammalian histone pre-mRNAs. Drosophila 3' end processing does not require ATP and occurs in the presence of EDTA. However, in contrast to mammalian processing, Drosophila processing generates the final product ending four nucleotides after the stem-loop. Cleavage of the Drosophila substrates is abolished by depleting the extract of the Drosophila stem-loop binding protein (dSLBP), indicating that both dSLBP and the stem-loop structure in histone pre-mRNA are essential components of the processing machinery. Recombinant dSLBP expressed in insect cells by using the baculovirus system efficiently complements the depleted extract. Only the RNA-binding domain plus the 17 amino acids at the C terminus of dSLBP are required for processing. The full-length dSLBP expressed in insect cells is quantitatively phosphorylated on four residues in the C-terminal region. Dephosphorylation of the recombinant dSLBP reduces processing activity. Human and Drosophila SLBPs are not interchangeable and strongly inhibit processing in the heterologous extracts. The RNA-binding domain of the dSLBP does not substitute for the RNA-binding domain of the human SLBP in histone pre-mRNA processing in mammalian extracts. In addition to the stem-loop structure and dSLBP, 3' processing in Drosophila nuclear extracts depends on the presence of a short stretch of purines located ca. 20 nucleotides downstream from the stem, and an Sm-reactive factor, most likely the Drosophila counterpart of vertebrate U7 snRNP (Dominski, 2002).

Development of an in vitro system based on nuclear extracts from human and mouse cells was a major advance that allowed a molecular analysis of 3' end processing of mammalian histone pre-mRNA. A Drosophila in vitro system based on the mammalian system has been developed using nuclear extract from Drosophila S-2 cells and Drosophila histone pre-mRNAs. This system was utilized for mapping the structural features in Drosophila histone pre-mRNA and dSLBP essential for 3' end processing (Dominski, 2002).

Drosophila cultured cells are a convenient and relatively inexpensive source of nuclear extracts proficient in transcription or splicing and have been used for large-scale purification of spliceosomal snRNPs. The nuclear extract from Drosophila S-2 cells and 0- to 20-h-old embryos are also very efficient in 3' end processing of all five Drosophila histone pre-mRNAs. Since Drosophila contains only one gene (present in multiple copies) for each of the five different histone proteins, it is essential that all five Drosophila histone pre-mRNAs be efficiently processed. In mammalian cells there are multiple nonallelic copies of each histone gene, and the processing efficiency of different pre-mRNAs encoded by these copies significantly varies in vivo and in vitro (Dominski, 2002).

Drosophila histone pre-mRNA processing in vitro has biochemical properties similar to processing in mammalian cells. The reaction does not require divalent ions or ATP and generates the final product without a significant lag time, suggesting that a relatively small number of factors assemble to form a functional processing complex. The presence of the cleaved 3' fragment indicates that generation of the mature 3' end in Drosophila histone mRNAs occurs through endonucleolytic cleavage and not by the activity of a 3' exonuclease. In dNE, histone pre-mRNAs are processed four nucleotides after the stem-loop, whereas in mammalian nuclear extracts cleavage occurs one nucleotide farther downstream. There is an adenosine residue four nucleotides after the stem-loop in all five Drosophila histone pre-mRNAs, whereas most mammalian histone mRNAs end in ACCCA, suggesting that cleavage after an A has been conserved in evolution (Dominski, 2002).

Nuclear extract from Drosophila S-2 cells was very active in cleaving histone pre-mRNAs containing the downstream element encoded by all five Drosophila histone genes. In sea urchins an invariant sequence (CAAGAAAGA) has been identified in the downstream element of all histone pre-mRNAs, and this sequence base pairs with the 5' end of sea urchin U7 snRNA. The Drosophila histone genes do not share any highly conserved sequences downstream of the stem-loop, although they are all generally purine-rich. Mutagenesis studies of the downstream sequence from Drosophila H3 pre-mRNA have revealed that a GAGAUA element plays a critical role in processing; substitution of this sequence with the complementary nucleotides (mutant M1) abolishes in vitro processing, whereas mutation of adjacent nucleotides has no effect. In addition to identifying the downstream processing element in Drosophila H3 pre-mRNA, the M1 mutant provides further evidence that the in vitro system reproduces a genuine processing event and is not a result of 3' to 5' exonucleases activity stalled by the stem-loop associated with dSLBP. The sequence requirements of the downstream element must be more complex than simply the presence of the purine-rich element at a proper distance from the stem-loop since the mouse H2a pre-mRNA contains a similar purine-rich sequence in the same location and is processed in the Drosophila extract very inefficiently, in contrast to all five Drosophila histone pre-mRNAs. Perhaps there are other sequence elements in Drosophila histone pre-mRNAs that are not conserved in pre-mRNAs of higher organisms and which contribute to high efficiency of processing in dNE (Dominski, 2002).

While the low efficiency of processing of mouse H2a pre-mRNA in Drosophila extract is puzzling, it is easier to explain the inability of the mNE to process Drosophila H3 pre-mRNA. The downstream element from Drosophila H3 pre-mRNA has a very limited complementarity to the 5' end of mouse U7 snRNA and in the optimal configuration the two RNAs can form only 10 bp over a 19-nucleotide region, with the longest uninterrupted stretch of duplex RNA consisting of only 4 bp. For comparison, the mouse H1t pre-mRNA, previously shown to be a poor and completely SLBP-dependent substrate, can form either 11 or 13 bp in two alternative alignments with the U7 snRNA, and the longest uninterrupted duplex consists of 7 and 6 bp, respectively. In contrast, the mouse H2a-614 pre-mRNA, a good mammalian processing substrate, forms 14 base pairs with mouse U7 snRNA interrupted by only one mismatch. Thus, given the requirement for an extensive duplex between U7 snRNA and the downstream element for mammalian 3' end processing, the inability of the mNE to process Drosophila H3 pre-mRNA is not surprising (Dominski, 2002).

The downstream purine-rich sequence identified in these studies as essential for processing in the dNE is most likely recognized by the Drosophila equivalent of U7 snRNP. This interpretation is supported by the finding that Sm antibodies, but not control monoclonal antibodies, reproducibly reduce the efficiency of histone pre-mRNA processing. However, despite sequencing the entire Drosophila genome, U7 snRNA has not yet been identified in this organism. Both the small size and the limited evolutionary conservation precludes a search for this RNA based on sequence similarity to known vertebrate and sea urchin U7 snRNAs (Dominski, 2002).

In mammalian extracts the importance of SLBP in 3' end processing in vitro varies considerably between multiple histone pre-mRNAs and depends on the strength with which U7 snRNA base pairs with the downstream element. When there is limited complementarity between the downstream element and 5' end of U7 snRNA, as in the case of mouse H1t pre-mRNA, there is a complete dependence of processing on SLBP. Processing of some mammalian histone mRNAs that are capable of extensive base pairing with the U7 snRNA can occur in the absence of SLBP. In contrast, in vitro processing of all five Drosophila histone pre-mRNAs is virtually completely dependent on the presence of SLBP. This dependence most likely results from the relatively short downstream element in Drosophila histone pre-mRNA, which in dH3 pre-mRNA appears to be ca. six nucleotides long, and the 5' end of a putative Drosophila U7 snRNA. In sea urchins the region of complementarity between pre-mRNA and U7 snRNA is limited to only 6 bp and the formation of rather short duplexes between the two RNAs and thus a requirement for additional interaction involving U7 snRNP and SLBP may be a general feature of 3' end processing in lower metazoans (Dominski, 2002).

A series of mutations in the dSLBP gene results in a large reduction in dSLBP concentration in vivo. In the mildest of the mutants, which are viable and female sterile and still express some dSLBP, there is a great reduction in the amount of histone mRNA synthesized during oogenesis, resulting in embryonic lethality due to a lack of histone proteins and the inability to complete the syncytial cell cycles. More severe mutants are zygotically lethal, with death occurring in the larval stages. Interestingly, zygotic mutants express a significant amount of polyadenylated histone mRNA during embryogenesis as a result of transcription past the stem-loop and usage of cryptic polyadenylation sites that are present 3' of each of the Drosophila histone genes. However, even the most severe dSLBP mutants generate, during embryogenesis, a substantial amount of histone mRNA that ends at or near the stem-loop. Whether these histone mRNAs are formed by a small amount of dSLBP remaining in these embryos (which in at least one mutant cannot be detected by biochemical assays) or whether there is an alternative mechanism for forming and stabilizing mRNAs ending at the stem-loop is not known. If the latter situation is true, then this mechanism does not function in nuclear extracts from Drosophila cultured cells (Dominski, 2002).

A striking feature of dSLBP not shared by vertebrate SLBPs is its hyperphosphorylation. dSLBP overexpressed in insect cells is quantitatively phosphorylated on four sites within the C-terminal region. Based on electrophoretic mobility, a similar level of phosphorylation is present both in embryonic dSLBP and dSLBP expressed in Drosophila cultured cells. Phosphorylation of dSLBP is essential for complete processing activity of the protein in vitro. Since the dephosphorylated and the phosphorylated dSLBP bind to the stem-loop with similar affinity, the phosphorylation must be required for interaction of dSLBP with other factors involved in histone pre-mRNA processing. In the last 13 amino acids of dSLBP there are four serines, which alternate with aspartic acid residues, and it is likely that dSLBP is phosphorylated on these four serines, creating a highly acidic C terminus. Full-length dSLBP also contains at least two partially phosphorylated sites. Detailed mutational analysis will be required to determine which of these sites are critical for histone pre-mRNA processing (Dominski, 2002).

Reversible changes in phosphorylation status of dSLBP would provide an attractive mechanism for regulating function of dSLBP during embryogenesis and/or the cell cycle. Dephosphorylation would convert the active o an inactive form that would effectively inhibit processing. In mammals SLBP accumulates to the highest level in S phase and is degraded by the proteasome pathway immediately after completion of DNA replication. It is not known whether dSLBP displays the same pattern of accumulation and disappearance during the cell cycle in Drosophila cells, but reversible changes in phosphorylation status could provide an equally efficient mechanism of adjusting dSLBP activity. While regulation of dSLBP phosphorylation is a possible attractive mechanism that could contribute to regulation of histone pre-mRNA processing, only hyperphosphorylated SLBP has been observed in Drosophila cultured cells and embryos. However, changes in dSLBP phosphorylation that affected only one or two sites would not necessarily have been detected, particularly if the modification did not alter the electrophoretic mobility (Dominski, 2002).

The initial characterization of histone pre-mRNA processing in sea urchins was made possible because one of the sea urchin histone pre-mRNAs was not processed in frog oocytes as a result of differences in the HDE. The Drosophila histone pre-mRNAs are not processed in mammalian extracts, and the mammalian mRNAs are processed very inefficiently in Drosophila extracts. Moreover, Drosophila and human SLBPs are not interchangeable: dSLBP does not function in 3' end processing in mammalian nuclear extracts, and human SLBP fails to complement dSLBP-depleted nuclear extract from Drosophila S-2 cells. Instead, each protein has a strong inhibitory effect on processing in the heterologous nuclear extract by competing with the endogenous SLBP for binding to the stem-loop in histone pre-mRNA. The processing activity of both dSLBP and human SLBP requires the RBD and the adjacent amino acids of the C-terminal region. Amino acid conservation between Drosophila and mammalian SLBPs is limited only to the RBD and does not extend into the C-terminal region, thus explaining the inability of each protein to substitute for each other. Interestingly, the RBDs are also not interchangeable. The hybrid H-D-H SLBP containing both flanking domains from human SLBP and the RBD from dSLBP does not support processing in mammalian nuclear extracts. The human RBD contains a nine-amino-acid region dispensable for RNA binding but necessary for processing. This region, together with the C-terminal residues, is involved in interaction of the SLBP-pre-mRNA complex with a novel 100-kDa zinc finger protein (hZFP100) associated with the U7 snRNP. The nine-amino-acid region includes the critical DR dipeptide, which is changed in dSLBP to ER. It is possible that this single amino acid substitution, while allowing dSLBP to function efficiently in dNE, is largely responsible for inability of the hybrid H-D-H protein to function in mammalian nuclear extract. These data are consistent with coevolution of the machinery independently in the vertebrate and invertebrate lineages (Dominski, 2002).

The data presented in this study suggest that there are many similarities between the processing machineries in Drosophila and higher organisms, including the requirement for SLBP and the purine-rich sequence downstream from the cleavage site. However, the components have diverged significantly during evolution and are no longer recognized in the heterologous systems. Formally, it is still possible that there is no U7 snRNA in Drosophila, since there is no U12 snRNA, although there are ATAC introns and a U11 snRNA. This would imply that the mechanism of histone pre-mRNA processing in Drosophila is substantially different from that in sea urchins and vertebrates. It might be significant that three other proteins required for mammalian histone pre-mRNA processing hZFP100 and two U7 specific proteins, Lsm10 and Lsm11, have no obvious homologues in Drosophila genomes. Undoubtedly, future studies with dNE will be very helpful in providing more information about the mechanism of histone pre-mRNA processing in this organism (Dominski, 2002).

Molecular mechanisms for the regulation of histone mRNA stem-loop-binding protein by phosphorylation

Replication-dependent histone mRNAs end with a conserved stem loop that is recognized by stem-loop-binding protein (SLBP), an RNA-binding protein involved in the histone pre-mRNA processing. The minimal RNA-processing domain of SLBP is phosphorylated at an internal threonine, and Drosophila SLBP (dSLBP) also is phosphorylated at four serines in its 18-aa C-terminal tail. This study shows that phosphorylation of dSLBP increases RNA-binding affinity dramatically, and structural and biophysical analyses of dSLBP and a crystal structure of human SLBP phosphorylated on the internal threonine were used to understand the striking improvement in RNA binding. Together these results suggest that, although the C-terminal tail of dSLBP does not contact the RNA, phosphorylation of the tail promotes SLBP conformations competent for RNA binding and thereby appears to reduce the entropic penalty for the association. Increased negative charge in this C-terminal tail balances positively charged residues, allowing a more compact ensemble of structures in the absence of RNA (Zhang, 2014).

Hierarchical recruitment of polycomb group silencing complexes

Polycomb group (PcG) proteins maintain the transcriptional silence of target genes through many cycles of cell division. This study provides evidence for the sequential binding of PcG proteins at a Polycomb response element (PRE) in proliferating cells in which the sequence-specific DNA binding Pho and Phol proteins directly recruit E(z)-containing complexes, which in turn methylate histone H3 at lysine 27 (H3mK27). This provides a tag that facilitates binding by a Pc-containing complex. In wing imaginal discs, these PcG proteins also are present at discrete locations at or downstream of the promoter of a silenced target gene, Ubx. E(z)-dependent H3mK27 is also present near the Ubx promoter and is needed for Pc binding. The location of E(z)- and Pc-containing complexes downstream of the Ubx transcription start site suggests that they may inhibit transcription by interfering with assembly of the preinitiation complex or by blocking transcription initiation or elongation (Wang, 2004).

Genome-wide activities of Polycomb complexes control pervasive transcription

Polycomb group (PcG) complexes PRC1 and PRC2 are well known for silencing specific developmental genes. PRC2 is a methyltransferase targeting histone H3K27 and producing H3K27me3, essential for stable silencing. Less well known but quantitatively much more important is the genome-wide role of PRC2 that dimethylates approximately 70% of total H3K27. H3K27me2 occurs in inverse proportion to transcriptional activity in most non-PcG target genes and intergenic regions and is governed by opposing roaming activities of PRC2 and complexes containing the H3K27 demethylase UTX. Surprisingly, loss of H3K27me2 results in global transcriptional derepression proportionally greatest in silent or weakly transcribed intergenic and genic regions and accompanied by an increase of H3K27ac and H3K4me1. H3K27me2 therefore sets a threshold that prevents random, unscheduled transcription all over the genome and even limits the activity of highly transcribed genes. PRC1-type complexes also have global roles. Unexpectedly, a pervasive distribution of histone H2A ubiquitylated at lysine 118 (H2AK118ub) was found outside of canonical PcG target regions, dependent on the RING/Sce subunit of PRC1-type complexes. It was shown, however, that H2AK118ub does not mediate the global PRC2 activity or the global repression and is predominantly produced by a new complex involving L(3)73Ah, a homolog of mammalian PCGF3 (H. G. Lee, 2015).

Interdependence of PRC1 and PRC2 for recruitment to Polycomb Response Elements

Polycomb Group (PcG) proteins are epigenetic repressors essential for control of development and cell differentiation. They form multiple complexes of which PRC1 and PRC2 are evolutionary conserved and obligatory for repression. The targeting of PRC1 and PRC2 is poorly understood and has been proposed to be hierarchical and involve tri-methylation of histone H3 (H3K27me3) and/or monoubiquitylation of histone H2A (H2AK118ub). This study tested this hypothesis using the Drosophila model. It was discovered that neither H3K27me3 nor H2AK118ub is required for targeting PRC complexes to Polycomb Response Elements (PREs). PRC1 can bind PREs in the absence of PRC2 but at many PREs PRC2 requires PRC1 to be targeted. It was shown that one role of H3K27me3 is to allow PcG complexes anchored at PREs to interact with surrounding chromatin. In contrast, the bulk of H2AK118ub is unrelated to PcG repression. These findings radically change the view of how PcG repression is targeted and suggest that PRC1 and PRC2 can communicate independently of histone modifications (Kahn, 2016).

A balancing act: heterochromatin protein 1a and the Polycomb group coordinate their levels to silence chromatin in Drosophila

Early in Drosophila development, Heterochromatin protein 1a (HP1a) collaborates with the Polycomb/trithorax groups of proteins to regulate gene expression and that the two chromatin systems do not act separately as convention describes. This study shows that HP1a affects the levels of both the Polycomb complexes and RNA polymerase II at promoters, as assayed by chromatin immunoprecipitation analysis. Deposition of both the repressive (H3K27me3) and activating (H3K4me3) marks promoted by the Polycomb/trithorax group genes at gene promoters is affected. Additionally, depending on which parent contributes the null mutation of the HP1a gene, the levels of the H3K27me3 and H3K9me3 silencing marks at both promoters and heterochromatin are different. Changes in levels of the H3K27me3 and H3K9me3 repressive marks show a mostly reciprocal nature. The time around the mid-blastula transition, when the zygotic genome begins to be actively transcribed, appears to be a transition/decision point for setting the levels. This study finds that HP1a affects the generation of the epigenetic marks of the Polycomb/trithorax groups of proteins, chromatin modifiers which are key to maintaining gene expression in euchromatin. At gene promoters, deposition of both the repressive H3K27me3 and activating H3K4me3 marks of histone modifications shows a dependence on HP1a. Around the mid-blastula transition, when the zygotic genome begins to be actively transcribed, a pivotal decision for the level of silencing appears to take place. This is also when the embryo organizes its genome into heterochromatin and euchromatin. A balance between the HP1a and Polycomb group silencing systems appears to be set for the chromatin types that each system will primarily regulate (Cabrera, 2015).

Propagation of Polycomb-repressed chromatin requires sequence-specific recruitment to DNA

Epigenetic inheritance models posit that during Polycomb repression, Polycomb Repressive Complex 2 (PRC2) propagates histone H3K27 tri-methylation (H3K27me3) independently of DNA sequence. This study shows that insertion of Polycomb Response Element (PRE) DNA into the Drosophila genome creates extended domains of H3K27me3-modified nucleosomes in the flanking chromatin and causes repression of a linked reporter gene. After excision of PRE DNA, H3K27me3 nucleosomes become diluted with each round of DNA replication and reporter gene repression is lost, whereas in replication-stalled cells, H3K27me3 levels stay high and repression persists. Hence, H3K27me3-marked nucleosomes provide a memory of repression that is transmitted in a sequence-independent manner to daughter strand DNA during replication. In contrast, propagation of H3K27 tri-methylation to newly incorporated nucleosomes requires sequence-specific targeting of PRC2 to PRE DNA (Laprell, 2017).

Direct interrogation of the role of H3K9 in metazoan heterochromatin function

A defining feature of heterochromatin is methylation of Lys9 of histone H3 (H3K9me), a binding site for heterochromatin protein 1 (HP1). Although H3K9 methyltransferases and HP1 are necessary for proper heterochromatin structure, the specific contribution of H3K9 to heterochromatin function and animal development is unknown. Using a recently developed platform to engineer histone genes in Drosophila, H3K9R mutant flies were generated, separating the functions of H3K9 and nonhistone substrates of H3K9 methyltransferases. Nucleosome occupancy and HP1a binding at pericentromeric heterochromatin are markedly decreased in H3K9R mutants. Despite these changes in chromosome architecture, a small percentage of H3K9R mutants complete development. Consistent with this result, expression of most protein-coding genes, including those within heterochromatin, is similar between H3K9R and controls. In contrast, H3K9R mutants exhibit increased open chromatin and transcription from piRNA clusters and transposons, resulting in transposon mobilization. Hence, transposon silencing is a major developmental function of H3K9 (Penke, 2016).

Ctr9, a key component of the Paf1 complex affects proliferation and terminal differentiation in the developing Drosophila nervous system

The Paf1 protein complex (Paf1C) promotes H3K4 and H3K36 trimethylation, H2BK123 ubiquitination, RNA Pol II transcriptional termination, and also RNA-mediated gene silencing. Paf1C contains five canonical protein components including Paf1 and Ctr9, that are critical for overall complex integrity, as well as , Leo1 and Cdc73/Hyrax. This study provide the first detailed phenotypic study of Ctr9 function in Drosophila. Ctr9 mutants die at late embryogenesis or early larval life, but can be partly rescued by nervous system re-expression of Ctr9. A number of phenotypes are observed in Ctr9 mutants, including increased neuroblast numbers, increased nervous system proliferation, as well as down-regulation of many neuropeptide genes. Analysis of cell cycle and regulatory gene expression reveals up-regulation of the E2f1 cell cycle factor, as well as changes in Antennapedia and Grainy head expression. Reduction of H3K4me3 modification was found in the embryonic nervous system. Genome-wide transcriptome analysis points to additional downstream genes that may underlie these Ctr9 phenotypes, revealing gene expression changes in Notch pathway target genes, cell cycle genes and neuropeptide genes. In addition, significant effects were found on the gene expression of metabolic genes. These findings reveal that Ctr9 is an essential gene that is necessary at multiple stages of nervous system development, and provides a starting point for future studies of the Paf1C in Drosophila (Bahrampour, 2016).

upSET, the Drosophila homologue of SET3, Is required for viability and the proper balance of active and repressive chromatin marks

Chromatin plays a critical role in faithful implementation of gene expression programs. Different post-translational modifications of histone proteins reflect the underlying state of gene activity, and many chromatin proteins write, erase, bind, or are repelled by these histone marks. One such protein is UpSET, the Drosophila homolog of yeast Set3 and mammalian KMT2E (MLL5). This study shows that UpSET is necessary for the proper balance between active and repressed states. Using CRISPR/Cas-9 editing, S2 cells were generated that are mutant for upset. Loss of UpSET was tolerated in S2 cells, but heterochromatin is misregulated, as evidenced by a strong decrease in H3K9me2 levels assessed by bulk histone post-translational modification quantification. To test whether this finding was consistent in the whole organism, the upset coding sequence was deleted using CRISPR/Cas-9; it was found to be lethal in both sexes in flies. This lethality could be rescued using a tagged upSET transgene; UpSET protein was found to localizes to transcriptional start sites of active genes throughout the genome. Misregulated heterochromatin is apparent by suppressed position effect variegation of a wm4 allele in heterozygous upset-deleted flies. This result applies to heterochromatin genes generally using nascent-RNA sequencing in the upset-mutant S2 lines. These findings support a critical role for UpSET in maintaining heterochromatin, perhaps by delimiting the active chromatin environment (McElroy, 2017).

Differences and similarities between Drosophila and mammalian 3' end processing of histone pre-mRNAs

Nuclear extracts from Drosophila Kc cells were used to characterize 3' end processing of Drosophila histone pre-mRNAs. Drosophila Stem-loop binding protein (SLBP) plays a critical role in recruiting the U7 snRNP (Dominski, 2003) to the pre-mRNA and is essential for processing all five Drosophila histone pre-mRNAs. The Drosophila processing machinery strongly prefers cleavage after a fourth nucleotide following the stem-loop and favors an adenosine over pyrimidines in this position. Increasing the distance between the stem-loop and the histone downstream element (HDE) does not result in a corresponding shift of the cleavage site, suggesting that in Drosophila processing the U7 snRNP does not function as a molecular ruler. Instead, SLBP directs the cleavage site close to the stem-loop. The upstream cleavage product generated in Drosophila nuclear extracts contains a 3' OH, and the downstream cleavage product is degraded by a nuclease dependent on the U7 snRNP, suggesting that the cleavage factor has been conserved between Drosophila and mammalian processing. A 2'O-methyl oligonucleotide complementary to the first 17 nt of the Drosophila U7 snRNA was not able to deplete the U7 snRNP from Drosophila nuclear extracts, suggesting that the 5' end of the Drosophila U7 snRNA is inaccessible. This oligonucleotide selectively inhibited processing of only two Drosophila pre-mRNAs and had no effect on processing of the other three pre-mRNAs. Together, these studies demonstrate that although Drosophila and mammalian histone pre-mRNA processing share common features, there are also significant differences, likely reflecting divergence in the mechanism of 3' end processing between vertebrates and invertebrates (Dominski, 2005).

Metazoan replication-dependent histone pre-mRNAs do not contain introns, and the only processing reaction necessary to generate mature histone mRNAs is a single endonucleolytic cleavage of the mRNA precursors (pre-mRNAs) to form the 3' end. Studies on 3' end processing were initially carried out in Xenopus oocytes using synthetic pre-mRNAs and sea urchin histone genes and later were facilitated by the development of an in vitro system based on nuclear extracts from mammalian cells. Replication-dependent histone pre-mRNAs contain two cis elements required for 3' end processing: a highly conserved stem-loop structure consisting of a 6-bp stem and a 4-nt loop and a less conserved histone downstream element (HDE) located ~15 nt 3' of the stem-loop. Mammalian histone pre-mRNAs are cleaved between the two elements, 5 nucleotides downstream of the stem-loop. The stem-loop is recognized by the stem-loop binding protein (SLBP), also referred to as the hairpin binding protein (HBP). The HDE interacts with the U7 snRNP, which contains an ~60-nt U7 snRNA, and this interaction is primarily mediated by base-pairing between the HDE and the 5' end of U7 snRNA. In vitro studies in mammalian nuclear extracts suggest that SLBP stabilizes binding of the U7 snRNP to the pre-mRNA and is essential in processing of only those pre-mRNAs that do not form sufficiently stable duplexes with the U7 snRNA. This role of SLBP in mammalian processing is most likely mediated by ZFP100, a 100-kDa zinc finger protein associated with the U7 snRNP and interacting with the SLBP/stem-loop complex. In addition to bridging the two factors bound to their respective sequence elements, ZFP100 may also play other roles in 3' end processing, possibly including the recruitment of the cleavage factor (Dominski, 2005).

Purification of the U7 snRNP from mammalian cells resulted in identification of two novel Sm-like proteins: Lsm10 and Lsm11, which replace the D1 and D2 Sm proteins present in the spliceosomal snRNPs. Lsm11 interacts in vitro with ZFP100 and plays a key role in recognizing the unique sequence of the Sm binding site in U7 snRNA. Orthologs of Lsm10 and Lsm11 are also found in the Drosophila U7 snRNP, demonstrating that the unique structure of the U7 snRNP in vertebrates and invertebrates is conserved. A counterpart of ZFP100 has not been yet identified in the Drosophila genome, suggesting that ZFP100 is either weakly conserved between vertebrates and invertebrates or processing of histone pre-mRNAs in Drosophila does not require this protein (Dominski, 2005).

Nuclear extracts from Drosophila S-2 and Kc cultured cells and embryos are capable of 3' end processing of presynthesized Drosophila histone pre-mRNAs. Nuclear extracts from Kc cells are also capable of cotranscriptional processing of histone pre-mRNAs. Unlike the auxiliary role played by SLBP in mammalian in vitro processing, Drosophila SLBP is indispensable for processing of all Drosophila histone pre-mRNAs. This observation suggests that Drosophila SLBP plays a much more important role in recruiting the U7 snRNP to the pre-mRNA than it does in the mammalian processing. This study uses an in vitro system based on Drosophila nuclear extracts to characterize 3' end processing of Drosophila histone pre-mRNAs and to define differences and similarities in processing between this model invertebrate processing system and processing in mammalian nuclear extracts (Dominski, 2005).

These studies demonstrate that although Drosophila and mammalian histone pre-mRNA processing occur with similar chemistry and both require SLBP and the U7 snRNP, the two mechanisms differ significantly in the relative importance of these trans-acting factors and in the specification of the cleavage site (Dominski, 2005).

Drosophila nuclear extracts cleave histone pre-mRNAs after the fourth nucleotide following the stem-loop and prefer an adenosine preceding the cleavage site. Consistent with this, all natural Drosophila histone pre-mRNAs contain an adenosine in this position. If the fourth nucleotide is changed to a pyrimidine, cleavage is also efficient after an adenosine at the third position but not after an adenosine located 5 nt downstream of the stem-loop, i.e., at the site exclusively utilized during mammalian processing. Sea urchin histone mRNAs, the only other invertebrate histone mRNAs with the characterized 3' ends, terminate with an ACCA consensus sequence. Thus, cleavage after the fourth nucleotide following the stem-loop may be a general feature of 3' end processing of invertebrate histone pre-mRNAs. Both Drosophila and mammalian processing machineries are similar in their extreme resistance to EDTA, generation of a 3' hydroxyl group at the end of the upstream cleavage product, and degradation of the downstream cleavage product by a U7 snRNP dependent activity. These results suggest that both processing machineries utilize the same or a highly related cleavage factor in 3' end processing of histone pre-mRNAs (Dominski, 2005).

In mammalian processing, the site of cleavage is determined by the position of the HDE, and moving the HDE, and, hence, the U7 snRNP, away from the stem-loop by as few as 4 nt results in a corresponding shift of the cleavage site. This observation led to the hypothesis that U7 snRNP recruits the cleavage factor to the pre-mRNA and acts as a molecular ruler to specify the cleavage site. SLBP bound to the stem-loop facilitates binding of the U7 snRNP to the HDE but does not play a direct role in recruitment of the cleavage factor. Consistent with this model, removal of SLBP, or using a substrate that cannot bind SLBP, reduces processing activity but does not abolish it (Dominski, 2005).

In contrast to mammalian processing, processing of Drosophila histone pre-mRNA is absolutely dependent on SLBP. In addition, increasing the distance between the stem-loop and the HDE by 4 or 8 nt in Drosophila histone pre-mRNA moved the cleavage site only 1 nt upstream from its normal position and did not abolish processing at the normal site. Larger insertions between the stem-loop and the HDE resulted in low efficiency cleavage further away from the stem-loop, but cleavage at these sites was still dependent on SLBP. This is in direct contrast to mammalian histone processing, where cleavage at the distant sites is independent of SLBP. Thus, in Drosophila processing the U7 snRNP does not function as a molecular ruler, but instead SLBP plays the critical role in specifying the cleavage site (Dominski, 2005).

To explain the observed differences between processing in Drosophila and mammalian nuclear extracts, it is proposed that within the Drosophila processing complex SLBP tightly interacts with the U7 snRNP, and this interaction is essential for bringing the U7 snRNP to the pre-mRNA. The two factors remain associated even if their respective binding sites are separated by a larger distance, likely by looping out the inserted nucleotides. The mutant pre-mRNAs are preferentially cleaved close to the stem-loop, reflecting the critical role of SLBP in forming the processing complex, although the precise position of the cleavage site and efficiency of processing depends on the size of the insert. In mammalian processing, the region between the stem-loop and the HDE is either rigidified, thus precluding looping out the inserted nucleotides, as previously suggested, or the interaction between SLBP and the U7 snRNP is relatively weak and disrupted by larger insertions, so binding of the U7 snRNP to the pre-mRNA depends solely on the base-pairing interaction. It is likely that in Drosophila processing the cleavage factor is recruited to histone pre-mRNA by interaction with both the U7 snRNP and SLBP, and neither factor is competent to carry out this function individually (Dominski, 2005).

In mammalian nuclear extracts, processing of histone pre-mRNAs is efficiently inhibited by relatively short 2'O-methyl oligonucleotides complementary to the 5' end of the mammalian U7 snRNA. These oligonucleotides, including a 10-mer, are also very efficient in depleting the U7 snRNP from nuclear extracts and were successfully used to affinity purify U7 snRNP from mammalian cells, demonstrating that the 5' end of the mammalian U7 snRNA is readily accessible. In contrast, two relatively long oligonucleotides, alphaDa, complementary to the first 17 nt of the Drosophila U7 snRNA, and alphaDb, complementary to nt 4-23, were not effective in depleting the U7 snRNP from Drosophila nuclear extracts. These results suggest that the 5' end of U7 snRNA is not accessible in the Drosophila U7 snRNP (Dominski, 2005).

Surprisingly, the alphaDa 2'O-methyl oligonucleotide abolished processing of the dH3* and dH1* pre-mRNAs (hybrid pre-mRNAs consisting of the stem-loop and cleavage site from the mouse H2a-614 pre-mRNA) but did not significantly affect processing of the other three Drosophila histone pre-mRNAs. Three additional oligonucleotides complementary to the regions of the U7 snRNP located closer to the Sm binding site effectively blocked processing of all five histone pre-mRNAs. It is not understood why processing of only two Drosophila pre-mRNAs is affected by the alphaDa oligonucleotide and which features of the HDEs make processing of the Drosophila pre-mRNAs either sensitive or resistant to this oligonucleotide. Selective inhibition of processing by the alphaDa oligonucleotide depending on the type of pre-mRNA used in the reaction suggests that blocking of the U7 snRNA must occur during processing. One possibility is that the U7 snRNP is initially recruited to the pre-mRNA solely by SLBP bound to the pre-mRNA, and later this interaction is followed by formation of a duplex between the HDE and the U7 snRNA, as a result of unmasking of the 5' end of U7 snRNA. The alphaDa oligonucleotide might block binding of the U7 snRNA to the HDE in the hybrid dH1* and dH3* pre-mRNAs, but not in the other pre-mRNAs, during this later step, while the other oligonucleotides block binding to all the HDEs (Dominski, 2005).

Overall, thes studies indicate that the structure of the 5' end of the Drosophila U7 snRNA and the mechanism of its initial interactions with the HDE differ significantly from the recognition of the HDE in processing of mammalian histone pre-mRNAs (Dominski, 2005).

In vitro processing of all five Drosophila histone pre-mRNAs is absolutely dependent on SLBP. This study has demonstrated that SLBP is essential for recruitment of the U7 snRNP to the pre-mRNA. The necessity of SLBP for recruitment of the U7snRNP to the Drosophila pre-mRNAs suggests that either Drosophila HDEs are unable to form a strong duplex with the U7 snRNA or that the interaction of the U7 snRNP with the SLBP/pre-mRNA complex is necessary to promote base-pairing by making the 5' end of U7 snRNA accessible (Dominski, 2005).

Both the 5' end of the Drosophila U7 snRNA and Drosophila HDEs are AU rich, allowing a number of possible base-pair schemes for making a duplex between the two RNAs. It is hypothesized that the most likely alignment used during processing is the one that allows formation of the largest number of base pairs between the purine core of the HDE and the CUCUUU sequence in the U7 snRNA and not necessarily the alignment that allows formation of the overall most stable duplex. The CUCUUU sequence is highly conserved among all known U7 snRNAs and is involved in recognition of the purine core in sea urchin and mammalian histone pre-mRNAs. A 3-nt mutation within the purine core of the hybrid dH3* pre-mRNA abolishes processing, whereas a 6-nt mutation within the AU-rich region immediately downstream of the purine core only partially inhibits processing. These results support the interpretation that base-pairing between the U7 snRNA and the purine core is critical, whereas formation of additional base in other regions increases the efficiency of Drosophila processing. It is also possible that the base-pairing interaction is limited to the purine core and the CUCUUU sequence in the U7 snRNA, whereas the AU-rich sequences in the U7 snRNA and the HDE are brought together by protein-protein interactions (Dominski, 2005).

This study demonstrated that the HDE of the hybrid dH3* pre-mRNA can abolish processing of the full-length substrate, presumably by sequestering the U7 snRNP, only when present at very high concentrations. Interestingly, this weak interaction of Drosophila HDEs with the U7 snRNP is sufficient to recruit a 5'-3' exonuclease that specifically degrades the downstream cleavage product in a U7 dependent manner. Thus, the endonucleolytic cleavage must require much stronger binding of the U7 snRNP to the pre-mRNA, while degradation of the DCP by an exonuclease may require only loose association of the HDE with the U7 snRNP (Dominski, 2005).

The most notable difference between histone pre-mRNA processing in Drosophila and mammalian nuclear extracts is the absolute dependence of Drosophila processing on SLBP and the role of SLBP in specifying the cleavage site close to the stem-loop. The Drosophila U7 snRNP does not function as a molecular ruler in processing and this feature most likely reflects a critical role of SLBP in recruiting the cleavage factor as well as the U7 snRNP, to histone pre-mRNA. These data suggest that SLBP and the U7 snRNP may form a tight complex on the histone pre-mRNA, and this complex remains stable even in the presence of large insertions between the stem-loop and the HDE (Dominski, 2005).

The similarities in the chemistry of the cleavage reaction, including preference for an adenosine preceding the cleavage site and generation of the 3'OH group in the presence of EDTA, as well as degradation of the downstream cleavage product by a U7-dependent 5'-3' exonuclease suggest that the cleavage factor has been conserved between Drosophila and mammalian processing. It will be of interest to determine whether there are factors unique to only one of these two types of organisms emphasizing long evolutionary distance and the divergence between vertebrates and invertebrates (Dominski, 2005).

U7 snRNA mutations in Drosophila block histone pre-mRNA processing and disrupt oogenesis

Metazoan replication-dependent histone mRNAs are not polyadenylated, and instead terminate in a conserved stem–loop structure generated by an endonucleolytic cleavage involving the U7 snRNP, which interacts with histone pre-mRNAs through base-pairing between U7 snRNA and a purine-rich sequence in the pre-mRNA located downstream of the cleavage site. Null mutations of the single Drosophila U7 gene were generated and U7 snRNA was demonstrated to be required in vivo for processing all replication-associated histone pre-mRNAs. Mutation of U7 results in the production of poly A+ histone mRNA in both proliferating and endocycling cells because of read-through to cryptic polyadenylation sites found downstream of each Drosophila histone gene. A similar molecular phenotype also results from mutation of Slbp, which encodes the protein that binds the histone mRNA 3' stem–loop. U7 null mutants develop into sterile males and females, and these females display defects during oogenesis similar to germ line clones of Slbp null cells. In contrast to U7 mutants, Slbp null mutations cause lethality. This may reflect a later onset of the histone pre-mRNA processing defect in U7 mutants compared to Slbp mutants, due to maternal stores of U7 snRNA. A double mutant combination of a viable, hypomorphic Slbp allele and a viable U7 null allele is lethal, and these double mutants express polyadenylated histone mRNAs earlier in development than either single mutant. These data suggest that SLBP and U7 snRNP cooperate in the production of histone mRNA in vivo, and that disruption of histone pre-mRNA processing is detrimental to development (Godfrey, 2006).

Chromosome duplication during the cell cycle requires the production of histones during S phase to package newly replicated DNA into chromatin. Bulk histone production during S phase is achieved through the biosynthesis of replication-dependent histone mRNAs, which are cell-cycle regulated and accumulate only in S phase. In animal cells these histone mRNAs are unique: The 3' end terminates in a conserved 26-nt sequence that forms a stem–loop rather than in a poly A+ tail. Since histone genes lack introns, the only processing step required for mature histone mRNA production is endonucleolytic cleavage of the pre-mRNA to form the 3' end of the mRNA. Much of the cell-cycle regulation of histone mRNAs is post-transcriptional and is mediated by the 3' end of the mRNA. Thus, a complete understanding of cell-cycle-regulated histone mRNA production requires a full understanding of the factors required for histone pre-mRNA processing (Godfrey, 2006).

The processing of histone pre-mRNAs requires two cis elements and a number of trans-acting factors. The cis elements are the stem–loop at the 3' end of histone mRNA and a purine-rich region downstream of the cleavage site, termed the histone downstream element (HDE). A protein called stem–loop binding protein (SLBP) or hairpin binding protein (HBP) specifically binds the 3' end of histone mRNA. SLBP is required for histone pre-mRNA processing in vivo and accompanies the mRNA to the cytoplasm, where it promotes the translation of the histone mRNA. The HDE binds U7 snRNP by base-pairing with the 5' end of U7 snRNA. In mammals, SLBP, the U7 snRNP, and a U7 snRNP-associated zinc finger protein called ZFP100 cooperate to recruit an endonuclease complex that cleaves the pre-mRNA. Recent evidence indicates that CPSF73, a component of the complex that mediates AAUAAA-directed cleavage prior to polyadenylation, is the likely endonuclease. This revealed some unexpected overlap in the machinery carrying out histone pre-mRNA processing and canonical polyadenylation (Godfrey, 2006).

The U7 snRNA is a small RNA (55–70 nt) that, like the spliceosomal snRNAs, contains both a trimethyl guanosine cap and an Sm binding site, which is essential for its function. The Sm site in these snRNAs stably binds a complex of seven related proteins of the LSm/Sm family to form the core snRNP particle. Proteins of the LSm/Sm family share a common tertiary structure called the Sm fold that assembles into hexameric or heptameric rings capable of binding single-stranded RNA. The U snRNPs contain a heptameric Sm ring, with each of the seven individual subunits making a specific contact with a residue in the Sm binding site of the snRNA. The heptameric Sm ring of spliceosomal snRNPs contains the proteins SmB/B', SmD1, SmD2, SmD3, SmE, SmF, and SmG. In contrast, the U7 snRNP contains five of these Sm proteins (B/B1, D3, E, F, G) and two novel Sm proteins called LSm10 and LSm11 that replace SmD1 and SmD2 of the spliceosomal snRNPs. The Sm site found in U7 snRNAs is distinct from the Sm site in spliceosomal snRNAs and is responsible for incorporation of LSm10 and LSm11 into the U7 snRNP. In addition to the Sm fold that participates in ring formation, LSm11 contains an NH2 terminal extension that makes contacts with ZFP100 and possibly other components of the histone pre-mRNA processing machinery (Godfrey, 2006).

The role of U7 snRNP in histone pre-mRNA processing has been examined primarily in nuclear extract systems that support the processing of synthetic histone pre-mRNAs, and by monitoring the processing of histone pre-mRNAs injected into Xenopus ooctyes. Complementary mutations in U7 snRNA and the HDE provided early evidence that base-pairing between the 5' end of U7 and the HDE was an important part of U7 snRNP function. Furthermore, blocking the 5' end of the U7 snRNA with a complementary oligonucleotide specifically inhibits processing of synthetic histone pre-mRNAs in nuclear extracts. However, the contribution of U7 snRNA to endogenous histone mRNA biosynthesis and whether this contribution is important for animal development have not been examined. To explore these issues, U7 snRNA mutations in Drosophila were generated and characterized (Godfrey, 2006).

Drosophila SLBP, U7 snRNA, and U7 snRNP specific proteins Lsm10 and Lsm11, have all been identified, and steps have been taken to characterize them genetically. Mutations in the Drosophila Slbp gene block normal histone pre-mRNA processing during embryonic development and result in production of polyadenylated histone mRNAs as a consequence of read-through past the normal processing site. This occurs because each of the five Drosophila histone genes contains cryptic polyadenylation sites downstream of the HDE that are utilized in the absence of SLBP. Null mutations of Slbp cause lethality during larval and pupal stages, presumably because of the histone processing defects, although the precise cause of lethality is not known. Slbp mutant cells are capable of replicating chromatin, likely because the inappropriate polyadenylated mRNAs are translated. A hypomorphic Slbp mutant allele that produces reduced amounts of SLBP protein results in the production of both normal and poly A+ histone mRNAs during embryogenesis, but does not cause lethality. However, these viable mutant females lay eggs that contain reduced amounts of histone mRNA and protein and do not develop. Thus, SLBP is required during both zygotic development and oogenesis (Godfrey, 2006).

This study compared mutations in the U7 snRNA gene, and the resulting phenotypes were compared with those caused by mutation of Slbp. The results indicate that U7 snRNA is required for normal histone mRNA biosynthesis during Drosophila development and that, like Slbp mutations, loss of U7 snRNA results in the production of polyadenylated histone mRNAs. However, unlike Slbp null mutants, U7 null mutants are viable, but both males and females are sterile. This difference in terminal phenotype is most likely because the maternal supply of U7 snRNA delays the onset of the histone processing defect in U7 mutants relative to Slbp mutants, which do not have a significant maternal supply of SLBP protein. Both U7 and SLBP are required for normal histone mRNA biosynthesis in the female germ line, and mutation of either gene disrupts oogenesis. These data indicate that loss of SLBP and U7 cause similar molecular phenotypes in Drosophila and suggest that early expression of this molecular phenotype prevents normal development (Godfrey, 2006).

Drosophila stem cells share a common requirement for the histone H2B ubiquitin protease scrawny: Scrawny interacts with PAF1 and likely opposes BRE1 action on H2B thus participating in a conserved pathway of chromatin regulation linking H2B ubiquitylation with H3K4me3 methylation

Stem cells within diverse tissues share the need for a chromatin configuration that promotes self-renewal, yet few chromatin proteins are known to regulate multiple types of stem cells. A Drosophila gene, scrawny (scny), encoding a ubiquitin-specific protease, is required in germline, epithelial, and intestinal stem cells. Like its yeast relative UBP10, Scrawny deubiquitylates histone H2B and functions in gene silencing. Consistent with previous studies of this conserved pathway of chromatin regulation, scny mutant cells have elevated levels of ubiquitinylated H2B and trimethylated H3K4. These findings suggest that inhibiting H2B ubiquitylation through scny represents a common mechanism within stem cells that is used to repress the premature expression of key differentiation genes, including Notch target genes (Buszczak, 2009).

Stem cells are maintained in an undifferentiated state by signals they receive within the niche and are subsequently guided toward particular fates upon niche exit. Within ES cells and during differentiation, cell state changes are controlled at the level of chromatin by alterations involving higher order nucleosome packaging and histone tail modifications. Polycomb group (PcG) and Trithorax group (trxG) genes influence key histone methylation events at the promoters of target genes, including H3K27 and H3K4 modifications associated with gene repression and activation, respectively, but few other genes with a specific role in stem cells are known (Buszczak, 2009).

Histone H2A and H2B mono-ubiquitylation play fundamental roles in chromatin regulation, and H2A ubiquitylation has been linked to PcG-mediated gene repression and stem cell maintenance. The mammalian Polycomb repressive complex 1 (PRC1) component RING1B is a H2A ubiquitin ligase that is required to block the elongation of poised RNA polymerase II on bivalent genes in ES cells. Mutations in the PRC1 component, BMI-1, the ortholog of Psc in the mammalian PRC1, complexes with RING1B, and causes multiple types of adult stem cells to be prematurely lost. The role of H2B ubiquitylation in stem cells is unclear, however. In yeast, ubiquitylation of Histone H2B by the RAD6 and BRE1 ligases controls H3K4 methylation (H3K4me3), a process that requires the polymerase accessory factor PAF1. Conversely, H2B deubiquitylation by the ubiquitin-specific protease (USP) family member UBP10 is required for silencing telomeres, rDNA and other loci. The Drosophila homolog of BRE1, dBRE1, also is needed for H3K4 methylation, suggesting that this pathway is conserved. Furthermore, the Drosophila ubiquitin-specific protease USP7 is part of a complex that selectively deubiquitylates H2B and genetically interacts with PcG mutations. Mutations in another USP family member, Nonstop, increase H2B ubiquitylation and cause axon targeting defects in the eye (Buszczak, 2009).

In order to gain further insight into the role of H2B ubiquitylation in stem cells, a novel Drosophila gene, scrawny (scny) (CG5505), was identified, whose encoded USP family protein shares homology with human USP36 and among yeast USPs closely matches UBP10 within the core protease domain. Strains bearing scny insertions, except for a viable GFP protein trap (CA06690), were female sterile or lethal, and proved to be allelic. Transposon excision or expression of a scny-RB cDNA reverts the phenotype of tested alleles. An anti-SCNY antibody raised against a domain common to all SCNY isoforms recognizes wild type and SCNY-GFP on a Western blot. SCNY protein levels in homozygous third instar larvae are greatly reduced in lethal mutants, and SCNY expression is also lower in stem cell-enriched ovarian tissue from adults homozygous for the sterile d06513 allele. Consistent with a role in gene silencing, several scny mutations act as dominant suppressors of position effect variegation (Buszczak, 2009).

Further studies strongly suggested that SCNY functions in vivo as an H2B-ubiquitin protease. Recombinant full-length SCNY protein, but not a version bearing a point mutation in the protease domain, efficiently deubiquitylates histone H2B in vitro. scnyf01742 homozygous tissue contains levels of Ub-H2B that are elevated at least twofold compared to wild type. As expected if Ub-H2B is required for H3K4 methylation, clones of homozygous scnye00340 mutant cells stain more strongly for H3K4me3 than heterozygous cells. Consistent with a direct rather than an indirect action on Ub-H2B levels, anti-SCNY antibodies co-immunoprecipitate H2B from Drosophila embryonic nuclear extracts. Moreover, epitope-tagged SCNY co-immunoprecipitates Drosophila PAF1, but not Cyclin T (or several other tested chromatin proteins) when co-expressed in S2 tissue culture cells. Together, these data support the view that SCNY participates in a conserved pathway of chromatin regulation linking H2B ubiquitylation with H3K4me3 methylation. Because the effects of scny mutation on Ub-H2B and H3K4me3 are opposite to those of dBre1 mutation, SCNY likely opposes dBRE1 action on H2B, just as UBP10 opposes BRE1 action on H2B in yeast (Buszczak, 2009).

Drosophila male and female gonads contain well characterized germline stem cells (GSCs) that allow the effects of genes on stem cell maintenance to be quantitatively analyzed. High levels of scny expression were observed in female and male GSCs using SCNY-GFP and identical staining was observed using anti-SCNY immunofluoresence. SCNY protein resides in cell nuclei and is enriched in nucleoli. In sterile or semi-fertile scny mutant adults, the numbers of germline stem cells surrounding the testis hub and within germaria were clearly reduced. The half-lives of female GSCs bearing clones of three different scny alleles were all sharply reduced. Later follicular development was also abnormal suggesting that scny continues to function after the stem cell stage. However, previous studies indicate that accelerated GSC loss is a specific phenotype, and hence that scny has a preferential requirement in GSCs (Buszczak, 2009).

A known mechanism of increased GSC loss is the premature activation of differentiation genes. Staining germaria with an antibody specific for multiple sites of histone H3 acetylation (H3-Ac) suggested that scny mutation affects the global chromatin organization of GSCs. Wild type GSCs contain lower levels of H3-Ac than slightly older germ cells within cysts. Presumptive GSCs located in the GSC niche in scny mutants frequently stained more strongly, suggesting that they have begun to upregulate general transcription. Some scny GSC-like cells also expressed bag-of-marbles (bam), a key cystoblast differentiation gene, and GSC-like cells in scnyd06513; bamΔ86 mutant females persist in the germarium . However, it could not be completely ruled out that the observed increases in H3-Ac levels and bam expression were a result rather than a cause of the premature differentiation and loss of scny GSCs (Buszczak, 2009).

To determine if scny is also required in a very different type of stem cell, the epithelial follicle stem cell (FSC), the persistence of individual scny mutant FSCs was quantitatively. The half-life of FSCs mutant for scnyl(3)02331 was reduced more than 10-fold, while the scnyf01742 mutation also caused a sharp decline. However, mutant follicle cells continued to develop normally at later stages. Thus, scny is preferentially required to maintain FSCs as well as GSCs (Buszczak, 2009).

The largest population of Drosophila stem cells are the hundreds of multipotent intestinal stem cells (ISCs) that maintain the adult posterior midgut. ISCs signal to their daughters via Delta-Notch signaling to specify enterocyte vs. enteroendocrine cell fate, but the pathway must remain inactive in the ISCs themselves to avoid differentiation. Most ISCs (those about to produce enterocytes) express high levels of the Notch ligand Delta, allowing them to be specifically distinguished from other diploid gut cells. This study found that SCNY-GFP is expressed in ISCs suggesting that SCNY plays a role in these stem cells as well. While 7-day old normal adult midguts contain a high density of ISCs, as revealed by Delta staining, it was found that corresponding tissue from 7-day-old scnyf01742 or scnyf01742/scnyl(3)02331 escaper adults possess very few Delta-positive cells. ISCs are present in near normal numbers at eclosion, but are rapidly lost in the mutant adults, indicating that scny is required for ISC maintenance (Buszczak, 2009).

It is suspected that inappropriate Notch pathway activation was responsible for the premature ISC loss in scny mutants. dBre1 mutations strongly reduce Notch signaling, suggesting that Notch target genes are particularly dependent on H2B mono-ubiquitylation and H3K4 methylation. Consequently, scny mutations, which have the opposite effects on Ub-H2B and H3K4me3 levels, might upregulate Notch target genes, stimulating ISCs to differentiate prematurely. This idea was tested by supplementing the food of newly eclosed scnyf01742/scnyl(3)02331 adults with 8 mM DAPT, a gamma-secretase inhibitor that blocks Notch signaling and phenocopies Notch mutation when fed to wild type animals. scnyf01742/scnyl(3)02331 DAPT-treated adults remained healthy and the guts of 7-day old animals still contained many ISCs, although not as many as wild type. Tumors like those produced in wild type animals fed DAPT were not observed. Thus, in these animals endogenous stem cell loss can be slowed by drug treatment (Buszczak, 2009).

These experiments provide strong evidence that a pathway involving the ubiquitin protease Scrawny and the ubiquitin ligase dBRE1 controls the levels of Ub-H2B, and H3K4me3 at multiple target sites in the Drosophila genome. Although, other ubiquitin proteases also act on Ub-H2B in Drosophila, the direct interaction between SCNY and H2B, and the strong effects of scny mutations argue that it plays an essential, direct role in silencing genomic regions critical for cellular differentiation, including Notch target genes. SCNY interacts with the RNA polymerase accessory factor complex component, PAF1. Upregulation of H2B ubiquitinylation and H3 methylation in yeast is mediated by the PAF1 complex and is associated with elongating RNA Pol II. Drosophila PAF1 is required for normal levels of H3K4me3 at the hsp70 gene, and another PAF1 complex member, RTF1, is needed for H3K4 methylation and Notch target gene expression. Indeed, the pathway connecting Ub-H2B, H3K4me3 and gene silencing appears to be conserved in organisms as distant as Arabidopsis. A human protein closely related to SCNY, USP36, is overexpressed in ovarian cancer cells, and the results suggest it may act as an oncogene by suppressing differentiation (Buszczak, 2009).

Above all, these experiments indicate that SCNY-mediated H2B deubiquitylation is required to maintain multiple Drosophila stem cells, including progenitors of germline, epithelial and endodermal lineages. In ES cells and presumably in adult stem cells, many differentiation genes contain promoter-bound, arrested RNA Pol II and are associated with Polycomb group proteins. It is envisioned that in the niche environment SCNY activity overrides that of dBRE1, keeping levels of Ub-H2B (and hence H3K4me3) low at key differentiation genes. Upon exit from the niche, the balance of signals shifts to favor H2B ubiquitylation, H3K4 trimethylation, and target gene activation. Thus, the control of H2B ubiquitylation, like H2A ubiquitylation, plays a fundamental interactive role in maintaining the chromatin environment of the stem cell state (Buszczak, 2009).

Paternal diet defines offspring chromatin state and intergenerational obesity

The global rise in obesity has revitalized a search for genetic and epigenetic factors underlying the disease. This study presents a Drosophila model of paternal-diet-induced intergenerational metabolic reprogramming (IGMR) and identifies genes required for its encoding in offspring. Intriguingly, as little as 2 days of dietary intervention in fathers elicits obesity in offspring. Paternal sugar acts as a physiological suppressor of variegation, desilencing chromatin-state-defined domains in both mature sperm and in offspring embryos. Requirements were identified for H3K9/K27me3-dependent reprogramming of metabolic genes in two distinct germline and zygotic windows. Critically, evidence is found that a similar system may regulate obesity susceptibility and phenotype variation in mice and humans. The findings provide insight into the mechanisms underlying intergenerational metabolic reprogramming and carry profound implications for understanding of phenotypic variation and evolution (Ost, 2014).

This study shows that acute dietary interventions, as short as 24 hr, have the capacity to modify F1 offspring phenotype via the male germline. Reprogramming occurs in response to dietary manipulations over a physiological range, and phenotypic outcomes require polycomb- and H3K9me3-centric plasticity in spatially and chromatin-state-defined regions of the genome. The eye color shifts in wm4 h offspring and the reduced fat body H3K9me3 staining in adult IGMR offspring supports the conclusions, first, that there are chromatin state changes and, second, that these are stable lifelong. These data are corroborated by selective derepression of Su(var)3-9, SETBD1, Su(var)4-20, and polycomb-sensitive transcripts; chromatin-state-associated transcriptional rearrangements genome wide; selective reprogramming of highly dynamic histone-mark-defined regions; and the fact that intergenerational metabolic reprogramming (IGMR ) itself is sensitive to a string of distinct H3K9me3-centric and polycomb mutants. Although nontrivial, ChIP-seq comparisons of repressive chromatin architecture in mature sperm and multiple defined offspring tissues will be important to establishing the ubiquitousness of these regulatory events and the nature of intergenerational signal itself. These data highlight how acutely sensitive intergenerational control can be to even normal physiological changes, and they identify some of the first genes absolutely required for transmission evolution (Ost, 2014).

First categorized simply as heterochromatin versus euchromatin, multiple empirical models now divide the genome into 5 to 51 chromatin states, depending on the analysis. Paternal high sugar increases gene expression preferentially of heterochromatic-embedded genes in embryos. Specifically, these genes are characterized by active deposition of H3K9me3 and H3K27me3, by long distance from class I insulators, and by sensitivity to fully intact expression of Su(var)3-9, Su(var)4-20, SetDB1, Pc, and E(z) . The data support a model where phenotype has been evolutionarily encoded directly into the chromatin state of relevant loci. Specifically, an abundance of genes important to both cytosolic and mitochondrial metabolism appear to be embedded into H3K9me3- and distinct polycomb-dependent control regions. Indeed, GO analysis of the five chromatin colors indicate a largely mutually exclusive picture, in which functional pathways are not randomly distributed across chromatin states. The paternal IGMR data set revealed clear and strong overlaps with pathways of black (lamin-associated) and blue (polycomb) chromatin and included many key metabolic pathways, including glycolysis, TCA cycle, mitochondrial OxPhos, chitin, and polysaccharide metabolism, changes that could well prime the system for altered functionality given the appropriate stimulus. Indeed, paternal IGMR phenotype is a susceptibility to diet-induced obesity and is most readily observable upon high-sugar diet challenge evolution (Ost, 2014).

The data support a trans-acting mechanism. In the wm4h experiments, male offspring inherited their X chromosome and thus the reporter from their unchallenged mothers, i.e., the reporter allele never encounters the initial signal but is reproducibly reprogrammed. Further, the failure of Su(var) 4-20SP and SetDB11473 mutants to elicit IGMR responses in their wild-type offspring indicate that wild-type haploid sperm carry the same insufficient reprogramming template as their syncytial mutant counterparts. cis- and trans-acting mechanisms are not mutually exclusive though. Signals transmitted via paternal chromosomes, though likely transmitted in cis, may be manifest via expression of paternal transcripts, which then act in trans. Paternal reductions of Su(var)3-9, SetDB1, and Hp1, for instance, would affect the maternal genome in trans evolution (Ost, 2014).

Despite their genetic similarity, isogenic or congenic animals reared under controlled conditions exhibit measurable variation in essentially all phenotypes. Such variability in genome output is thought to arise largely from probabilistic or chance developmental events in early. This study mapped a mechanism that couples acute paternal feeding and zygotic chromatin state integrity directly to phenotypic output of the next generation. These same signatures predict obesity susceptibility in isogenic mouse and human obesity cohorts. Because acute circadian fluctuations in feeding are essentially constant over evolutionary timescales, they are the perfect mechanistic input upon which a system could evolve to ensure defined phenotypic variation within a given population evolution (Ost, 2014).

dRYBP counteracts chromatin-dependent activation and repression of transcription

Chromatin dependent activation and repression of transcription is regulated by the histone modifying enzymatic activities of the trithorax (trxG) and Polycomb (PcG) proteins. To investigate the mechanisms underlying their mutual antagonistic activities this study analyzed the function of Drosophila Ring and YY1 Binding Protein (dRYBP), a conserved PcG- and trxG-associated protein. dRYBP is ubiquitylated and binds ubiquitylated proteins. Additionally dRYBP was shown to maintain H2A monoubiquitylation, H3K4 monomethylation and H3K36 dimethylation levels and does not affect H3K27 trimethylation levels. Further it was shown that dRYBP interacts with the repressive SCE (Ring) and dKDM2 (Lysine (K)-specific demethylase 2) proteins as well as the activating dBRE1 protein. Analysis of homeotic phenotypes and post-translationally modified histones levels show that dRYBP antagonizes dKDM2 and dBRE1 functions by respectively preventing H3K36me2 demethylation and H2B monoubiquitylation. Interestingly, the results show that inactivation of dBRE1 produces trithorax-like related homeotic transformations, suggesting that dBRE1 functions in the regulation of homeotic genes expression. These findings indicate that dRYBP regulates morphogenesis by counteracting transcriptional repression and activation. Thus, they suggest that dRYBP may participate in the epigenetic plasticity important during normal and pathological development (Fereres, 2014).

Phf7 controls male sex determination in the Drosophila germline

Establishment of germline sexual identity is critical for production of male and female germline stem cells, as well as sperm versus eggs. This study identified PHD Finger Protein 7 (PHF7) as an important factor for male germline sexual identity in Drosophila. PHF7 exhibits male-specific expression in early germ cells, germline stem cells, and spermatogonia. It is important for germline stem cell maintenance and gametogenesis in males, whereas ectopic expression in female germ cells ablates the germline. Strikingly, expression of PHF7 promotes spermatogenesis in XX germ cells when they are present in a male soma. PHF7 homologs are also specifically expressed in the mammalian testis, and human PHF7 rescues Drosophila Phf7 mutants. PHF7 associates with chromatin, and both the human and fly proteins bind histone H3 N-terminal tails with a preference for dimethyl lysine 4 (H3K4me2). It is proposed that PHF7 acts as a conserved epigenetic 'reader' that activates the male germline sexual program (Yang, 2012).

Sex determination is key to sexual reproduction, and both somatic cells and germ cells need to establish sex-specific developmental fates. Germline sexual development is essential for the production of two distinct gametes, and underlies important differences in the regulation of male versus female fertility. In some species, germline stem cells are present in both males and females to sustain constant gamete production, but are regulated differently throughout development. In other species such as humans, sex-specific germ cell development produces a germline stem cell population only in males, whereas females have a much more limited capacity in making eggs. Defects in germline sexual development lead to a failure in gametogenesis, thus the study of germline sex determination is essential for understanding normal reproductive potential and treating infertility (Yang, 2012).

In some animals, such as mammals and Drosophila, the sex chromosome compositions of the soma and germline are interpreted independently, and the 'sex' of the germline must match that of the soma for proper germ cell development to occur. For example, patients with Klinefelter's Syndrome have an XXY sex chromosome constitution and are almost always infertile. These individuals develop somatically as males due to the presence of a Y chromosome but the germline suffers from severe atrophy, including the loss of premeiotic germline and germline stem cells. This is due to the presence of two X chromosomes in the germ cells, as the limited spermatogenesis in these patients is from germ cells that have lost one of the X chromosomes. In Drosophila, XX females that are somatically transformed into males exhibit a similar germline loss due to a conflict in sexual identity between the masculinized soma and XX germline. Thus, fruit flies are a valuable model organism for studying how germ cells establish a proper sexual identity by coordinating intrinsic signals and those coming from the soma (Yang, 2012).

In Drosophila, the presence of two X chromosomes promotes female somatic identity by activating an alternative splicing cascade that acts through Sex lethal (SXL) and Transformer (TRA), and ultimately leads to production of either the male or female forms of the transcription factors Doublesex (DSX) and Fruitless (FRU). DSX and FRU are responsible for virtually all sexually dimorphic somatic traits in Drosophila, with DSX being the key factor in the somatic gonad. In contrast, the germline does not determine its sex with this cascade and factors like TRA and DSX are not required in germ cells. Although SXL is required to promote female germ cell identity, its targets and mechanism of action in the germline are not known. The transcription factor OVO and the ubiquitin protease Ovarian Tumor (OTU) are also required in the female germline and thought to function upstream of SXL. Even less is known about how sexual identity is specified in male germ cells. Male germ cells receive a signal through the JAK/STAT pathway that promotes their sexual identity, but the downstream factors that are subsequently activated are not known. Similarly, how male germ cells respond to their own sex chromosome constitution is also not known (Yang, 2012).

This study reports a histone code reader, Plant Homeodomain (PHD) Finger 7 (PHF7), that acts in the Drosophila germline to promote male sexual identity. PHF7 is specifically expressed in male germ cells from early stages of development and is restricted to male germline stem cells (GSCs) and spermatogonia. Phf7 is required for GSC maintenance and proper entry into spermatogenesis. Interestingly, expression of Phf7 in female germ cells causes ablation of the female germline. Moreover, Phf7 affects sexual compatibility between germline and soma. Loss of Phf7 in XY germ cells alleviates the germline loss typically observed when XY germ cells are surrounded by a female soma, and expression of Phf7 can induce spermatogenesis in XX germ cells nurtured by male soma. These findings indicate that Phf7 is an essential factor in determining sexual development in the Drosophila germline, and suggest that activation of the male identity occurs through interaction with the germline epigenome (Yang, 2012).

The data indicate that Phf7 acts to promote a male identity in the germline. Loss of Phf7 function affected male GSC maintenance and spermatogenesis, but had no effect in females. Phf7-mutant GSCs exhibited a more female-like pattern of spectrosome localization, and male (XY) germ cells mutant for Phf7 were more compatible with a female soma than were wild-type male germ cells. Further, expression of PHF7 was able to masculinize the female germline: PHF7 expression induced apoptosis in developing XX germ cells and interacted with mutations in otu in a manner that indicates XX germ cells that express PHF7 are more male-like. Strikingly, PHF7 expression was able to induce spermatogenesis in XX germ cells when they are present in a male soma, something that XX germ cells are normally not able to do. Taken together, these results indicate that Phf7 promotes and is sufficient to induce male identity in the germline (Yang, 2012).

Sex determination is thought to be initiated early during development, and sex-specific differences in the male and female germline are first observed during embryogenesis. The data indicate that Phf7 plays a role in early germline sexual development, rather than a late role to regulate germ cell differentiation and gametogenesis. First, PHF7 expression is observed in the embryonic gonad and, in the adult, PHF7 is found in the GSCs and early gonia and disappears dramatically as gonia transition to spermatocytes. Further, forced PHF7 expression disrupts early female germ cell development, around the time when they are first forming GSCs. Expression of PHF7 after the early cystoblast stage (Bam-Gal4, UAS-Gal4) had no effect on the female germline, indicating that it can only affect early stages of female germ cell development. Phf7 mutants show defects in male GSC behavior and maintenance, and in the initial progression to form spermatocytes, but it is possible that these defects are due to even earlier problems in male sexual identity (Yang, 2012).

Germline sexual identity is determined by both the germ cell sex chromosome constitution and signals from the surrounding soma. Phf7 expression is activated in XX germ cells when in contact with a male soma and repressed in XY germ cells when contacting a female soma. However, in a female somatic environment, XY germ cells are somewhat more likely than XX germ cells to express Phf7, indicating that Phf7 may also respond to the sex chromosome constitution of the germ cells in addition to being regulated by the soma. Further, exogenous expression of Phf7 is required to promote spermatogenesis in XX germ cells when in a male soma. Thus, the Phf7 expression that is normally initiated in such germ cells by the male soma must either not be maintained, or may be insufficient to overcome the influence of the XX sex chromosome genotype (Yang, 2012).

It is likely that Phf7 is not acting alone to control male sexual identity. Phf7 mutant males are still able to undergo spermatogenesis, but at a much reduced capacity. This appears to be the null phenotype for Phf7 as ther mutants have lost significant portions of the coding sequence. Further, when PHF7 is expressed in XX germ cells present in a male soma, these germ cells can undergo spermatogenesis, but the penetrance of this phenotype is low. Interestingly, the rescue of spermatogenesis in these XX germ cells follows an 'all or nothing' pattern; either the rescue is largely complete to give full testes and sperm production, or little rescue is observed. Therefore, there appears to be a threshold that must be crossed to promote male germline sexual identity, and that once this threshold is met, those germ cells either take over the testis, or induce other germ cells to also follow the male pathway. The simplest explanation for both the incomplete block to spermatogenesis in Phf7 mutants and the incomplete rescue of spermatogenesis by Phf7 in XX males is that an additional factor (or factors) exists that promotes male identity in addition to Phf7. Such a factor could function parallel to Phf7 in a single pathway, or represent independent input regarding germline sex determination (e.g., independent signals from the soma that influence germline sex) (Yang, 2012).

PHD fingers, such as those found in PHF7, are best known for their ability to specifically bind histones that have been modified on their N-terminal tails, in particular methylated H3K4. This study shows that both Drosophila and human PHF7 can directly associate with dimethylated H3K4, indicating that PHF7 is indeed a histone code reader. It is uncommon for PHD domains to associate preferentially with H3K4me2 over H3K4me3, but this specificity has been observed previously, and is likely important for how PHF7 modulates expression of its targets. Both di- and trimethylated H3K4 are found at actively transcribed genes, but H3K4me2 is normally localized at the 5′ end of coding sequences, downstream of H3K4me3, which is near promoters. The two marks are also regulated by different demethylases. A few recent studies have started to dissect effects of H3K4me2 on gene transcription, but the exact mechanisms are not well understood. Some PHD finger proteins also contain other domains, such as those that modify histones enzymatically. This does not appear to be the case for PHF7, and the region of homology between PHF7 homologs of different species is restricted to the PHD domains. However, individual PHD fingers can bind modified histone tails independently, and it is yet unclear which PHD finger in PHF7 contacts H3K4me2 and what activities the others might have. The logic of how PHF7 is recruited to specific loci and affects chromatin structure and gene activity are interesting questions for future work (Yang, 2012).

Another point of interest is how a reader of such a common epigenetic mark would have a sex-specific role in regulating male germline identity. It has been observed that mutation of an H3K4me2 demethylase in Caenorhabditis elegans, which leads to increased dimethylation at H3K4, results in ectopic activation of male-specific germline genes. A similar mutation in Drosophila causes female germline developmental defects, which may be related to the germline atrophy observed when PHF7 expression was upregulated in female germ cells. These data are consistent with the hypothesis that H3K4me2 has a role in regulating the male germline genome. Interestingly, another germline chromatin factor, No child left behind (NCLB), has been identifed that is expressed in germ cells of both sexes but required for GSC function only in males. Thus, NCLB may cooperate with PHF7 in regulating the male GSC transcriptional program (Yang, 2012).

Based on sequence homology, orthologs of Phf7 are present in a wide range of mammalian species. Human and mouse PHF7 share extensive homology to Drosophila PHF7 throughout the N-terminus where the PHD fingers are present, and the results confirm that human PHF7 recognizes H3K4me2, similar to the fly protein. Interestingly, EST profiling indicates strong testis biases for Phf7 expression in many species, including humans, mice, rats, and dogs. Moreover, several studies that performed genome-wide RNA profiling from purified mouse germline populations indicate that mouse Phf7 expression is present in spermatogonia and is further induced in spermatocytes. Remarkably, human PHF7 was able to rescue fecundity defects in male flies mutant for Phf7. Thus, the sequence conservation observed between mammalian and Drosophila Phf7 represents true functional orthology (Yang, 2012).

As in Drosophila, germline sex determination in mouse is regulated at an early stage and is controlled by important signals from the soma, which for the mouse include retinoic acid and FGF9. Such signals regulate the timing of meiotic entry, which is different between the sexes, and also influence sex-specific programs of germline gene expression, such as expression of the key male-specific factor nanos2. Significant changes in germ cell chromatin occur during this critical time in germ cell development, including changes in the H3K4 methylation state. Thus, Phf7 represents a prime candidate for interpreting these chromatin changes in a sex-specific manner to regulate male-specific gene expression. It will be of great interest to determine whether Phf7 plays a critical role in mouse and human male germ cell development, as is proposed for Drosophila (Yang, 2012).

Asymmetric division of Drosophila male germline stem cell shows asymmetric histone distribution

A long-standing question concerns how stem cells maintain their identity through multiple divisions. It has been reported that pre-existing and newly synthesized histone H3 are asymmetrically distributed during Drosophila male germline stem cell (GSC) asymmetric division. This study shows that phosphorylation at threonine 3 of H3 (H3T3P) distinguishes pre-existing versus newly synthesized H3. Converting T3 to the unphosphorylatable residue alanine (H3T3A) or to the phosphomimetic aspartate (H3T3D) disrupts asymmetric H3 inheritance. Expression of H3T3A or H3T3D specifically in early-stage germline also leads to cellular defects, including GSC loss and germline tumors. Finally, compromising the activity of the H3T3 kinase Haspin enhances the H3T3A but suppresses the H3T3D phenotypes. These studies demonstrate that H3T3P distinguishes sister chromatids enriched with distinct pools of H3 in order to coordinate asymmetric segregation of "old" H3 into GSCs and that tight regulation of H3T3 phosphorylation is required for male germline activity (Xie, 2015).

Epigenetic phenomena are heritable changes in gene expression or function that can persist throughout many cell divisions without alterations in primary DNA sequences. By regulating differential gene expression, epigenetic processes are able to direct cells with identical genomes to become distinct cell types in humans and other multicellular organisms. However, with the exception of DNA methylation, little is known about the molecular pathways leading to epigenetic inheritance (Xie, 2015).

Prior research has shown that epigenetic events play particularly important roles in ensuring both proper maintenance and differentiation of several stem cell populations. Many types of adult stem cells undergo asymmetric cell division to generate a self-renewed stem cell and a daughter cell that will subsequently differentiate. Mis-regulation of this balance leads to many human diseases, ranging from cancer to tissue dystrophy to infertility. However, the mechanisms of stem cell epigenetic memory maintenance as well as how loss of this memory contributes to disease remain unknown (Xie, 2015).

During the asymmetric division of the Drosophila male germline stem cell (GSC), the pre-existing histone 3 (H3) is selectively segregated to the self-renewed GSC daughter cell whereas newly synthesized H3 is enriched in the differentiating daughter cell known as a gonialblast (GB) (Tran, 2012). In contrast, the histone variant H3.3, which is incorporated in a replication-independent manner, does not exhibit such an asymmetric pattern. Furthermore, asymmetric H3 inheritance occurs specifically in asymmetrically dividing GSCs, but not in the symmetrically dividing progenitor cells. These findings demonstrate that global asymmetric H3 histone inheritance possesses both molecular and cellular specificity. The following model is proposed to explain these findings (Xie, 2015).

First, the cellular specificity exhibited by the H3 histone suggests that global asymmetric histone inheritance occurs uniquely in a cell-type (GSC) where the mother cell must divide to produce two daughter cells each with a unique cell fate. Because this asymmetry is not observed in symmetrically dividing GB cells, asymmetric histone inheritance is proposed to be a phenomenon specifically employed by GSCs to establish unique epigenetic identities in each of the two daughter cells. Second, as stated previously, a major difference between H3 and H3.3 is that H3 is incorporated to chromatin during DNA replication, while H3.3 variant is incorporated in a replication-independent manner. Because this asymmetric inheritance mode is specific to H3, a two-step model is proposed to explain asymmetric H3 inheritance: (1) prior to mitosis, pre-existing and newly synthesized H3 are differentially distributed on the two sets of sister chromatids, and (2) during mitosis, the set of sister chromatids containing pre-existing H3 is segregated to GSCs, while the set of sister chromatids enriched with newly synthesized H3 is segregated to the GB that differentiates (Tran, 2012; Tran, 2013; Xie, 2015 and references therein)

This study reports that a mitosis-enriched H3T3P mark acts as a transient landmark that distinguishes sister chromatids with identical genetic code but different epigenetic information, shown as pre-existing H3-GFP and newly synthesized H3-mKO. By distinguishing sister chromatids containing different epigenetic information, H3T3P functions to allow these molecularly distinct sisters to be segregated and inherited differentially to the two daughter cells derived from one asymmetric cell division. The selective segregation of different populations of histones likely allows these two cells to assume distinct fates: self-renewal versus differentiation. Consequently, loss of proper epigenetic inheritance might lead to defects in both GSC maintenance and GB differentiation, suggesting that both cells need this active partitioning process to either 'remember' or 'reset' their molecular properties (Xie, 2015).

The temporal and spatial specificities of H3T3P make it a great candidate to regulate asymmetric sister chromatid segregation. First, H3T3P is only detectable from prophase to metaphase, the window of time during which the mitotic spindle actively tries to attach to chromatids through microtubule-kinetochore interactions. Second, the H3T3P signal is enriched at the peri-centromeric region, where kinetochore components robustly crosstalk with chromatin-associate factors. Third, H3T3 shows a sequential order of phosphorylation, first appearing primarily on sister chromatids enriched with pre-existing H3 and then subsequently appearing on sister chromatids enriched with newly synthesized H3 as the GSC nears metaphase. The distinct temporal patterns shown by H3T3P are unique to GSCs and would allow the mitotic machinery to differentially recognize sister chromatids bearing distinct epigenetic information; an essential step necessary for proper segregation during asymmetric GSC division. Furthermore, the tight temporal control of H3T3 phosphorylation suggests that rather than serving as an inherited epigenetic signature, H3T3P may act as transient signaling mark to allow for the proper partitioning of H3. It is hypothesized that H3T3P needs to be under tight temporal control in order to ensure proper H3 inheritance and germline activity (Xie, 2015).

These studies have shown that H3T3P is indeed subject to stringent temporal controls during mitosis. The H3T3P mark is undetectable during G2 phase. Upon entry to mitosis, sister chromatids enriched with pre-existing H3-GFP histone begin to show H3T3 phosphorylation prior to sister chromatids enriched with newly synthesized H3-mKO. As the cell continues to progress toward metaphase, H3T3P signal begins to appear on sister chromatids enriched with newly synthesized H3-mKO. Such a tight regulation of H3T3P is compromised when levels of H3T3P are altered due to the incorporation of mutant H3T3A or H3T3D. Incorporation of the H3T3A mutant results in a significant decrease in the levels of H3T3P on sister chromatids throughout mitosis, such that neither sister becomes enriched with H3T3P as the GSC progresses toward metaphase. Conversely, incorporation of the H3T3D mutant would result in seemingly elevated levels of H3T3P early in mitosis. Although H3T3A and H3T3D act in different ways, both mutations significantly disrupt the highly regulated temporal patterns associated with H3T3 phosphorylation, the result of which is randomized H3 inheritance patterns and germ cell defects in testes expressing either H3T3A or H3T3D (Xie, 2015).

To further evaluate the extent of H3T3A and H3T3D roles in the segregation of sister chromatids enriched with different populations of H3 during mitosis, all possible segregation patterns were modeled in male GSCs, and these estimates were compared to the experimental results. To simplify the calculations, two important assumptions were made: first, nucleosomal density was assumed to be even throughout the genome. This assumption allows the inference that the overall fluorescent signal contributed by each chromosome is proportional to their respective number of DNA base pairs. Second, by quantifying pre-existing H3-GFP asymmetry in anaphase and telophase GSCs, it was estimated that the establishment of H3-GFP asymmetry is ∼4-fold biased, i.e., 80% on one set of sister chromatids and 20% on the other set of sister chromatids, based on quantification of GFP signal in anaphase and telophase GSCs (Tran, 2012). With these two simplifying assumptions, both GFP and mKO ratios were caculated among all 64 possible combinations. If asymmetry is designed as a greater than 1.5-fold difference in fluorescence intensity, then based on a model of randomized sister chromatid segregation, it is estimated that a symmetric pattern should appear for 53.1% (34/64) of GSC-GB pairs whereas both conventional and inverted asymmetric patterns should occur with equal frequencies and account for 18.7% (12/64) of total GSC-GB pairs. The remaining 9.4% (6/64) of GSC-GB pairs should produce histone inheritance patterns with a 1.45- to 1.55-fold difference in signal intensity (Xie, 2015).

This estimation is close to the experimental data in both H3T3A- and H3T3D-expressing testes. Of the 64 quantified post-mitotic GSC-GB pairs in nos>H3T3A testes, ~71.9% showed symmetric inheritance pattern. Conventional and inverted asymmetric patterns were detected at 9.4% and 12.5%, respectively, and 6.3% at the borderline. Similarly, of the 57 quantified post-mitotic GSC-GB pairs in nos>H3T3D testes, ∼79.0% showed symmetric inheritance pattern. Conventional and inverted asymmetric patterns were detected at 7.0% and 10.5%, respectively with 3.5% of pairs at the borderline. Some differences between predicted ratios and the experimental data could be due to the simplified assumptions, the limited sensitivity of the measurement, and/or some coordinated chromatid segregation modes that bias the eventual read-out. In summary, comparison between the modeling ratios and the experimental data suggest that loss of the tight control of H3T3 phosphorylation in GSCs randomizes segregation of sister chromatids enriched with different populations of H3 (Xie, 2015).

If the temporal separation in the phosphorylation of H3T3 on epigenetically distinct sister chromatids facilitates their proper segregation and inheritance during asymmetric cell division, it is likely that mutations of the Haspin kinase will also affect the temporal control of H3T3 phosphorylation. In the context of H3T3A, where the levels of H3T3P are already reduced, a further decrease in H3T3P by reducing Haspin levels should limit the GSC's ability to distinguish between sister chromatids enriched with distinct H3. Indeed, haspin mutants enhance the phenotypes in nos>H3T3A testes. A different situation appears in the context of H3T3D where sister chromatids experience seemingly elevated levels of H3T3P at the start of mitosis. These elevated H3T3P levels may be exacerbated by the phosphorylation activity of the Haspin kinase. Therefore, it is conceivable that by halving the levels of the Haspin kinase, H3T3 phosphorylation should be reduced to a level more closely resembling wild-type. In this way, some of the temporal specificity that is lost in the H3T3D mutant is restored, resulting in suppression of the phenotypes observed in nos>H3T3D testes. An exciting topic for future study would be to further explore how exactly Haspin phosphorylates H3T3 in the context of chromatin and whether H3T3A and H3T3D mutations act synergistically or antagonistically in regulating asymmetric sister chromatids segregation through differential phosphorylation of a key histone residue (Xie, 2015).

It would also be interesting to understand the potential connection between asymmetric histone inheritance and another phenomenon reported by several investigators: selective DNA strand segregation. Recent development of the chromosome orientation fluorescence in situ hybridization (CO-FISH) technique allows study of selective chromatid segregation at single-chromosome resolution. Using this technique in mouse satellite cells, it has been demonstrated that all chromosomes are segregated in a biased manner, such that pre-existing template DNA strands are preferentially retained in the daughter cell that retains stem cell identity. Interestingly, this biased segregation becomes randomized in progenitor non-stem cells. Using CO-FISH in Drosophila male GSCs, sex chromosomes have been shown to segregate in a biased manner. Remarkably, sister chromatids from homologous autosomes have been shown to co-segregate independent of any specific strand preference. Such findings hint at a possible epigenetic source guiding the coordinated inheritance of Drosophila homologous autosomes. In many cases of biased inheritance, researchers have speculated about the existence of a molecular signature that would allow the cell to recognize and segregate sister chromatids bearing differential epigenetic information. However, the identity of such a signature has remained elusive. The work represented in this paper provides experimental evidence demonstrating that a tightly-controlled histone modification, H3T3P, is able to distinguish sister chromatids and coordinate their segregation (Xie, 2015).

Epigenetic processes play important roles in regulating stem cell identity and activity. Failure to appropriately regulate epigenetic information may lead to abnormalities in stem cell behaviors, which underlie early progress toward diseases such as cancer and tissue degeneration. Due to the crucial role that such processes play in regulating cell identity and behavior, the field has long sought to understand whether and how stem cells maintain their epigenetic memory through many cell divisions. Yhe results of this study suggest that the asymmetric segregation of pre-existing and newly synthesized H3-enriched chromosomes may function to determine distinct cell fates of GSCs versus differentiating daughter cells (Xie, 2015).

Linking H3K79 trimethylation to Wnt signaling through a novel Grappa-containing Dot1-containing complex

Epigenetic modifications of chromatin play an important role in the regulation of gene expression. KMT4/Dot1 is a conserved histone methyltransferase capable of methylating chromatin on Lys79 of histone H3 (H3K79). This study reports the identification of a multisubunit Dot1 complex (DotCom), which includes several of the mixed lineage leukemia (MLL) partners in leukemia such as ENL, AF9/MLLT3, AF17/MLLT6, and AF10/MLLT10, as well as the known Wnt pathway modifiers TRRAP, Skp1, and β-catenin. The human DotCom is indeed capable of trimethylating H3K79 and, given the association of β-catenin, Skp1, and TRRAP, a role was sought for Dot1 in Wnt/Wingless signaling in an in vivo model system. Knockdown of Dot1 in Drosophila (Grappa) results in decreased expression of a subset of Wingless target genes. Furthermore, the loss of expression for the Drosophila homologs of the Dot1-associated proteins involved in the regulation of H3K79 shows a similar reduction in expression of these Wingless targets. From yeast to human, specific trimethylation of H3K79 by Dot1 requires the monoubiquitination of histone H2B by the Rad6/Bre1 complex. This study demonstrates that depletion of Bre1, the E3 ligase required for H2B monoubiquitination, leads specifically to reduced bulk H3K79 trimethylation levels and a reduction in expression of many Wingless targets. Overall, this study describes for the first time the components of DotCom and links the specific regulation of H3K79 trimethylation by Dot1 and its associated factors to the Wnt/Wingless signaling pathway (Mohan, 2010).

In eukaryotic organisms, gene expression patterns are spatiotemporally regulated in a manner that allows for specification of diverse cell types and their differentiation. This spatiotemporal expression is coordinated in part by transcription factors and chromatin modifiers, and by the activity of several signaling pathways, which contribute to gene expression by regulating the transcription factors. Understanding the relationship between chromatin events and signaling pathways is crucial to understanding gene regulation, development of the organism, and disease pathogenesis (Mohan, 2010).

The nucleosome, the basic unit of chromatin, consists of histones H2A, H2B, H3, and H4, and 146 base pairs (bp) of DNA. Crystal structure studies have demonstrated that the N-terminal tails of each histone protrude outward from the core of the nucleosome. These histone tails are subject to various post-translational modifications, including methylation, ubiquitination, ADP ribosylation, acetylation, phosphorylation, and sumoylation, and such modifications are involved in many biological processes involving chromatin such as transcription, genome stability, replication, and repair (Mohan, 2010).

Histones are methylated on either the lysine and/or arginine residues by different histone methyltransferases (HMTases). Histone lysine methylation can occur as mono-, di-, or trimethylated forms, and several lysine residues of histones have been shown to be multiply methylated. This includes methylation on Lys4, Lys9, Lys27, Lys36, and Lys79 of histone H3, and Lys20 of histone H4. Almost all of the lysine HMTases characterized to date contain a SET domain, named after Drosophila Su(var)3-9, Enhancer of zeste [E(z)], and trithorax (trx). SET domain-containing enzymes can catalyze the methylation of specific lysines on histones H3 and H4, and many SET domain-containing enzymes, such as Trithorax and Enhancer of zeste, are central players in epigenetic regulation and development (Mohan, 2010).

Histone H3 at Lys79 (H3K79) can be mono-, di-, and trimethylated by Dot1, which to date is the only characterized non-SET domain-containing lysine HMTase. Dot1 is conserved from yeast to humans. In yeast, telomeric silencing is lost when Dot1 is overexpressed or inactivated, as well as when H3K79 is mutated. Unlike other histone methylation patterns, the pattern of di- and trimethylation of H3K79 in yeast appears to be nonoverlapping. It was also first discovered in yeast that monoubiquitination of histone H2B on Lys123 (H2BK123) by the Rad6/Bre1 complex is required for proper H3K79 trimethylation by Dot1. In vivo analysis of the pattern of H2B monoubiquitination in yeast demonstrated that the H3K79 trimethylation pattern overlaps with that of H2B monoubiquitination, and that the H3K79 dimethylation pattern and H2B monoubiquitination appear to be nonoverlapping. This observation resulted in the proposal that the recruitment of the Rad6/Bre1 complex and the subsequent H2B monoubiquitination could dictate diversity between H3K79 di- and trimethylation on chromatin on certain loci within the genome. In addition to a role in the regulation of telomeric silencing in yeast, Dot1 has also been shown to be involved in meiotic checkpoint control and in double-strand break repair via sister chromatid recombination. A relationship has been found between cell cycle progression and H3K79 dimethylation, but not trimethylation, by Dot1. Consequently, to date, very little is known about a specific biological role of histone H3K79 trimethylation (Mohan, 2010).

In Drosophila, H3K79 methylation levels correlate with gene activity. Mutations in grappa, the Dot1 ortholog in Drosophila, show not only the loss of silencing, but also Polycomb and Trithorax-group phenotypes, indicating a key role for H3K79 methylation in the regulation of gene activity during development. Similarly, Dot1 in mammals has been implicated in the embryonic development of mice, including a role in the structural integrity of heterochromatin. Genome-wide profiling studies in various mammalian cell lines have suggested that Dot1 as well as H3K79me2 and H3K79me3 localize to the promoter-proximal regions of actively transcribed genes, and correlate well with high levels of gene transcription (Steger, 2008). It has also been proposed that Dot1 HMTase activity is required for leukemia pathogenesis (Mohan, 2010 and references therein).

The highly conserved Wnt/Wingless (Wnt/Wg) signaling pathway is essential for regulating developmental processes, including cell proliferation, organogenesis, and body axis formation. Deregulation or ectopic expression of members of the Wnt pathway has been associated with the development of various types of cancers, including acute myeloid and B-cell leukemias. In the canonical Wnt/Wg pathway, a cytoplasmic multiprotein scaffold consisting of Glycogen synthase kinase 3-β (GSK3-β), Adenomatous polyposis coli (APC), Casein kinase 1 (CK1), Protein phosphatase 2A, and Axin constitutively marks newly synthesized β-catenin/Armadillo for degradation by phosphorylation at the key N-terminal Ser and Thr residues. Binding of the Wnt ligands to the seven-transmembrane domain receptor Frizzled (Fz) leads to recruitment of an adaptor protein, Disheveled (Dvl), from the cytoplasm to the plasma membrane. Axin is then sequestered away from the multiprotein Axin complex, resulting in inhibition of GSK3-β and subsequent stabilization of hypophosphorylated β-catenin levels in the cytoplasm. Stabilized β-catenin translocates into the nucleus and binds to members of the DNA-binding T-cell factor/lymphoid enhancer factor (TCF/LEF) family, resulting in the recruitment of several chromatin-modifying complexes, including transformation/transcription domain-associated protein (TRRAP)/HIV Tat-interacting 60-kDa protein complex (see Tip60) histone acetyltransferase (HAT), ISWI-containing complexes, and the SET1-type HMTase mixed lineage leukemia 1/2 (MLL1/MLL2) complexes, thereby activating the expression of Wnt/Wg target genes (Mohan, 2010 and references therein).

Although much is known about Dot1 as an H3K79 HMTase, biochemical studies isolating to homogeneity a Dot1-containing complex have not been successful during the past decade. This study reports the first biochemical isolation of a multisubunit complex associated with Dot1, which has been called DotCom. DotCom is comprised of Dot1, AF10, AF17, AF9, ENL, Skp1, TRRAP, and β-catenin. This complex is enzymatically active and can catalyze H3K79 dimethylation and trimethylation. Indeed, nucleosomes containing monoubiquitinated H2B are a better substrate for DotCom in the generation of trimethylated H3K79. Given the association of Skp1, TRRAP, and β-catenin with DotCom, and the fact that these factors have been linked to the Wnt signaling pathway in previous studies, this study investigated the role of the Drosophila homolog of Dot1, dDot1 (Grappa), for the regulation of Wg target genes. RNAi of dDot1 leads to a reduced expression of a subset of Wg target genes, including senseless, a high-threshold Wingless target gene. Furthermore, reduction by RNAi in the levels of the Drosophila homologs of other components of DotCom that regulate the pattern of H3K79 methylation in humans also showed a similar reduction in senseless expression and other Wg target genes. Importantly, DotCom requires monoubiquitination of H2B for H3K79 trimethylation, and, in Drosophila, the loss of Bre1, the E3 ubiquitin ligase, leads to reduction of H3K79 trimethylation and decreased expression of the senseless gene. Taken together, these data support a model in which monoubiquitinated H2B provides a regulatory platform for a novel Dot1 complex to mediate H3K79 trimethylation, which is required for the proper transcriptional control of Wnt/Wg target genes (Mohan, 2010).

Although H3K79 methylation is a ubiquitous mark associated with actively transcribed genes, and its presence is a clear indicator for the elongating form of RNA polymerase II, Dot1 itself has a very low abundance and is very hard to detect in cells. This indicates that Dot1 is an active enzyme with a very high specific activity toward its substrate, H3K79. Due to the low abundance of Dot1 in cells, its molecular isolation and biochemical purification have been hindered for the past decade. This study reports the biochemical isolation of a Dot1-containing complex (DotCom) and demonstrate a specific link between H3K79 trimethylation by DotCom and the Wnt signaling pathway. The study reports (1) the identification and biochemical isolation of a large macromolecular complex (~2 MDa) containing human Dot1, in association with human AF10, AF17, AF9, ENL, Skp1, TRRAP, and β-catenin; (2) the biochemical demonstration that the human DotCom is capable of trimethylating H3K79, and the analysis of the role of histone H2B monoubiquitination in the enhancement of this H3K79 trimethylase activity of the human DotCom; (3) identification of the role of the components of DotCom in the regulation of its H3K79 methylase activity; (4) demonstration of a role for the Drosophila homolog of Dot1 and its associated factors in the Wnt signaling pathway; and, finally, (5) the identification of a specific requirement of H3K79 trimethylation, but not mono- or dimethylation, in the regulation of Wnt target transcription, thereby linking H3K79 trimethylation to Wnt signaling (Mohan, 2010).

Dot1 was initially isolated from yeast, and these studies demonstrated that the enzyme is capable of mono-, di-, and trimethylating H3K79. Subsequent molecular and biochemical studies demonstrated that prior H2B monoubiquitination by the Rad6/Bre1 complex is required for proper H3K79 trimethylation by yeast Dot1. A recent analysis of the human homolog of Dot1 suggested that its HMTase domain is not capable of trimethylating H3K79, and that this enzyme can only dimethylate its substrate. It has also been demonstrated that reconstitution of monoubiquitinated H2B into chemically defined nucleosomes, followed by enzymatic treatment with Dot1, resulted only in dimethylation of H3K79. Since these observations are in contrast with the published studies in yeast, this study tested the enzymatic activity of purified human DotCom toward monoubiquitinated and nonmonoubiquitinated nucleosomes. The studies demonstrate that the human DotCom can indeed trimethylate H3K79, and that monoubiquitination of histone H2B enhances this enzymatic property of the human DotCom. Since the enzymatic studies employ antibodies generated toward mono-, di-, and trimethylated H3K79 to identify the products of the enzymatic reactions containing human Dot1, it was important to make certain that the observations are not the result of cross-reactivity between these antibodies. Therefore recombinant nucleosomes were generated and treated with human Dot1 in the presence and absence of SAM, and the products were analyzed by MS. The chemical analysis of the products from this enzymatic reaction confirmed that human Dot1 is capable of trimethylating H3K79. The hDot1-treated nucleosome samples were digested with Endoproteinase Arg-C because previous unpublished work on analyzing yeast histone modifications by MudPIT had shown that the trimethylated peptide containing H3K79 was not detected when digesting with trypsin. Notably, McGinty (2008) performed their digestions with trypsin, which might explain their failure to detect this modification by MS (Mohan, 2010).

These studies identified several factors—including ENL, AF9, AF17, AF10, SKP1, TRA1/TRAPP, and β-catenin—as components of the human DotCom. To test the role of these factors in regulating Dot1’s catalytic activity, their levels were reduced via RNAi. These studies demonstrated that AF10 functions with Dot1 to regulate its catalytic properties in vivo. Significant differences in Dot1’s H3K79 HMTase activity were not detected in vivo when reducing the levels of ENL, AF9, and AF17. Factors that significantly alter the H3K79 methylation pattern by Dot1 are also linked to its transcriptional regulatory functions at Wnt target genes (Mohan, 2010).

Since Dot1 also appears to interact with β-catenin, and given the known role for β-catenin, Skp1, and TRRAP in the Wnt signaling pathway, the role for Dot1 and the components of its complex were tested in Wnt signaling. Drosophila is an outstanding model system for the study of the Wnt signaling pathway. Given the power of genetics and biochemistry in Drosophila, the role of dDot1 and the members of its complex in wingless signaling were tested. From this study, it was learned that down-regulation of Drosophila Dot1 and Drosophila AF10 had the most significant effects in the regulation in the expression of the Wg target senseless. Given the fact that the molecular studies demonstrated that Dot1 and AF10 have the strongest effect in the regulation of H3K79 methylation in vivowe wanted to determine whether a specific form of H3K79 methylation is required for Wnt target gene expression was tested (Mohan, 2010).

Histone H2B monoubiquitination is required for proper H3K79 trimethylation. The E2/E3 complex Rad6/Bre1 is required for the proper implementation of H2B monoubiquitination on chromatin, and this complex is highly conserved from yeast to humans. Deletion of the Drosophila homolog of Bre1 results in the loss of H2B monoubiquitination and the specific loss of H3K79 trimethylation. Interestingly, reduction in the levels of H3K79 trimethylation results in a defect in expression of one of the Wnt target genes, senseless, although the H3K79 mono- and dimethylation in this mutant background appear to be normal. In addition to senseless, the role of H3K79 methylation at other Wnt targets was tested, and the same effect was observed for Notum and CG6234. Overall, these studies demonstrate a link between H3K79 trimethylation by the DotCom and the Wnt signaling pathway (Mohan, 2010).

Wnt/Wg signaling serves a critical role in tissue development, proliferation of progenitor cells, and many human cancers. The key player in the Wnt pathway is β-catenin, which is shuttled into the nucleus at the onset of activation of the pathway. Various proteins that interact with β-catenin in the nucleus— such as CBP/p300, TRRAP, MLL1/MLL2, Brg1, telomerase, Hyrax, Pygopus, and CDK8— modulate the transcriptional output of Wg/Wnt target genes. These proteins probably provide the context specificity to Wnt response directing proliferation or differentiation effects of Wnt signaling. The finding that dDotCom is required for expression of a subset of Wg targets suggests that dDotCom might also facilitate Wg-regulated programs of transcriptional regulation in specific contexts. As most human cancers have elevated levels of Wnt signaling and require Wnt signaling for continued proliferation, DotCom might play a role in supporting the high rate of expression of Wnt target genes in such cancers (Mohan, 2010).

Several studies have found interactions between Dot1 and many translocation partners of MLL. While these associations suggest a link between Dot1 methylation and leukemogenesis, it was not clear how Dot1 methylation would participate in this process. Recently, GSK3, a regulator of β-catenin and Wnt signaling, was found to be essential for proliferation of MLL-transformed cells and for progression of a mouse model of MLL-based leukemia (Wang, 2008). These studies linking Dot1 H3K79me3 with Wnt signaling provide insight into the role of Wnt signaling and Dot1 methylation in MLL translocation-based leukemia (Mohan, 2010).

Epigenetic silencers and Notch collaborate to promote malignant tumours by Rb silencing

Cancer is both a genetic and an epigenetic disease. Inactivation of tumour-suppressor genes by epigenetic changes is frequently observed in human cancers, particularly as a result of the modifications of histones and DNA methylation. It is therefore important to understand how these damaging changes might come about. By studying tumorigenesis in the Drosophila eye, two Polycomb group epigenetic silencers, Pipsqueak and Lola, have been identified that participate in this process. When coupled with overexpression of Delta, deregulation of the expression of Pipsqueak and Lola induces the formation of metastatic tumours. This phenotype depends on the histone-modifying enzymes Rpd3 (a histone deacetylase), Su(var)3-9 and E(z), as well as on the chromodomain protein Polycomb. Expression of the gene Retinoblastoma-family protein (Rbf ) is downregulated in these tumours and, indeed, this downregulation is associated with DNA hypermethylation. Together, these results establish a mechanism that links the Notch-Delta pathway, epigenetic silencing pathways and cell-cycle control in the process of tumorigenesis (Ferres-Marco, 2006).

H3K4 methylation is thought to be permissive for maintaining and propagating activated chromatin and is thought to neutralize repressor tags by precluding binding of the HDAC complex and impairing SUV39H1-mediated H3K9 methylation. Thus, H3K4me3 depletion may contribute to tumour formation by permitting aberrant chromatin silencing. It was found that a 50% reduction in dosage of the HDAC gene Rpd3 or of Su(var)3-9 decreased the tumour phenotype dominantly. In contrast, reducing the activity of the H3K4 histone methyltransferase genes Trx (known as ALL1 or MLL in humans) or Ash1, which would be expected to deplete the H3K4me3 tag further, did not visibly enhance the tumours (Ferres-Marco, 2006).

E(z) when complexed with the Extra sex combs (Esc) protein becomes a histone methyltransferase. The E(z)-Esc complex and its mammalian counterpart Ezh2-Eed show specificity for H3K27 but may also target H3K9. The complex also contains the HDAC Rpd3, and this association with Rpd3 is conserved in mammals. H3K27 methylation facilitates binding of the chromodomain protein Pc (HPC in humans), which then creates a repressive chromatin state that is a stable silencer of genes (Ferres-Marco, 2006).

Although loss of E(z) does not cause proliferation defects within discs, halving the E(z) gene dosage dominantly suppressed tumorigenesis, indicating that histone methylation by the E(z)-Esc complex is also a prerequisite for the excessive proliferation of these tumours. Accordingly, Esc- or Pc- mutations also notably reduced the tumours (Ferres-Marco, 2006).

Together, these findings suggest that the development of these tumours involves, at least in part, changes in the structure of chromatin brought about by covalent modifications of histones. These changes probably switch the target genes from the active H3K4me3 state to a deacetylated H3K9 and H3K27 methylation silent state (Ferres-Marco, 2006).

In vivo analysis of Drosophila SU(Z)12 function

Polycomb group (PcG) proteins are required to maintain a stable repression of the homeotic genes during Drosophila development. Mutants in the PcG gene Suppressor of zeste 12 (Su(z)12) exhibit strong homeotic transformations caused by widespread misexpression of several homeotic genes in embryos and larvae. Su(z)12 has also been suggested to be involved in position effect variegation and in regulation of the white gene expression in combination with zeste. To elucidate whether SU(Z)12 has any such direct functions the binding pattern to polytene chromosomes was investigated and the localization to other proteins was compared. SU(Z)12 was found to bind to about 90 specific eukaryotic sites, however, not the white locus. Staining was found at the chromocenter and the nucleolus. The binding along chromosome arms is mostly in interbands and these sites correlate precisely with those of Enhancer-of-zeste and other components of the PRC2 silencing complex. This implies that SU(Z)12 mainly exists in complex with PRC2. Comparisons with other PcG protein-binding patterns reveal extensive overlap. However, SU(Z)12 binding sites and histone 3 trimethylated lysine 27 residues (3meK27 H3) do not correlate that well. Still, it was shown that Su(z)12 is essential for tri-methylation of the lysine 27 residue of histone H3 in vivo, and that overexpression of SU(Z)12 in somatic clones results in higher levels of histone methylation, indicating that SU(Z)12 is rate limiting for the enzymatic activity of PRC2. In addition, the binding pattern of Heterochromatin Protein 1 (HP1) was analyzed and it was found that SU(Z)12 and HP1 do not co-localize (S. Chen, 2008).

In Drosophila, Polycomb group (PcG) proteins are negative regulators of homeotic gene expression and play an important role in maintaining silenced states during development of the fly. The regulatory regions of homeotic genes, such as Ultrabithorax (Ubx) and Abdominal-B (Abd-B), contain specific cis-regulatory elements needed for PcG complexes to mediate this silencing effect. Several such Polycomb response elements (PREs) have been identified, not only in the Antennapedia (AntpC) and Bithorax complexes (BxC) but also in the regulatory regions of many genes mainly encoding transcription factors. PREs appear to be complex DNA elements targeted by several proteins, not only PcG but also trithorax group (TrxG) proteins, proposed to have an opposing effect to PcG, i.e. to maintain an active state of homeotic genes. Recently, a genome-wide analysis of PcG targets in Drosophila identified over 200 genes that are simultanously bound by the three PcG proteins Polycomb (PC), Enhancer of zeste (E(Z)) and Posterior sex combs (PSC). However, none of these proteins bind directly to DNA but are recruited either by DNA-binding proteins bound to PREs or by specifically modified histones. The Frontabdominal-7 (Fab-7) region of the Abd-B locus, e.g., the iab-7 PRE, which is approximately 230 bp in length, contains three DNA motifs that are recognized by five proteins: Zeste (Z), GAGA factor (GAGA), Pipsqueak (PSQ), Pleiohomeotic (PHO) and Pho-like (PHO-L). Interestingly, both PcG repressors and TrxG activators appear to bind at PREs in both the repressed and the active state. Instead, transcription of non-coding RNA through the PREs has been shown to be important for initiation of the correct transcriptional status of homeotic genes during development. Earlier work suggested that PRE transcription counteracts PcG-dependent silencing of Hox genes, while more recent work show that non-coding RNA is not present in cells expressing Ubx, indicating that transcription through PREs promotes gene silencing. The silencing ability of a given PRE is, however, also strongly dependent on the genomic context, homologous pairing or proximity to other PRE sequences (S. Chen, 2008).

The TrxG gene zeste encodes a non-essential transcription factor, which binds to DNA not only within PREs but also in regulatory regions of many genes, e.g. the white locus. The neomorphic zeste 1 (z 1 ) mutation causes an amino acid exchange, which renders the mutated protein extremely sticky. This results in transcriptional repression of paired white + (w + ) genes in z 1 mutants. Thus, in this zeste- white interaction, the X-linked white gene, either paired in trans (e.g., in females) or in cis (e.g. in males carrying a tandem duplication of white), will be repressed, resulting in a yellow eye colour. Whenever unpaired, the white gene is not repressed by z 1 , resulting in wild-type eye pigmentation. The zeste gene is also implicated to cause transvection, i.e., the ability of regulatory elements on one chromosome to affect the expression of the homologous gene in a somatically paired chromosome. A third type of pairing sensitive effects by z 1 is found in transgenic lines, where PRE-induced silencing of a mini-white gene often is pairing dependent. However, homologous pairing is not always required for PRE-induced silencing of w + expression and in such cases the z 1 allele has no influence on the repression (S. Chen, 2008 and references therein).

The biochemical characterization of Drosophila PcG proteins suggests that there are at least two distinct multi-protein complexes, each containing several PcG proteins. The Polycomb repressive complex 1 (PRC1) contains PC, PSC, polyhomeotic (PH) and Sex combs extra (SCE or dRING) and some additional accessory proteins such as Z, Sex comb on midleg (SCM), and general transcription factors. A second complex (PRC2) consists of E(Z), Extra sex combs (ESC), Suppressor of zeste-12 (SU(Z)12) and NURF55 (Chromatin associated factor-1 subunit, CAF-1), as well as some accessory proteins; Polycomb-like (PCL), RPD3 and SIR2. The core proteins E(Z), ESC and SU(Z)12 are conserved both in mammals and plants. The E(Z) protein contains a SET domain which specifically methylates lysine residues of histone 3. The SU(Z)12 protein contains a zinc finger and a well conserved region called the VEFS box, which has been shown to bind to EZH2 protein (the human E(Z) homolog) and Heterochromatin protein 1α (HP1α) in mammalian cells). Recently, in vitro binding between mammalian SUZ12 and MEP50 was reported. MEP50 binds selectively to histone H2A and interacts with the arginine methyltransferase PRMT5 and H2A, mediating transcriptional repression of target genes. So far the SU(Z)12 protein has not been ascribed any specific function in the PRC2 complex, apart from increasing the ability of the E(Z) protein to tri-methylate the lysine 27 residue of histone H3 (3meK27 H3) in vitro and forming, with NURF55, the minimal nucleosome-binding module of PRC2. 3meK27 H3 is a target of the chromodomain of the PC protein in the PRC1 complex. These two main silencing complexes together with their accessory molecules seem to inhibit transcription by preventing nucleosome remodeling and, by binding to promoter regions, block the transcription initiation machinery. The precise molecular mechanisms are, however, still poorly understood (S. Chen, 2008).

The first Su(z)12 mutant allele was first identified as a suppressor of the zeste- white interaction and was also shown to be important for correct maintenance of the silenced state of the Ubx gene during development and to suppress position-effect variegation (PEV)). The biochemical studies of the PcG complexes have definitely linked SU(Z)12 to PcG-mediated gene silencing; however, the involvement in position effect variegation and in regulation of the white gene expression in combination with z 1 is less well investigated. To elucidate whether Su(z)12 directly regulates the white gene expression genetic interaction studies were used with Su(z)12 alleles and it could be revealed that the dominant derepression of white expression caused by Su(z)12 mutants is dependent on the repressive action of the z 1 allele, but in order to observe this derepression either the white gene has to be paired (in females) or contain insertions of transposable elements at the white gene (in males). Another way to find out whether SU(Z)12 has other functions, apart from gene silencing via PREs, is to investigate protein binding and nuclear localization and compare with binding patterns of the other PRC2 subunits. Therefore, SU(Z)12 binding to polytene chromosomes was analyzed. It was found that SU(Z)12 binds to at about 90 specific loci, however, not detectably at the white locus. Neither is any increase of 3meK27 H3 binding found at the white locus in repressed strains, indicating that other repressive mechanisms are acting at white. Results with HP1 suggest that heterochromatin silencing may play a role. Moreover, it is concluded that there is a complete co-localization between SU(Z)12 and E(Z) and that the overlap with other PcG proteins is high, indicating that SU(Z)12 in salivary gland tissue always is in complex with PRC2. However, there was a surprisingly low degree of co-localization with 3meK27 H3. In order to rule out the action of other H3-K27 methylases, somatic Su(z)12 knock-out clones were generated and it was concluded that SU(Z)12 is essential for function of the PRC2 complex in tri-methylation of lysine 27 in histone H3 in vivo (S. Chen, 2008).

Regulation of the white gene by zeste is complex; first, the Z1 mutant protein can form large protein aggregates that cooperatively bind to several ZBS within the regulatory region of white, second, Z protein is involved in recruiting both silencing and activating maintenance complexes (when bound to PREs) and finally the white mutants studied here contain insertions of various mobile elements, which might inflict heterochromatic properties to the white gene or interfere with dosage compensation in males. Using genetic interaction studies, this study found that Su(z)12 + is required for the Z1 mediated repression of white, but no physical binding of SU(Z)12 protein at the white locus is found in polytene chromosomes. Neither is any increased 3meK27 H3 binding there. One explanation for the derepression of white expression by Su(z)12 mutants could be that there are tissue specific differences in PcG silencing, and that in eye discs the white gene is silenced by PcG, but not in salivary glands. This is not likely, since there is no PRE at the white locus, only a set of ZBS, which is not sufficient to recruit silencing complexes. Another alternative is thus that the derepression observed in Su(z)12 mutants are secondary effects. Possibly, lower levels of SU(Z)12 can down-regulate other PcG proteins, which is accompanied by a simultaneous depletion of Z1 proteins, relieving white repression. Alternatively, Z1 can recruit still other silencing mechanisms to the white locus (S. Chen, 2008).

Su(z)12 mutant derepression is observed in homozygous z 1 females but only in a few white alleles in males. Insertions of mobile elements at the white locus in these mutants might superimpose heterochromatic silencing, repressing transcription further. However, these specific transposon insertions on their own accord do not visibly repress white transcription. Interestingly, a novel weak binding of HP1 at the white locus in polytene chromosomes was found in one of the strains that are derepressed by Su(z)12 mutations, suggesting that the inserted transposable element could recruit heterochromatic silencing proteins, adding to the silencing already present. The loss of one copy of the Su(var)205 gene slightly relieves this silencing. It is concluded that the z 1 repression in conjunction with transposable element insertions results in a yellow eye colour, allowing detection of the derepression caused by Su(z)12 mutants. This shows that a combination of repressive mechanisms is acting at the white gene in the studied mutants, but that PcG and heterochromatin silencing are not the main ones (S. Chen, 2008).

It was not possible to show any co-localization between SU(Z)12 and HP1 proteins on Drosophila polytene chromosomes (except at telomeres and the chromocenter), which is also confirmed by the binding pattern of HP1 on polytene chromosomes. This is in contrast to the in vitro findings with the mammalian counterparts, where the region between the Zinc finger and the VEFS box in human SU(Z)12 protein directly binds to mammalian HP1α in vitro. The human HP1α protein is showing a somewhat higher homology to the Drosophila HP1b protein sequence than to HP1 encoded by Su(var)205. Therefore, Drosophila HP1 may not be the functional homologue of HP1α, which could explain why no co-localization was found with SU(Z)12 and HP1 in Drosophila. It would be interesting to further study the role of HP1b in gene silencing (S. Chen, 2008).

Müller and coworkers reported that SU(Z)12 is essential for the binding of PRC2 to nucleosomes, and that SU(Z)12 and NURF55 together constitute the minimal nucleosome-binding complex. These two subunits alone show a better binding to nucleosomes in vitro than a complex also containing E(Z). This study has found that SU(Z)12 and E(Z) always co-localize to polytene chromosomes, and that NURF55 is also present at these sites. Since a FLAG-tagged ESC protein also completely co-localizes with E(Z) this indicates that the complete core PRC2 complex is present at these sites and is functional, and that only NURF55 can bind to chromatin without the other subunits. E(Z) and ESC have also been found to bind to the chromocenter so probably the role of SU(Z)12 as suppressor of PEV is still connected to the function of the PRC2 complex. This refutes the hypothesis that SU(Z)12 has functions outside of the PRC2 complex (S. Chen, 2008).

It was surprising to find that there are SU(Z)12 binding sites that show no or very weak 3meK27 H3 binding on polytene chromosomes and that there are sites that contain high levels of 3meK27 H3 where PRC2 proteins are not present. Furthermore, it was unexpected to find high levels of 3meK27 H3 in puffs, which contain actively transcribing genes. Most 3meK27 H3 is otherwise seen in interbands, which are considered to contain regulatory regions for transcriptional activation of condensed chromatin in adjacent bands. ChIP analyses have revealed that 3meK27 H3 is binding to large domains covering entire genes that are silenced, while PRC2 proteins bind to more restricted regions. In the genome wide analysis of PcG targets performed by Pirrotta and co-workers, there is a very high co-localization between PcG proteins and 3meK27 H3, and also good correlation between 3meK27 H3 and transcriptional silence. However, 3 of the 149 binding sites reported lack either PcG binding or H3 methylation mark. Probably there are more examples since the report only includes sites where all four or three of the proteins (PC, E(Z), PSC and 3meK27 H3) bind simultaneously with a twofold enrichment. Furthermore, they use cell cultures for their analysis, which might give different results compared to differentiated tissues like salivary glands. The result obtain in the current study could also be caused by physical disruption of sub-nuclear compartments (like PcG bodies) where PRC2 complexes and chromosome regions with high levels of trimethylated histones normally reside. Yet another alternative could be that a dynamic reorganization of chromatin in polytene chromosomes occurs, leading to translocation of PcG proteins. It has been shown that PcG chromosome-association profiles can change during development. Further options could be that there are other yet-unidentified HMTs that can induce 3meK27 H3 formation, or that SU(Z)12, E(Z) and ESC are subunits in larval complexes with other functions than to methylate H3 residues (S. Chen, 2008).

Somatic knock-out of SU(Z)12 in wing discs results in a complete abolishment of 3meK27 H3, showing the vital role of SU(Z)12 for the function of the PRC2 complex. This is in agreement with the results reported by Cao (2004) for mammalian cell lines. The Su(z)12 knock-out clones are generally very small in size, indicating a role for SU(Z)12 in cell proliferation. Twin spots show an increase in histone methylation compared to heterozygous mutant cells. Clones over-expressing SU(Z)12+ also exhibit high level of H3 methylation compared to normal cells. This implies that SU(Z)12 is rate limiting and that SU(Z)12 over-expression facilitates assembly of PRC2 subunits, stabilizes existing PRC2 complexes or in some way augments HMT activity of these. It is known that most PcG proteins bind to some PcG genes, e g. Psc/Su(z)2 and ph (Schwartz, 2006) and this study also sees binding of SU(Z)12 at both these loci. Therefore, an alternative explanation could be that increased SU(Z)12 levels induces positive feedback, activating transcription of genes encoding PRC2 subunits. Indeed, examples of such stimulatory effects between PcG genes have been found. esc and E(z) mutants significantly decrease Pc, Psc and ph transcription levels indicating a stimulatory effect. Similarly, ASX, E(PC) and PCL positively regulates Psc transcription, while PH, PC and PSC negatively regulate Psc (S. Chen, 2008).

In humans the Suz12 locus is a region of frequent translocation in endometrial stromal sarcomas, which generates a fusion protein between JAZF1 and SUZ12. This suggests that over-expression of SUZ12 fusion protein has a causal role in the pathogenesis of this tumour (Koontz, 2001). Furthermore, SU(Z)12 is up-regulated in various other tumours (Kirmizis, 2003). The finding that over-expression of SU(Z)12 increases the HMT activity, possibly by inducing a positive feedback regulation of the other PRC2 genes, emphasize the importance of maintaining a balance between activity and silencing of e.g. tumour suppressor genes (S. Chen, 2008).

The H3K27me3 demethylase dUTX is a suppressor of Notch- and Rb-dependent tumors in Drosophila

Trimethylated lysine 27 of histone H3 (H3K27me3) is an epigenetic mark for gene silencing and can be demethylated by the JmjC domain of UTX. Excessive H3K27me3 levels can cause tumorigenesis, but little is known about the mechanisms leading to those cancers. Mutants of the Drosophila H3K27me3 demethylase dUTX display some characteristics of Trithorax group mutants and have increased H3K27me3 levels in vivo. Surprisingly, dUTX mutations also affect H3K4me1 levels in a JmjC-independent manner. A disruption of the JmjC domain of dUTX results in a growth advantage for mutant cells over adjacent wild-type tissue due to increased proliferation. The growth advantage of dUTX mutant tissue is caused, at least in part, by increased Notch activity, demonstrating that dUTX is a Notch antagonist. Furthermore, the inactivation of Retinoblastoma (Rbf in Drosophila) contributes to the growth advantage of dUTX mutant tissue. The excessive activation of Notch in dUTX mutant cells leads to tumor-like growth in an Rbf-dependent manner. In summary, these data suggest that dUTX is a suppressor of Notch- and Rbf-dependent tumors in Drosophila melanogaster and may provide a model for UTX-dependent tumorigenesis in humans (Herz, 2010).

Mammalian UTX, UTY, and JmjD3 and Drosophila UTX (dUTX) are histone demethylases that specifically demethylate di- and trimethylated lysine 27 on histone H3 (H3K27me2 and H3K27me3, respectively). The catalytic domain of this activity is the Jumonji C (JmjC) domain, located at the C terminus of these proteins. The N-terminal domains of UTX, UTY, and dUTX contain several tetratricopeptide repeats (TPRs) thought to be required for protein-protein interactions (Herz, 2010).

H3K27me3 is a histone mark for Polycomb (Pc)-mediated genomic silencing and transcriptional repression and is associated with animal body patterning, X-chromosome inactivation, genomic imprinting, and stem cell maintenance. H3K27 methylation is catalyzed by Polycomb repressive complex 2 (PRC2), which in Drosophila is composed of the catalytic subunit enhancer of zeste [E(z)] (EZH2 in mammals), extra sex combs (Esc), suppressor of zeste 12 [Su(z)12], and nucleosome remodeling factor 55 (Nurf55). H3K27me3 is recognized by the chromodomain of Pc, which is a component of a different silencing complex, called PRC1, which, in addition to Pc, contains Polyhomeotic (Ph), posterior sex combs (Psc), and dRING. The wild-type function of UTX is to demethylate H3K27me3 and, thus, to antagonize Polycomb-mediated silencing (Herz, 2010).

UTX is also a component of mixed-lineage leukemia complex 3 (MLL3) and MLL4. MLL complexes are histone methyltransferases for H3K4. The function of UTX in MLL3 and MLL4 is unknown. However, it appears that UTX is not required for the H3K4 methyltransferase activity of MLL3 and MLL4. The best-characterized targets of H3K27me3/Pc-mediated silencing are homeotic genes, which are critical regulators of animal patterning. However, many other genes are also enriched for H3K27 methylation and Pc binding. Furthermore, elevated H3K27me3 levels due to an increased activity of the methyltransferase EZH2 could be a leading cause of certain human cancers. Recently, mutations that inactivate UTX, and which are thus expected to cause increased H3K27me3 levels, have been linked to the development and progression of human cancer. However, the precise mechanisms by which this occurs are largely unknown (Herz, 2010).

Notch is the receptor of a highly conserved signaling pathway involved in many biological processes, including lateral inhibition, stem cell maintenance, and proliferation control. The binding of Delta or Serrate, the two ligands in Drosophila melanogaster, triggers the proteolytic processing of Notch, resulting in the release and translocation of the Notch intracellular domain (NICD) into the nucleus, where it regulates gene expression. Aberrant, oncogenic Notch signaling has been linked to tumor development in humans, including T-cell acute lymphoblastic leukemias (T-ALLs), pancreatic cancer, medulloblastoma, and mucoepidermoid carcinoma. Thus, an improved understanding of Notch signaling will have significant implications for human health (Herz, 2010).

In Drosophila, the Notch signaling pathway also controls the growth of the eye primordium and wing margin formation during development. Although the mechanistic details are unclear, one way by which Notch signaling controls proliferation during Drosophila eye development is through the negative regulation of the Retinoblastoma (Rb) family member Rbf. Rbf inactivation has also been implicated in Notch-induced eye tumors in Drosophila. Rb is a tumor suppressor that negatively regulates cell cycle progression through the inhibition of the transcription factor E2F. Rb binds directly to E2F and represses its transcriptional activity. The release of Rb activates E2F to induce the transcription of cell cycle regulators such as cyclin E and PCNA. Therefore, the inactivation of Rbf by increased Notch signaling can trigger increased proliferation, which may lead to cancerous growth (Herz, 2010).

This study genetically characterizes loss-of-function mutations of dUTX. dUTX mutants display some of the characteristics of Trithorax group mutants and have increased H3K27me3 levels in vivo. Surprisingly, dUTX mutations also affect H3K4me1 levels in a JmjC-independent manner. dUTX mutant tissue has an H3K27me3-dependent growth advantage over wild-type tissue due to increased proliferation in the developing eye. The growth advantage of dUTX mutant tissue is caused by increased Notch activity, demonstrating that dUTX is a Notch antagonist. The inactivation of Rbf contributes to the growth advantage of dUTX mutant tissue. Moreover, an excessive activation of Notch in dUTX mutant cells leads to tumor-like growth in an Rbf-dependent manner. In summary, these data suggest that dUTX is a suppressor of Notch- and Rbf-dependent tumors in Drosophila and may provide a model for UTX-dependent tumorigenesis in humans (Herz, 2010).

Based on the enzymatic activity of the JmjC catalytic domain as H3K27me3 demethylases, UTX proteins are predicted to counteract Polycomb function. Consistently, it was found that dUTX mutants display genetic characteristics of Trithorax group genes. In vitro studies have shown that dUTX and UTX demethylate H3K27me2 and H3K27me3. However, dUTX mutants affect the global levels of only H3K27me3 but not of H3K27me2. Nevertheless, this observation does not mean that dUTX does not demethylate H3K27me2 in vivo. There may be fewer genes regulated by dUTX at the H3K27me2 level such that the global levels are not detectably altered in dUTX mutants (Herz, 2010).

Interestingly, dUTX mutants also affect global levels of H3K4me1, which are significantly reduced in mutant tissue. Mammalian UTX is a component of the MLL3 and MLL4 methyltransferase complexes, and based on the reduction of H3K4me1 levels, it is predicted that dUTX is also a component of the Drosophila equivalent of the MLL3/MLL4 methyltransferase complex, which contains Trithorax-related (Trr) as a histone methyltransferase. The function of UTX in MLL3 and MLL4 complexes is currently unknown. It was suggested previously that UTX is not required for H3K4 methylation, but in these studies, only H3K4me2 and H3K4me3 were investigated. Consistently, the global levels of H3K4me2 and H3K4me3 are not affected in dUTX mutant clones. The data demonstrate that dUTX is required for the monomethylation of H3K4. Interestingly, the JmjC demethylase domain of dUTX is not required for H3K4me1 methylation, suggesting that other domains of dUTX, such as the TPR domains, may be necessary for mediating this function. The finding that the global levels of H3K4me2 and H3K4me3 are not affected in dUTX mutants is also quite interesting, as it implies that the monomethylation of H3K4 is not required for the di- or trimethylation of H3K4 (Herz, 2010).

The epigenetic control of gene expression has been best studied for the control of homeotic gene expression, which is established during embryogenesis and maintained throughout animal life. However, not only homeotic genes are regulated through epigenetic modifications. Other genes in different developmental processes are also subject to epigenetic control. In this study, by analyzing the dUTX mutant phenotype, a role was establised of H3K27me3 levels in cell cycle control. The data suggest that increased H3K27me3 levels in dUTX clones cause the epigenetic silencing of several genes involved in Notch signaling. This includes both positive and negative regulators of Notch signaling activity as well as target genes that are either positively or negatively regulated by the Notch pathway. Such an incoherent control of gene expression by the Notch pathway has been reported previously, suggesting that the final outcome of Notch activity may be determined by the relative expression levels of positive or negative regulators. Because this study determined that the overrepresentation phenotype of dUTX clones is caused by elevated levels of Notch signaling, it appears that the silencing of Notch inhibitors is dominant over the silencing of Notch activators, resulting in a net increase of Notch activity. However, this increased Notch activity may be specific for the cell cycle phenotype of dUTX mutants, since increased Notch activity was not found for other Notch-dependent paradigms, such as E(spl)m8-lacZ. This is also consistent with the finding that E(spl) genes contain increased H3K27me3 levels in dUTX mutants. Thus, the wild-type function of dUTX is to restrict the cell cycle through the negative control of Notch. Therefore, the data link H3K27me3-dependent Notch activity with enhanced tissue growth, implying that dUTX is a Notch antagonist regarding the cell cycle and explaining the overrepresentation phenotype of dUTX mutant clones (Herz, 2010).

However, this phenotype is subtle compared to that of mutants in growth control pathways such as the Hippo pathway. Nevertheless, the overgrowth of dUTX clones is strongly potentiated by the additional activation of Notch. The expression of Delta in dUTX clones causes a strong tumor-like growth phenotype. Thus, dUTX functions as a suppressor of Notch-induced tumors under normal conditions. This synergistic interaction between the loss of dUTX and increased Notch activity is a clear example that tumor development requires several hits for progression (Herz, 2010).

The overrepresentation phenotype of dUTX clones can be dominantly enhanced by the genetic loss of Rbf, suggesting that the reduction of Rbf contributes to the overrepresentation phenotype. However, the reduction of Rbf activity in dUTX clones is not caused by direct epigenetic silencing at the Rbf locus. No increased H3K27me3 levels was found at the Rbf locus in dUTX mutants, and mRNA levels of Rbf were unchanged. Instead, Rbf is negatively regulated by the Notch pathway during eye growth. Thus, the increased activity of Notch in dUTX clones leads to a partial inactivation of Rbf and increased proliferation, causing the overrepresentation phenotype. Currently, it is unknown how Notch regulates Rbf (Herz, 2010).

The control of cell cycle progression by UTX proteins is likely conserved in mammals. A parallel study performed by Wang showed that the loss of mammalian UTX also results in elevated levels of proliferation (Wang, 2010). Consistent with the current work, that study also implicated the inactivation of Rb function in increased proliferation in response to UTX knockdown. Similar to the current study, Rb itself is not subject to increased H3K27m3 silencing, but the promoters of several genes in the Rb network were found to be occupied and likely controlled by UTX (Wang, 2010). Thus, although the mechanisms of Rb control by UTX proteins (Notch in this study and the Rb network in the study reported previously by Wang are distinct, both studies established the control of the Rb pathway as a common element of cell cycle control by UTX proteins. Wang also demonstrated a link between UTX and Rb during vulval development in Caenorhabditis elegans. Thus, these studies combined suggest a well-conserved function of UTX proteins for Rb control (Herz, 2010).

Although these studies establish a link between UTX genes and Rb for cell cycle control, it should be noted that the loss of dUTX (and likely mammalian UTX) affects many genes. While the deregulation of individual genes may not cause a significant phenotype on its own, the combined deregulation may disrupt gene regulatory networks, which accounts for the growth phenotype of dUTX mutants. Thus, while aberrant Notch signaling was identified as an important element of the overrepresentation phenotype of dUTX mutants, other genes and signal transduction pathways may also contribute to this phenotype. For example, this study also identified genes involved in growth control by the Hippo pathway (four-jointed [fj] and warts) associated with increased H3K27me3 levels in dUTX mutants and showed reduced transcript levels for fj. Thus, it is possible that the Hippo pathway and other genes contribute to the overrepresentation phenotype of dUTX mutants (Herz, 2010).

These observations have important implications for the initiation and development of human tumors. Increased levels of H3K27me3 due to the elevated activity of the H3K27me3 methyltransferase EZH2 have been associated with human cancer. Furthermore, mutations that inactivate UTX have been linked to human cancer, and low UTX activity correlates with poor patient prognosis. This study establishes that increased levels of H3K27me3 affect Notch activity, which in turn affects Rbf activity. Rb is a well-known tumor suppressor, the loss of which causes human tumors. Therefore, tumors associated with the loss of UTX and, thus, increased H3K27me3 levels may be caused by decreased Rb activity. It should also be noted that aberrant Notch signaling is the cause of several human cancers, including T-cell acute lymphoblastic leukemias (T-ALLs), pancreatic cancer, medulloblastoma, and mucoepidermoid carcinoma. In summary, these data demonstrate that the appropriate control of H3K27 methylation is critical for normal tissue homeostasis, and increased H3K27me3 levels may contribute to cancer through the inactivation of Rb (Herz, 2010).

Drosophila UTX coordinates with p53 to regulate ku80 expression in response to DNA damage

UTX is known as a general factor that activates gene transcription during development. This study demonstrates an additional essential role of UTX in the DNA damage response, in which it upregulates the expression of ku80 in Drosophila, both in cultured cells and in third instar larvae. UTX mediates the expression of ku80 by the demethylation of H3K27me3 at the ku80 promoter upon exposure to ionizing radiation (IR) in a p53-dependent manner. UTX interacts physically with p53, and both UTX and p53 are recruited to the ku80 promoter following IR exposure in an interdependent manner. In contrast, the loss of utx has little impact on the expression of ku70, mre11, hid and reaper, suggesting the specific regulation of ku80 expression by UTX. Thus, these findings further elucidate the molecular function of UTX (Zhang, 2013).

To understand the mechanism underlying UTX function in tumorgenesis, this study explored whether UTX is involved in DNA damage response in Drosophila. This study found that UTX plays an essential role in DNA damage response by upregulation of ku80, which is uniquely required for p53 activated ku80 expression. In addition, the gene activity of utx is correlated with loss of histone demethylation at H3K27, suggesting that UTX could function as a histone demethylase and serve a gene-specific co-activator of p53 gene activation. This study therefore provides an example that p53 target genes expression may be regulated at the level of histone modifications (Zhang, 2013).

It is clear that p53 plays a pivotal role in the DNA damage response (DDR). One of the functions of p53 is to activate its target gene after DNA damage as transcription factor. For instance, p53 has been best characterized in regualting expression of cell cycle genes and apoptosis gene. However, the precise regulation mechanism of p53 is still not clear. It is interesting that in Drosophila ku80 upregulation mediated by p53 requires UTX, but not other genes in related to DNA repair and apoptosis. However, reduced H3K27me3 levels were found in apoptotic genes, which raises the possibility that there could be additional histone demethylases participating in DDR pathways that coordinate with p53 regulating expression of hid and reaper after DNA damage, and remaining to be determined in further studies. In contrast, reduced H3K27me3 levels were not detected in the ku70 promoter region following IR treatment. Further analysis revealed that the H3K27me3 level in the ku70 promoter region was lower than at the ku80 promoter. The expression of ku70 is independent of UTX, possibly due to the extremely low levels of H3K27me3 in the ku70 promoter region, which might not require demethylation for the expression of ku70 to occur. Thus, the data demonstrate the complexity of the function of p53 in the activation of target genes in response to DNA damage, particularly in terms of histone modification and the action of different demethylases (Zhang, 2013).

UTX has been reported to participate in many biological processes, including cell fate determination and animal development, largely depending on the transcriptional regulation of the target genes of UTX. UTX appears to play an important role in orchestrating several histone markers, including acetylation at H3K27 and ubiquitination at H2A, and mediates derepression of polycomb (Pc) target genes, such as HOX genes, by affecting Pc recruitment. These roles are consistent with UTX being a histone demethylase specific for H3K27. However, sporadic mutations of UTX have been linked to many types of human cancers and it remains to be elucidated whether this is also sufficiently explained by its enzymatic activity. Indeed, several studies have proposed a role of UTX independent of its demethylase activity in chromatin remodeling and embryonic development. This study found UTX is also involved in DDR by upregulation of ku80 in Drosophila after IR. Although there are no available data demonstrating that ku80 mRNA levels are increased following DSBs in human cells, the current data provide evidence that UTX functions to maintain genome stability and sheds light on the mechanism underlying the function of UTX in human cancer. Recent studies suggest that loss of polycomb-mediated silencing might promote the upregulation of DNA repair genes and facilitate the recovery of cells from genotoxic insults. UTX might therefore be required for various cell defense mechanisms under environmental stress, thereby contributing to tumor suppression (Zhang, 2013).

Propagation of Polycomb-repressed chromatin requires sequence-specific recruitment to DNA

Epigenetic inheritance models posit that during Polycomb repression, Polycomb Repressive Complex 2 (PRC2) propagates histone H3K27 tri-methylation (H3K27me3) independently of DNA sequence. This study shows that insertion of Polycomb Response Element (PRE) DNA into the Drosophila genome creates extended domains of H3K27me3-modified nucleosomes in the flanking chromatin and causes repression of a linked reporter gene. After excision of PRE DNA, H3K27me3 nucleosomes become diluted with each round of DNA replication and reporter gene repression is lost, whereas in replication-stalled cells, H3K27me3 levels stay high and repression persists. Hence, H3K27me3-marked nucleosomes provide a memory of repression that is transmitted in a sequence-independent manner to daughter strand DNA during replication. In contrast, propagation of H3K27 tri-methylation to newly incorporated nucleosomes requires sequence-specific targeting of PRC2 to PRE DNA (Laprell, 2017).

The ability of certain histone-modifying enzymes to bind to the modification they generated has led to models where such enzymes might propagate modified chromatin domains by a positive feedback loop, independently of the underlying DNA sequence. Two paradigms of chromatin states have been proposed to be maintained by such an epigenetic inheritance mechanism: constitutive heterochromatin with histone H3 lysine 9 di- and tri-methylation (H3K9me2/3) generated by Suv39/Clr4 enzymes, and Polycomb-repressed chromatin marked with H3K27me3 by PRC2. In both chromatin states, these histone modifications are essential for repressing gene transcription. To date, there is compelling evidence that H3K9me2/3- and H3K27me3-modified nucleosomes are transmitted to daughter strand DNA during replication. However, the steps required to propagate these modifications are much less understood. Fission yeast Clr4 has the capacity to propagate ectopically induced H3K9me2/3 domains over many cell divisions by an H3K9me2/3-based positive feedback loop but only in cells mutated for H3K9me2/3 demethylase activity. In the case of PRC2, allosteric activation of the enzyme induced by binding to H3K27me3 has been proposed to be the foundation for propagating H3K27me3 chromatin. In mammalian cells, transient DNA-tethering of PRC2 generates short ectopic H3K27me3 domains that were at least partially maintained for several cell divisions after release of DNA-tethered PRC2. However, in Drosophila, where PRC2 and other Polycomb group (PcG) protein complexes are targeted to PREs, repression imposed by insertion of PRE DNA next to a reporter gene was lost upon excision of PRE DNA. This study investigated how insertion and excision of PRE DNA at ectopic sites in Drosophila affects binding of PcG proteins and H3K27me3 at the molecular level (Laprell, 2017).

Two previously described strains were analyzed that each carried a single copy of the >PRE>dppWE-TZ reporter gene, integrated at different chromosomal locations. >PRE>dppWE-TZ contains a 1.6 kilobase (kb) DNA fragment of the bxd PRE from the HOX gene Ultrabithorax (Ubx), flanked by FRT recombination sites (>PRE>) to permit excision of PRE DNA by Flp-mediated recombination. Adjacent to the >PRE> cassette, the construct contains a reporter gene comprising the wing imaginal disc enhancer from decapentaplegic (dpp) (E), linked to the hsp70 TATA box minimal promoter (T) and LacZ sequences encoding β-galactosidase (Z) . In the presence of the >PRE> cassette, the transgene was silenced and no β-galactosidase activity could be detected in wing imaginal discs of >PRE>dppWE-TZ transgenic animals. In contrast, >dppWE-TZ transgenic animals, generated by excision of the >PRE> cassette in the germ line, showed strong β-galactosidase expression in the characteristic pattern driven by the dpp enhancer. The observation that silencing of the intact >PRE>dppWE-TZ reporter gene is lost in mutants lacking PRC2 function, prompted determination of the H3K27 methylation profile and binding of PcG proteins across the transgene. In both lines, the transgene had inserted into a genomic location normally devoid of H3K27me3 and PcG protein binding. Chromatin immunoprecipitation (ChIP) assays were performed on batches of wing imaginal discs from >PRE>dppWE-TZ and the corresponding >dppWE-TZ transgenic animals, and the immunoprecipitates were analyzed by quantitative real-time PCR (qPCR). For qPCR, primer pairs were used that selectively amplified transgene sequences and sequences in the genomic regions flanking the transgene insert. As controls, primer pairs were used amplifying sequences at the endogenous bx PRE in Ubx that are known to be bound by PcG proteins (C2) or enriched for H3K27me3 (C1 and C3) and at two regions elsewhere in the genome (C4 and C5) without PcG protein binding or H3K27me3 (Laprell, 2017).

The PRC1 subunits Polycomb (Pc), Polyhomeotic (Ph) and the PRC2 subunit E (z) were specifically enriched at the transgene PRE in animals carrying >PRE>dppWE-TZ and, as expected, no binding was detected in >dppWE-TZ animals. In both >PRE>dppWE-TZ transgenic lines, H3K27me3 was present at high levels across a domain that extended about 4-5 kb to either side of the >PRE> cassette, spanning almost the entire construct. No enrichment of H3K27me3 was detectable at the >dppWE-TZ transgene. At >PRE>dppWE-TZ, PRC2 thus tri-methylates H3K27 across a chromatin interval that spans about 8-10 kb (Laprell, 2017).

To estimate to what extent nucleosomes at the >PRE>dppWE-TZ transgene are tri-methylated at H3K27, the H3K27me2 profile was determined. H3K27me2 levels across the >PRE>dppWE-TZ transgene were much lower than at C4 and C5 and comparable to the levels at Ubx (regions C1-C3) that is repressed and predominantly tri-methylated at H3K27 in wing imaginal discs. Conversely, across >dppWE-TZ, H3K27me2 levels were much higher and comparable to those seen at C4 and C5. This suggest that the nucleosomes across the >PRE>dppWE-TZ transgene are predominantly tri-methylated at H3K27 (Laprell, 2017).

Excision of the >PRE> cassette from >PRE>dppWE-TZ transgenic animals by heat-shock induced expression of Flp during larval development results in appearance of β-galactosidase expression in the dpp pattern 12 hours after the heat shock. Efficiency of PRE excision was measured and it was found that 8 hours after a single 1-hour heat shock, excision had occurred in about 95% of wing imaginal disc cells. The delayed increase of β-galactosidase expression over time suggests a gradual rather than abrupt loss of repression. ChIP analyses were performed on chromatin prepared from batches of entire wing imaginal discs dissected from >PRE>dppWE-TZ transgenic animals 12, 32 or 56 hours after Flp-induction. This allowed monitoring the consequences of PRE excision in cells that had undergone at least one (+12 hours), at least two (+32 hours), or more than four (+56 hours) cell divisions. 12 hours after Flp-induction, H3K27me3 levels were at least two-fold reduced across the entire transgene and further reduced by at least two-fold at the 32 hours time point. 56 hours after Flp-induction, H3K27me3 levels across the transgene were nearly as low as in >dppWE-TZ animals derived from >dppWE-TZ germ cells. The histone H3 profile was unaltered at all time points, suggesting that PRE excision does not cause global disruption of nucleosome occupancy across the transgene. The loss of H3K27me3 after PRE excision suggests that PRC2 is unable to propagate H3K27me3 across the >dppWE-TZ transgene in the absence of PRE DNA (Laprell, 2017).

In parallel, Pc protein binding was monitored after PRE excision. Pc, unlike Ph or other PRC1 subunits, is not only bound at PREs but also associates with the chromatin flanking PREs likely reflecting its interaction with H3K27me3-modified nucleosomes. 12 hours after PRE excision, Pc binding at the transgene was already almost reduced to background levels (Laprell, 2017).

The H3K27me3 profile at the >PRE>dppWE-UZ transgene that contains a 4.1 kb fragment of the Ubx promoter instead of the hsp70 minimal promoter was then analyzed. At >PRE>dppWE-UZ, the H3K27me3 domain spans about 12 kb and is thus about 4 kb longer than at >PRE>dppWE-TZ. Nevertheless, after PRE excision, H3K27me3 at dppWE-UZ was lost at a rate comparable to that seen at dppWE-TZ. Ubx promoter DNA thus does not enable H3K27me3 propagation. It is concluded that even at a domain that spans 12 kb and therefore comprises about 60 nucleosomes, PRC2 is unable to propagate H3K27me3 in the absence of PRE DNA (Laprell, 2017).

The H3K27me3 profile and reporter gene repression was then analyzed after PRE excision in animals in which DNA replication had been blocked. Larvae were reared in liquid medium containing Aphidicolin, an inhibitor of DNA polymerases A and D, which resulted in a complete block of DNA replication in imaginal discs. In larvae reared in Aphidicolin-containing medium, Flp-induced PRE excision from >PRE>dppWE-TZ was as efficient as under normal growth conditions but 12 hours after excision, H3K27me3 levels at the transgene were undiminished compared to +PRE control larvae. In larvae reared in liquid medium without Aphidicolin, PRE excision resulted in the expected two-fold reduction of H3K27me3 levels after 12 hours. Together, this suggests that the loss of H3K27me3 nucleosomes after PRE excision in proliferating cells reflects their dilution as they become transmitted to DNA daughter strands during replication. Unlike under normal growth conditions, Aphidicolin-treated larvae lacked detectable β-galactosidase expression 12 hours after PRE excision. When these animals were permitted to recover in medium lacking Aphidocolin, they resumed DNA replication and began expressing β-galactosidase. If DNA replication is blocked and H3K27me3 levels stay high, repression is thus also sustained in the absence of PRE DNA, possibly by PRC1 (Laprell, 2017).

Finally, PRE excision was induced from >PRE>dppWE-TZ in larvae that were hemizygous for UtxΔ, a null mutation in the single H3K27me3 demethylase in Drosophila. 12 hours after Flp-induction, H3K27me3 levels at the transgene were reduced about two-fold, like in wild-type animals. This suggest that demethylation of H3K27me3 by Utx does not contribute to the disappearance of H3K27me3 from transgene chromatin after PRE excision (Laprell, 2017).

These results lead to the following conclusions. First, PRE cis-regulatory DNA provides the genetic basis not only for generating but also for propagating H3K27me3-modified chromatin. This argues against a simple epigenetic model where PRC2 binding to parental H3K27me3 nucleosomes after replication would suffice to propagate H3K27 tri-methylation in daughter strand chromatin. PRC2 needs to be recruited to PRE DNA first, before allosteric activation through interaction with H3K27me3 nucleosomes in flanking chromatin may then facilitate methylation of newly incorporated nucleosomes. Secondly, following PRE excision and replication, parental H3K27me3 nucleosomes remain associated with the same underlying DNA in daughter cells and thus provide epigenetic memory. However, while in replication-stalled cells high H3K27me3 levels permit to sustain repression also in the absence of PRE DNA, their dilution in proliferating cells is accompanied with loss of repression after one cell division. H3K27me3 nucleosomes therefore only appear to provide short-term epigenetic memory of the repressed state. Hence, DNA targeting of PRC2 after replication to replenish H3K27me3 is critical to preserve repression (Laprell, 2017).

Drosophila HOX and other large-size PcG target genes often contain multiple PREs and H3K27me3 domains that span dozens of kilobases. Deletion of single PREs from these genes typically results in only minor diminution of the H3K27me3 profile and misexpression is less severe than misexpression of the native genes in PcG mutants. Furthermore, when the same >PRE> cassette that was used in this study was excised from a Ubx-LacZ reporter gene with more extended Ubx upstream regulatory sequences, repression was lost with a longer delay\, suggesting that additional elements with PRE properties in those Ubx sequences permitted to sustain repression through more cell divisions. The evolution of PRE DNA sequences and of their frequency and arrangement within target genes may thus ultimately determine stability and heritability of H3K27me3 chromatin and PcG repression (Laprell, 2017).

Drosophila Kdm4 demethylases in histone H3 lysine 9 demethylation and ecdysteroid signaling

The dynamic regulation of chromatin structure by histone post-translational modification is an essential regulatory mechanism that controls global gene transcription. The Kdm4 family of H3K9me2,3 and H3K36me2,3 dual specific histone )emethylases has been implicated in development and tumorigenesis. This study shows that Drosophila Kdm4A and Kdm4B, both members of the JHDM3 histone demethylase family are together essential for mediating ecdysteroid hormone signaling during larval development. Loss of Kdm4 genes leads to globally elevated levels of the heterochromatin marker H3K9me2,3 and impedes transcriptional activation of ecdysone response genes, resulting in developmental arrest. It was further shown that Kdm4A interacts with the Ecdysone Receptor (EcR) and colocalizes with EcR at its target gene promoter. These studies suggest that Kdm4A may function as a transcriptional co-activator by removing the repressive histone mark H3K9me2,3 from cognate promoters (Tsurumi, 2013).

This study have discovered a role for Kdm4 in the transcriptional regulation of a subset of ecdysone pathway components. Furthermore, an interaction was demonstrated between Kdm4A and EcR in vivo, providing evidence that Kdm4 demethylases may act as co-activators of EcR. A genetic approach has allowed facilitated the detection of a previously uncharacterized, but essential, role of Kdm4 in development, and has identified a direct Kdm4 target gene in euchromatin. Interestingly, Human Kdm4 members interact with the nuclear hormone receptors, Androgen Receptor (AR) and Estrogen Receptor α (ERα), and has been proposed to serve as co-activators, suggesting a molecular mechanism by which Kdm4 can act as an oncogene in prostate and breast cancers. Kdm4B was shown to be a direct target gene of ERα, yielding a feed-forward loop for an augmented hormonal response. The results indicate that a similar epigenetic mechanism exists in Drosophila, where a nuclear hormone receptor requires the Kdm4 family of demethylases to remove H3K9 methylation at the promoter of a target gene. Taken together, the Kdm4 family of demethylases may function as transcriptional co-factors required for transcriptional activation by nuclear hormone receptors (Tsurumi, 2013).

Previous studies have shown that the Trithorax-related (Trr) H3K4 methyltransferase, the Nurf nucleosome remodeling complex component, Nurf301, the Brahma (Brm)-containing chromatin remodeler, and the histone acetyltransferase CREB-binding protein (CBP) are also co-activators of EcR, indicating that activation of ecdysone pathway genes requires substantial regulation of the chromatin environment. Since H3K4 hyper-methylation at promoters is a marker of active transcription, and since H3K9 hypo-methylation also promotes upregulation of gene expression, it is feasible that synchronizing these two events would lead to more robust target gene activation. The mammalian Kdm4B (JMJD2B) forms a complex with the mixed-lineage leukemia (MLL) 2 H3K4 methyltransferase and serves as a co-activator of Estrogen Receptor. The complex couples H3K9 demethylation with H3K4 methylation in order to facilitate ERα target gene activation. Similar functional cross-talk between H3K9 demethylation and H3K4 methylation has been described in S. pombe, where the Lsd1 H3K9 demethylase and the Set1 H3K4 methyltransferase were found in a complex. Since, in Drosophila, the Nurf301 subunit, Brm and CBP were also found to interact with EcR, nucleosome remodeling may cooperate as well in the rapid and dynamic activation of ecdysone regulated genes (Tsurumi, 2013).

These studies of the Kdm4A and Kdm4B homozygous double mutants demonstrate a requirement for these genes in the ecdysone pathway. This observation is similar to results obtained with mutant alleles of Nurf301 and trr, two seemingly ubiquitous chromatin regulators, where specific downregulation of ecdysone signaling genes has been detected. Additionally, this study is consistent with the reports that adult male Kdm4A mutants display abnormal courtship behavior and concomitant downregulated fru, a gene speculated to be a direct downstream target of EcR (Beckstead, 2005; Dalton, 2009). The specific defects in ecdysone signaling, rather than general transcription, exhibited by the double mutants indicate that either Kdm4 may not be essential for regulating all genes, or that the aberrant expression of ecdysone responsive genes is the earliest manifestation of loss of Kdm4. However, this study does not rule out the possibility that Kdm4 proteins regulate other crucial transcription factors that in turn regulate ecdysone pathway components by secondary effects. Further molecular and genomic studies are required to resolve this issue (Tsurumi, 2013).

H3K9 demethylation-dependent transcriptional activation of BR-C was demonstrated. It is possible however, that H3K36 demethylation also contributes to ecdysone pathway component regulation. Previous studies have shown that HP1a is recruited to developmental puffs in polytene chromosomes and that it stimulates H3K36 demethylation by Kdm4A. Perhaps H3K36 demethylation in the gene body and subsequent displacement of the HDAC complex is important for transcriptional elongation or for the activation of downstream nested promoters of ecdysone pathway components. Moreover, H3K36 plays a role in exon splice choice and thus ecdysone pathway genes that produce multiple splice variants may require Kdm4 regulation. However, immunostaining experiments show that HP1a and Kdm4A signals are mostly non-overlapping. Thus, it seems that HP1a's involvement in the demethylase activities of Kdm4 toward H3K9 or H3K36 would have to be transient and dynamic (Tsurumi, 2013).

In summary, this study has shown that double homozygous mutants of the two Kdm4 genes in Drosophila display developmental delays and lethality, with compromised activation of ecdysone related genes. Furthermore, it was found that BR-C may be a direct target of H3K9 demethylation, and that the interaction between Kdm4A and EcR may be important in transcriptional activation of BR-C. These results provide insight into the physiological functions and mechanistic roles of Kdm4 in vivo. The interaction between Kdm4 and EcR awaits further investigation. It is conceivable that EcR directs the recruitment of Kdm4A to the promoter of its target genes, or alternatively, that EcR allosterically regulates the demethylase activity of Kdm4A, allowing removal of H3K9m2,3 only upon hormone signaling (Tsurumi, 2013).

Polycomb repressive complex 2-dependent and -independent functions of Jarid2 in transcriptional regulation in Drosophila

Jarid2 was recently identified as an important component of the mammalian Polycomb repressive complex 2 (PRC2), where it has a major effect on PRC2 recruitment in mouse embryonic stem cells. Although Jarid2 is conserved in Drosophila, it has not previously been implicated in Polycomb (Pc) regulation. Therefore, Drosophila Jarid2 and its associated proteins were purified, and it was found that Jarid2 associates with all of the known canonical PRC2 components, demonstrating a conserved physical interaction with PRC2 in flies and mammals. Furthermore, in vivo studies with Jarid2 mutants in flies demonstrate that among several histone modifications tested, only methylation of histone 3 at K27 (H3K27), the mark implemented by PRC2, was affected. Genome-wide profiling of Jarid2, Su(z)12 (Suppressor of zeste 12), and H3K27me3 occupancy by chromatin immunoprecipitation with sequencing (ChIP-seq) indicates that Jarid2 and Su(z)12 have very similar distribution patterns on chromatin. However, Jarid2 and Su(z)12 occupancy levels at some genes are significantly different, with Jarid2 being present at relatively low levels at many Pc response elements (PREs) of certain Homeobox (Hox) genes, providing a rationale for why Jarid2 was never identified in Pc screens. Gene expression analyses show that Jarid2 and E(z) (Enhancer of zeste, a canonical PRC2 component) are not only required for transcriptional repression but might also function in active transcription. Identification of Jarid2 as a conserved PRC2 interactor in flies provides an opportunity to begin to probe some of its novel functions in Drosophila development (Herz, 2012).

Different and distinct gene expression patterns are established during development, which need to be maintained and regulated. This is important to allow for the integrity of cell identity and thus the functional preservation of tissues and organs. However, at the same time, transcribed loci must be equipped with an intrinsic flexibility to regulate these expression patterns and initiate changes if necessary. The core components that are required for the maintenance of gene expression or gene repression have been characterized quite extensively to date . Trithorax and Polycomb group genes play antagonistic roles in determining whether a gene is transcriptionally turned on or off, respectively. In Drosophila, so far four distinct complexes, pleiohomeotic repressive complex (PhoRC), Polycomb repressive complex 2 (PRC2), Polycomb repressive complex 1 (PRC1), and recently Polycomb repressive deubiquitinase (PR-DUB) have been described to play a role in Polycomb group-mediated gene repression. However, little is known about the factors involved in controlling recruitment and activity of these complexes on chromatin or about the mechanisms that drive such changes. It should be expected that quite a significant number of proteins would convey Polycomb group-mediated transcriptional changes in order to allow an uncoupling of individual gene activity from that of a group of Polycomb group-controlled genes. Functional redundancy might account for part of the problem to discover such candidates. Furthermore, biochemical approaches might be hindered by the fact that such context-specific and more gene-specific recruiters are contained in only a minor fraction of Polycomb repressive complexes (Herz, 2012).

Recently, Jarid2 the founding member of the JmjC domain-containing protein family, which plays important developmental roles in mice and Drosophila, has been characterized as a component of PRC2 in embryonic stem (ES) cells. The consensus indicates that in ES cells, PRC2 recruitment to many of its targets requires Jarid2. However, levels of bulk histone 3 trimethylated at K27 (H3K27me3) in ES cells depleted of Jarid2 were reported to be only slightly changed at best. This also holds true when individual PRC2 target genes are analyzed. Even though core components of the PRC2 complex were lost from chromatin in the absence of Jarid2, H3K27me3 was not reproducibly affected to a similar degree. Additionally, gene expression analyses in Jarid2-/- ES cells did not confirm a genome-wide derepression of PRC2 target genes as would be expected for any core component of PRC2 (Landeira, 2010; Herz, 2012 and references therein).

To further address whether Jarid2 constitutes a core PRC2 component, is involved in recruitment of PRC2 to chromatin, and regulates H3K27 methylation in Drosophila, a Jarid2 complex was purified from flies and a global in vivo analysis of was performed of Suppressor of zeste 12 [Su(z)12] and H3K27me3 occupancy in Jarid2 mutant animals. The data confirm that Drosophila Jarid2 purifies with the core members of the PRC2 complex. In imaginal discs, global H3K27me3 levels are only weakly but reproducibly affected under Jarid2 mutant and Jarid2-overexpressing conditions. These genome-wide studies suggest that in Drosophila, under physiological conditions, Jarid2 does not appear to be a canonical component of the PRC2 complex as PRC2 recruitment is not altered on most target genes in Jarid2 mutant animals. Interestingly, overexpression of Jarid2 results in reduced Su(z)12 binding and changed chromatin compaction on polytene chromosomes, highlighting a possible role for Jarid2 in altering chromatin architecture. Genome-wide, Jarid2 and Su(z)12 binding correlate very well. However, certain loci, such as Homeobox (Hox) genes, differ significantly from this pattern. Here, Jarid2 occupancy on Polycomb response elements (PREs) is often very low where usually the highest enrichment for Su(z)12 can be observed. Gene expression analyses suggest a PRC2-dependent and -independent role for Jarid2 in transcriptional regulation. Jarid2 appears to be involved in the regulation of a certain number of PRC2 target genes and also transcriptionally controls a subset of genes independently of PRC2. These data not only imply a function for Jarid2 and PRC2 in transcriptional repression but also support a possible role for both Jarid2 and PRC2 in active transcription on genes that are occupied by these factors (Herz, 2012).

This study describes the purification of a Jarid2 complex in Drosophila. Consistent with previous results in mammalian systems, Jarid2 was found to be a component of PRC2. Evidence is provided that in imaginal discs and on polytene chromosomes, Jarid2 is required to fine-tune global H3K27me3 levels. Jarid2 might accomplish this by modulating the activity of the core complex [E(z), Su(z)12, Esc, and Caf1]. The data indicate that Jarid2 could play an inhibitory role in the implementation of H3K27me3 as Jarid2 mutant imaginal disc clones display a global increase and as overexpression of Jarid2 results in a reduction in H3K27me3. Despite having a JmjC domain, Jarid2 has been predicted and reported to be catalytically inactive as a histone demethylase. Therefore, it is unlikely but not impossible that it could function in this manner toward H3K27me3, thereby counteracting PRC2 activity. Even if Jarid2 would be inactive as a histone demethylase, it might still be able to bind to chromatin and prevent spreading of the H3K27me3 mark, such as opposing a possible positive spreading effect of Esc (EED in mammals) (Herz, 2012).

Furthermore, even though Jarid2 could be purified with the PRC2 core members and its occupancy generally correlates very well with canonical PRC2 components such as Su(z)12, it does not appear to play a significant role in regulating PRC2 recruitment in a physiological context, as assessed by Jarid2 mutant animal studies. Apparent differences with published mammalian studies, which imply a major role for Jarid2 in recruitment of PRC2, could be explained by variation in the mechanisms employed or by the fact that the recruitment of PRC2 in ES cells generally differs from that in differentiated tissues. For example, PREs have been known to be highly effective in recruiting PRC2 to target sites in Drosophila. In mammals, attempts have been made to identify functionally analogous sequences but with only limited success. Indeed, it seems more likely that the recruitment of PRC2 in mammals not only requires specific sequences but is also more dependent on additional factors (proteins and RNA), which might explain why PRC2 recruitment is more strongly affected in Jarid2-depleted cells and why PRC1 recruitment in some instances appears to be dependent on PRC2 (H3K27me3). However, the data in Drosophila salivary glands suggest that recruitment of PRC2 (and methylation of H3K27) is not a prerequisite for targeting of PRC1, and the generality of this mechanism is also increasingly questioned in the mammalian system. Nonetheless, when Jarid2 is overexpressed in Drosophila, changes in chromosome compaction can be observed. Under these conditions, Jarid2 extensively occupies the chromosomes, and Su(z)12 localization and H3K27me3 are negatively affected). It is possible that increasing Jarid2 levels beyond a certain physiological level might interfere with PRC2 integrity. Larger amounts of Jarid2 might alter the stoichiometry of the PRC2 subunits, resulting in destabilization of the PRC2 complex on chromatin (Herz, 2012).

Jarid2 also behaves differently from other canonical PRC2 members in Drosophila, as is evident from its binding pattern on certain Hox genes. At Hox genes, occupancy of PRE sites by canonical PRC2 members is one of the highest in the whole genome. In contrast, Jarid2 displays relatively low occupancy on many of these loci, implying a minor or different function for Jarid2 in controlling transcription of these well-described PRC2 targets. It is also possible that at these loci Jarid2 has a more transient association or even that it is less accessible to interact with the antibodies that were have generated. However, these findings are also in agreement with modifier screens that have been performed in Drosophila to identify major regulators of Polycomb group-mediated phenotypes but that were unable to capture Jarid2 (Herz, 2012).

Additionally, the data suggest that Jarid2 appears to control PRC2-dependent transcription, although not necessarily in the same way as expected for canonical PRC2 members. For example, in contrast to the mammalian findings, this study observed that PRC2-mediated transcriptional regulation by Jarid2 in Drosophila is generally independent of changes in Su(z)12 occupancy and does not correlate with changes in H3K27me3 enrichment. However, it needs to be stressed that most Jarid2/PRC2 cobound genes with altered expression patterns in Jarid2 mutants and E(z)-RNAi larvae contain no or low levels of H3K27me3, which is in contrast to the mammalian system where PRC2 components are usually found only at genes with high H3K27me3 enrichment. Nonetheless, in Drosophila, genes with high H3K27me3 enrichment exist that change in transcription in Jarid2 mutants and E(z)-RNAi animals, demonstrating that H3K27me3 is not necessarily instructive for transcriptional repression per se. To date most of the evidence ascribing to H3K27me3 the role of a repressive mark is based on correlation from the observation that PRC2 components colocalize with H3K27me3 and that the respective genes seem to be transcriptionally silenced. The data imply that this might generally be the case but that there are also exceptions to the rule. That certain H3K27me3 patterns can also be connected to transcriptionally active genes in mammals has just recently been reported (Young, 2011; Herz, 2012 and references therein).

Finally, the results imply that Jarid2 and PRC2 are not only involved in maintenance of gene repression but could also function in active transcriptional processes such as transcriptional activation of elongation. This is in agreement with previous reports and demonstrates that PRC2 has cellular functions that extend beyond what was learned from its role at Hox genes. Importantly, the current studies also suggest that despite a very good correlation of Jarid2 and Su(z)12 occupancies, Jarid2 might function in transcriptional repression and activation independently of the canonical PRC2 complex [E(z)] and vice versa. This distinction in target genes between Jarid2 and canonical PRC2 components [E(z)] provides additional confirmation that Jarid2 in some respects behaves fundamentally differently than the canonical PRC2 complex. Together with the varied functions proposed for Jarid2 in mammals, these studies highlight the diverse aspects of Jarid2 function in PRC2-mediated gene regulation (Herz, 2012).

The Trithorax group protein Lid is a trimethyl histone H3K4 demethylase required for dMyc-induced cell growth

The Myc oncoprotein is a potent inducer of cell growth, cell cycle progression, and apoptosis. While many direct Myc target genes have been identified, the molecular determinants of Myc’s transcriptional specificity remain elusive. A genetic screen was carried out in Drosophila and the Trithorax group protein Little imaginal discs (Lid) was identified as a regulator of dMyc-induced cell growth. Lid was originally identified in intergenic noncomplementation with a mutation in ash1, a trithorax group gene (Gildea, 2000; full text of article). Lid binds to dMyc and is required for dMyc-induced expression of the growth regulatory gene Nop60B. The mammalian Lid orthologs, Rbp-2 (JARID1A) and Plu-1 (JARID1B), also bind to c-Myc, indicating that Lid-Myc function is conserved. Lid is a JmjC-dependent trimethyl H3K4 demethylase in vivo, and this enzymatic activity is negatively regulated by dMyc, which binds to Lid’s JmjC domain. Because Myc binding is associated with high levels of trimethylated H3K4, it is proposed that the Lid-dMyc complex facilitates Myc binding to, or maintenance of, this chromatin context (Secombe, 2007). Identication of Lid as a histone H3 trimethyl-Lys4 demethylase has also been reported by Lee (2007) and Eissenberg (2007).

Lid is a 1838-amino-acid protein possessing numerous conserved motifs including an ARID (A/T-rich interaction domain), implicated in binding A/T-rich DNA; a single C5HC2 zinc finger; three PHD fingers (plant homeobox domain), implicated in forming protein-protein interactions; and Jumonji N and C (JmjN and JmjC) domains. JmjC-containing proteins have recently been shown to act as histone demethylase enzymes in a Fe2+ and -ketoglutarate-dependent manner (Klose, 2006). To test whether Lid can demethylate histones in vivo, Lid was overexpressed in fat body and in wing disc cells and the levels of mono-, di-, and trimethylated histone H3K4 and H3K27 were examined. Di- and trimethylated histone H4K20 and trimethylated histone H3K9 and H3K36 were also examined. Overexpression of Lid specifically decreased the levels of the trimethylated form of H3K4 but had no effect on the other methylated histones examined in either GFP-marked fat body or wing disc cells. Significantly, expression of Lid in the wing disc reduced trimethyl H3K4 in a dose-dependent manner, with two copies of the UAS-Lid transgene reducing trimethyl H3K4 more efficiently than one copy. Moreover, levels of trimethylated H3K4 are increased in wing discs from lid homozygous mutant animals, consistent with the model that Lid regulates the levels of this histone modification during normal development. To determine whether the JmjC domain of Lid is required for the observed H3K4 demethylation, transgenic flies were generated carrying a Gal4-inducible form of full-length Lid containing Ala substitutions at His637 and Glu639 (Lid-JmjC*) that abolishes the protein’s ability to bind the Fe2+ cofactor required for demethylase activity. Similar mutations have been shown to disrupt the demethylase function of the JmjC domains of JHDM2A, JHDM3A, JHDM1, and JMJD2A. Unlike wild-type Lid, expression of full-length Lid-JmjC* did not decrease levels of trimethylated H3K4 in fat body or in wing disc cells, demonstrating that an active JmjC domain is required for Lid-mediated H3K4 demethylation. Interestingly, expression of Lid-JmjC* resulted in increased levels of trimethyl H3K4 in the fat body, perhaps due to a dominant interfering effect on wild-type Lid function in these cells. Taken together, these results demonstrate that Lid is a trimethyl H3K4 demethylase that modifies nucleosomal histone H3 in vivo. The global regulation of H3K4 trimethylation status by Lid is not, however, likely to be mediated by recruitment by dMyc, since no effect was observed of reduced or increased dMyc expression on trimethyl H3K4 levels in either fat body or wing disc cells (Secombe, 2007).

Forty other genomic regions were identified that enhanced or suppressed the GMR-Gal4, UAS-dMyc (GMM) phenotype when heterozygous. Two of these regions delete genes encoding known regulators of dMyc stability, such as ago, or are involved in Myc transactivation, such as Pcaf. Specific mutations in both of these genes have been shown to enhance or suppress the GMM rough eye phenotype, respectively. Interestingly, none of the known direct transcriptional targets of dMyc were identified as genetic modifiers of the GMM phenotype, suggesting that the GMM phenotype arises from modulation of multiple genes and provides a powerful tool to identify proteins directly required for dMyc function in vivo (Secombe, 2007).

TrxG proteins are renowned for their essential role in maintaining homeotic (hox) gene expression during development, with mutations in many TrxG genes resulting in lethality due to homeotic transformations. Six TrxG protein complexes have been identified to date. While one function of these complexes is to antagonize Polycomb group (PcG) repression to maintain active hox gene expression, TrxG proteins are also recruited to other developmentally important genes to either activate or repress their transcription in a context-dependent manner. Based on the suppression of the GMM phenotype, the physical interaction between Lid and dMyc, and the requirement of Lid for dMyc-dependent activation of Nop60B, it is predicted that Lid acts as a dMyc coactivator involved in cell growth. The interaction between endogenous Lid and dMyc proteins is also likely to be essential for normal larval development since reducing the gene dose of lid is lethal in combination with the dmyc hypomorphic allele dmP0. In addition, genetically reducing lid enhances a small bristle phenotype induced by expression of a dMyc RNAi transgene. The original small discs phenotype described for lid mutants also suggests a role for Lid in the regulation of cell growth or proliferation during larval development. Unfortunately, this phenotype occurs at a frequency far too low (<1% of lid mutant larvae) to allow characterization (Secombe, 2007).

It is expected that the function of the Lid-Myc complex is evolutionarily conserved, since the human orthologs of Lid, Rbp-2 (JARID1A) and Plu-1 (JARID1B), bind strongly to c-Myc and dMyc in vitro, and both have been implicated in transcriptional regulation. Originally described as a binding partner for the tumor suppressor protein Retinoblastoma (RB), Rpb-2 has been shown to behave as a coactivator for RB at some promoters while antagonizing RB function at others (Benevolenskaya, 2005). Rbp-2 has also been identified as a transcriptional coactivator for nuclear hormone receptors (NRs) (Chan, 2001) and for the LIM domain transcription factor Rhombotin-2 (Mao, 1997). In addition, Plu-1 acts as a transcriptional corepressor for BF1 and PAX9 (Lu, 1999; Tan, 2003). While the transcriptional repression activities of Rbp-2 and Plu-1 are likely to be linked to a conserved trimethyl H3K4 demethylase activity, the molecular mechanism by which they activate transcription remains unclear. The mechanism by which Lid functions is currently being addressed by carrying out genetic screens using phenotypes generated by gain or loss of lid function (Secombe, 2007).

Coimmunoprecipitation analyses revealed that dMyc is likely to form multiple distinct complexes comprising TrxG proteins: One includes the Brm (SWI/SNF) nucleosome remodeling complex, and another contains Lid and Ash2. Consistent with the physical interaction observed between dMyc and Brm, components of the Brm complex suppress the GMM phenotype when genetically reduced, indicating that they are required for dMyc-induced cell growth. An interaction between Myc and the Brm complex has been observed in mammalian cells, where c-Myc interacts with the Brm (Brg1) subunit Ini1, and expression of a dominant-negative Brg1 allele inhibits c-Myc-dependent activation of a synthetic E-box reporter. However, the interaction between dMyc and the Brm complex described in this study, using Drosophila, provides the first demonstration of a biological significance for this complex (Secombe, 2007).

The second dMyc-TrxG complex identified includes Lid and Ash2, with Ash2 being immunoprecipitated with both anti-dMyc and anti-Lid antisera. In addition, decreased levels of Ash2 suppress, and increased levels of Ash2 levels enhance, the GMM phenotype, suggesting that Lid and Ash2 are limiting for dMyc-induced cell growth. In Schizosaccharomyces pombe, the orthologs of Ash2 and Lid (Ash2p and Lid2p) interact in vivo. While Ash2 has no known enzymatic activity, it is an integral component of several conserved complexes, including the SET1 histone methyltransferase complex (TAC1 in Drosophila; MLL in mammals) that is essential for methylation of histone H3K4. Biochemical purification of SET1, Lid2p, and Ash2p complexes from S. Pombe has demonstrated that the Lid2p-Ash2p complex is distinct from the SET1-Ash2 complex. Reducing the gene dose of the SET1 ortholog trx does not affect the GMM phenotype, consistent with the Drosophila Lid-Ash2-dMyc complex also being independent of TAC1 methyltransferase complex. The observation that Ash2 is a component of both H3K4 methylating (MLL) and demethylating (Lid) complexes is intriguing and suggests that it may be a crucial modulator of H3K4 methylation status. Whether Ash2 is required for Lid-mediated H3K4 demethylation is currently being tested (Secombe, 2007).

Lid is the first enzyme characterized that specifically demethylates trimethylated histone H3K4 in vivo. Based on the similarity between Lid and its mammalian orthologs Rbp-2 and Plu-1, it is expected that this demethylase activity to be conserved. The enzymatic activity of Lid requires a functional JmjC domain; however, Lid's specificity for a trimethylated lysine target is likely to be determined by the presence of a conserved N-terminal JmjN domain. Evidence to date suggests that proteins that possess both a JmjN and a JmjC domain prefer di- or trimethylated lysine substrates, whereas JmjC proteins that lack a JmjN domain demethylate mono- or dimethylated lysines. Indeed, analysis of the crystal structure of JMJD2A, which targets trimethylated H3K9 and K36, has revealed that the JmjN domain makes extensive contacts within the catalytic core of the JmjC domain, presumably accounting for the differences in target specificity between JmjC and JmjN/JmjC proteins (Secombe, 2007).

Trimethylated H3K4 is often found surrounding the transcriptional start site of active genes and is strongly correlated with binding by c-Myc. The trimethyl H3K4 demethylase activity of Lid would predict that Lid/Rbp-2 proteins may act as transcriptional repressors in a similar manner to LSD1, which demethylates mono- and dimethylated H3K4. Consistent with this hypothesis, it is observed that a large number of genes are derepressed in microarrays of homozygous lid mutant wing discs. However, expression of dMyc abrogates Lid's enzymatic activity, indicating that Lid is not acting as a demethylase when bound to dMyc. This is consistent with the observation that expression of Lid-JmjC* (a Gal4-inducible form of full-length Lid containing Ala substitutions at His637 and Glu639 that abolishes the protein’s ability to bind the Fe2+ cofactor required for demethylase activity) enhances the GMM eye phenotype. Indeed, Lid behaves as a dMyc coactivator based on the requirement for Lid in dMyc-induced expression of the growth regulator Nop60B. Both activation and repression functions have been previously suggested for Rbp2. Interestingly, LSD1's demethylase activity is also negatively regulated by an associated protein, BHC80, in a similar manner to the inhibition of Lid's enzymatic activity by dMyc. Dynamic regulation of histone demethylase activity is therefore likely to be a common feature of regulated gene expression in vivo (Secombe, 2007).

Recently, analysis of c-Myc target gene promoters revealed a strong dependence on trimethylated H3K4 for E-box-dependent c-Myc binding. Based on this observation, it is tempting to speculate that although Lid is likely to be enzymatically inactive when complexed with dMyc, Lid may retain its ability to recognize trimethylated H3K4 (perhaps through its JmjN domain) and thereby facilitate appropriate E-box selection. The inhibition of Lid demethylase activity by dMyc may also result in maintenance of local H3K4 trimethylation to permit binding of additional dMyc molecules or other transcription factors. The maintenance of trimethylated H3K4 by dMyc may allow binding of the NURF chromatin remodeling complex that specifically recognizes trimethylated H3K4. NURF binding, through its large BPTF subunit, has been correlated with spatial control of Hox gene expression and is thought to link H3K4 methylation to ATP-dependent chromatin remodeling. Finally, considering the fact that Lid contains multiple domains potentially involved in DNA binding and protein interaction, it is likely that interaction of Lid/Rbp-2 with Myc in Drosophila and mammalian cells will promote association of other proteins with the Myc-Lid complex, allowing further diversification of Myc function (Secombe, 2007).

Yorkie promotes transcription by recruiting a histone methyltransferase complex

Hippo signaling limits organ growth by inhibiting the transcriptional coactivator Yorkie. Despite the key role of Yorkie in both normal and oncogenic growth, the mechanism by which it activates transcription has not been defined. This paper reports that Yorkie binding to chromatin correlates with histone H3K4 methylation and is sufficient to locally increase it. Yorkie can recruit a histone methyltransferase complex through binding between WW domains of Yorkie and PPxY sequence motifs of NcoA6, a subunit of the Trithorax-related (Trr) methyltransferase complex. Cell culture and in vivo assays establish that this recruitment of NcoA6 contributes to Yorkie's ability to activate transcription. Mammalian NcoA6, a subunit of Trr-homologous methyltransferase complexes, can similarly interact with Yorkie's mammalian homolog YAP. The results implicate direct recruitment of a histone methyltransferase complex as central to transcriptional activation by Yorkie, linking the control of cell proliferation by Hippo signaling to chromatin modification (Oh, 2014).

Transcriptional activators increase transcription through recruitment of transcriptional proteins or through chromatin modification. Each of these encompasses a wide range of specific mechanisms, including interaction with core subunits of RNA polymerase, interaction with Mediator proteins, interaction with chromatin remodeling complexes, or interaction with complexes that influence posttranslational modifications of histones, such as acetylation or methylation. Previous studies have observed that Yki and YAP could interact with Mediator subunits, ATP-dependent chromatin remodeling complexes, and other transcription factors such as GAGA. Nonetheless, based on the results described in this study, it is argued that a key mechanism by which Yki activates transcription is increasing H3K4 methylation through recruitment of the Trr HMT complex. Most notably, point mutations in Yki that specifically impair its ability to interact with NcoA6 abolish its transcriptional activity, and this transcriptional activity is restored by fusion with NcoA6. Moreover, the essential role of Yki as a transcriptional coactivator for its DNA binding partner Sd can be bypassed by fusing NcoA6 directly with Sd (Oh, 2014).

These observations tie Yki's transcriptional activity most directly to NcoA6, and the argument that this reflects a necessary and sufficient role for H3K4 methylation in transcriptional activation by Yki rests in part on the identity of NcoA6 as a component of the Trr HMT complex. This argument receives further support from several additional observations: the strong, genome-wide correlation between Yki's association with chromatin and H3K4 methylation; the increased H3K4 methylation when Yki competent to interact with NcoA6 is targeted to a novel chromosomal location; the similar decreases in expression of Yki target genes when either NcoA6 or Trr are reduced by RNAi in cultured cells or in vivo; and the recent biochemical demonstration that H3K4 methylation of chromatin by MLL2, a Trr-homologous complex in mammals, could increase transcription in in vitro assays (Oh, 2014).

NcoA6 and Trr have previously been linked to transcriptional activation by nuclear hormone receptors. NcoA6 is believed to play an analogous role in transcriptional activation by nuclear receptors, i.e., its direct binding to these transcription factors recruits the Trr HMT complex or its mammalian homologs. However, a distinct structural motif (LxxLL) within NcoA6 mediates interactions with nuclear receptors. Thus, NcoA6 appears to act as a multifunctional adaptor protein that can link different classes of transcriptional activators to Trr/MLL2/3 HMT complexes, which as is established in this study are involved not only in transcriptional activation induced by nuclear receptors but also by Yki and its mammalian homologs (Oh, 2014).

Crosstalk between Hippo signaling and other pathways has been observed at the level of transcription factors, including physical interactions between Yki, YAP and TAZ, and β-catenin and SMADs, which are transcriptional effectors of Wnt and BMP signaling, respectively. Thus, the current observations raise the possibility that Trr-dependent H3K4 methylation could also contribute to transcriptional activation by these pathways (Oh, 2014).

In humans, NCOA6 has been identified as a gene commonly amplified and overexpressed in breast, colon, and lung cancers (it is also known as Amplified in breast cancer). In mice, gene-targeted mutations have implicated NcoA6 in promoting growth during development and wound healing. These roles in promoting growth are reminiscent of YAP, which is similarly required for growth during embryonic development and wound repair and linked to these cancers when amplified or activated. Thus, while functional studies linking mammalian NCOA6 to cell survival, growth, and cancer have previously been interpreted as a reflection of its role as a coactivator of transcription mediated by nuclear hormone receptors, the current results, together with analysis of MLL2 binding by ChIP-seq, argue that at least part of its effects reflect its role as a cofactor of YAP (Oh, 2014).

A notable feature of Hippo signaling is the recurrence of WW domains or PPxY motifs in multiple pathway components. Within Yki, YAP, and TAZ, the WW domains serve a dual role. They facilitate inhibition, as major negative regulators, including Warts/Lats, Expanded (in Drosophila), and Angiomotin (in mammals), utilize PPxY motifs to bind Yki, YAP, and TAZ and promote their cytoplasmic localization. Conversely, they also facilitate activation, through binding to Wbp2 and, as is shown in this study, NcoA6. It seems unlikely to be coincidental that key positive and negative partners of Yki/YAP/TAZ bind the same structural motifs. Rather, this shared recognition of the same motifs may have evolved to ensure tight on/off regulation of Yki/YAP/TAZ-dependent transcription (Oh, 2014).

Drosophila UTX is a histone H3 Lys27 demethylase that colocalizes with the elongating form of RNA polymerase II

Histone H3 methylation at Lys27 (H3K27 methylation) is a hallmark of silent chromatin, while H3K4 methylation is associated with active chromatin regions. This study reports that a Drosophila JmjC family member, concertina, light, and rolled) are dependent on their genomic localization for proper transcriptional regulation, as their expression is reduced when their genomic loci are rearranged to lie next to a euchromatic breakpoint or when heterochromatin component genes are mutated. By qRT-PCR assay, it was demonstrated that concertina, light, and rolled are repressed in third instar larval salivary glands upon reduction of H1 levels. Thus, H1 is also required for activation of heterochromatic genes within the context of pericentric heterochromatin (Lu, 2009).

It has been proposed that heterochromatin-associated proteins function to support normal transcription of heterochromatic genes when those genes are at their normal chromosomal sites and that position effects result when these genes are deprived of such essential proteins by displacement away from heterochromatin 'compartments.' Similarly, H1 may contribute to the formation of a particular chromatin structure that interferes with activation of euchromatic genes but to which heterochromatic genes have become adapted. The loss of H1 would deplete the nucleus of this particular chromatin conformation, releasing silenced genes from repression while simultaneously depriving the resident heterochromatin genes of their functional context. Interestingly, mutations of rolled, similar to H1 depletion, lead to late larval or early pupal lethality and defective imaginal disc formation. It remains to be seen whether one of the effects contributing to the lethality of H1-depleted animals is down-regulation of specific heterochromatic genes (Lu, 2009).

As a control, a limited analysis of possible effects of H1 abrogation was performed on expression of several euchromatic genes. So far, no euchromatic in vivo transcriptional target for H1 has been found in Drosophila larvae. However, this lack of apparent effect can be explained by the limited sample size (four genes) and the choice of targets. Only abundant, ubiquitous genes, were assayed, whose transcription units in the wild-type animals (without H1 abrogation) may be positioned within chromatin that already contains little or no H1. In the future, it will be important to extend this analysis to tissue-specific, tightly regulated genes and to perform this experiment in an unbiased, genome-wide (microarray) format (Lu, 2009).

Although the Drosophila polytene chromosome has served as a model to study chromatin structure, remarkably little is known about its spatial organization or the molecular mechanisms that maintain the alignment of sister chromatids. Previous studies suggested that interchromatid cohesion is generated and maintained in the banded regions. H1 is widely distributed in euchromatic arms of polytene chromosomes; however, it localizes predominantly to bands of compacted chromatin. H1 depletion disrupts the normal band-interband structure of polytene chromosomes. Thus, H1 functions to establish or maintain the parallel alignment of band chromosome fibrils. When depleted by RNAi, residual H1 protein is not distributed uniformly in polytene chromosomes. Remarkably, the residual H1 maxima correlate with the persistent band-interband structure over short fragments of the H1-depleted polytene chromosomes. This result emphasizes the requirement for H1 in polytene chromatid alignment/adhesion. Similarly, the dissociation of the normal single chromocenter in polytene chromosomes into several foci of HP1 localization in the H1 knockdown larvae may also be related to the loss of adhesion (Lu, 2009).

Linker histone H1 is an abundant protein component of chromatin. It binds to DNA outside the core particle region, and its function in internucleosomal interactions and chromatin condensation is widely accepted. It is possible that internucleosomal interactions directly mediated by H1 can occur in trans between two distinct chromatin fibrils and, thus, play a role in adhesion of sister chromatids in polytene chromosomes. In that case, genomic regions of intrinsically higher H1 density (bands) would then cluster ('align') in polytene chromosomes. This direct mechanism is consistent with the partial conservation of the polytene chromosome banding structure of H1-depleted salivary gland cells in regions that contain elevated levels of residual H1. However, a possibility that H1 activity in chromatid alignment is mediated through interactions with other molecules important for chromatin structure maintenance, such as H3S10 kinase JIL-1, cannot be excluded (Lu, 2009).

Although JIL-1 hypomorphic or null alleles exhibit a defect in polytene chromosome alignment comparable with that observed in H1 knockdown alleles, other functions of these proteins are remarkably dissimilar. Unlike H1, JIL-1 localizes to gene-active interbands and counteracts the function of Su(var)3-9. JIL-1 is also an enhancer of PEV. Furthermore, in JIL-1 alleles, polytene chromosome arms are highly condensed and interband regions are missing, with the male X chromosome affected the most severely. None of these phenotypes are observed in H1 knockdown animals. On the contrary, H1-depleted polytene chromosomes are rather extended, probably due to the dispersal of normally compacted band regions. However, both H1 and JIL-1 appear to contribute to polytene fibril alignment. It is possible that the polytene chromosome structure is established through interplay between antagonistic effects mediated by several effectors, such as H1 and JIL-1 (or its substrates). In the future, it will be interesting to elucidate fine details of these putative functional interactions between H1 and JIL-1 (Lu, 2009).

Although H1 is clearly required for chromatid alignment in endoreplicating cells, it is likely dispensable or less critical for sister chromatid alignment in G2-M of proliferating cells. Mutations that affect Drosophila genes coding for the Rad21 subunit of cohesin, CAP-G subunit of condensin, and Orc2 and Orc5 subunits of the origin recognition complex have been shown previously to affect sister chromatid alignment and segregation in vivo. Mutations in these genes result in massive missegregation of chromosomes during mitosis, which was not observed in H1-depleted animals. In contrast, these mutations do not cause any abnormalities in polytene chromosome structure. Thus, adhesion of replicating chromatin in dividing and endoreplicating cells in Drosophila is likely to be maintained through distinct mechanisms (Lu, 2009).

In conclusion, this study demonstrated that the linker histone H1 is essential for normal development in Drosophila and required for proper chromosome structure and function. Specifically, H1 is involved in the establishment of repressive pericentric heterochromatin and deposition/maintenance of the several histone modification marks that are localized in proximal heterochromatin. Furthermore, reduced H1 expression results in defective polytene chromosome structure with dissociation of the chromocenter and an almost complete loss of the banding pattern in the chromosome arms. Thus, linker histone H1 plays an essential role in the architecture and activity of metazoan chromosomes (Lu, 2009).

The role of piRNA-mediated epigenetic silencing in the population dynamics of transposable elements in Drosophila melanogaster

The piwi-interacting RNAs (piRNA) are small RNAs that target selfish transposable elements (TEs) in many animal genomes. Until now, piRNAs' role in TE population dynamics has only been discussed in the context of their suppression of TE transposition, which alone is not sufficient to account for the skewed frequency spectrum and stable containment of TEs. On the other hand, euchromatic TEs can be epigenetically silenced via piRNA-dependent heterochromatin formation and, similar to the widely known "Position-effect variegation", heterochromatin induced by TEs can "spread" into nearby genes. This study hypothesized that the piRNA-mediated spread of heterochromatin from TEs into adjacent genes has deleterious functional effects and leads to selection against individual TEs. Unlike previously identified deleterious effects of TEs due to the physical disruption of DNA, the functional effect investigated in this study is mediated through the epigenetic influences of TEs. The repressive chromatin mark, H3K9me, was found to be elevated in sequences adjacent to euchromatic TEs at multiple developmental stages in Drosophila melanogaster. Furthermore, the heterochromatic states of genes depend not only on the number of and distance from adjacent TEs, but also on the likelihood that their nearest TEs are targeted by piRNAs. These variations in chromatin status probably have functional consequences, causing genes near TEs to have lower expression. Importantly, stronger selection against TEs were found that lead to higher H3K9me enrichment of adjacent genes, demonstrating the pervasive evolutionary consequences of TE-induced epigenetic silencing. Because of the intrinsic biological mechanism of piRNA amplification, spread of TE heterochromatin could result in the theoretically required synergistic deleterious effects of TE insertions for stable containment of TE copy number. The indirect deleterious impact of piRNA-mediated epigenetic silencing of TEs is a previously unexplored, yet important, element for the evolutionary dynamics of TEs (Y. C. Lee, 2015).

Drosophila ptip is essential for anterior/posterior patterning in development and interacts with the PcG and trxG pathways

Development of the fruit fly Drosophila depends in part on epigenetic regulation carried out by the concerted actions of the Polycomb and Trithorax group of proteins, many of which are associated with histone methyltransferase activity. Mouse PTIP is part of a histone H3K4 methyltransferase complex and contains six BRCT domains and a glutamine-rich region. This study describes an essential role for the Drosophila ortholog of the mammalian Ptip (Paxip1) gene in early development and imaginal disc patterning. Both maternal and zygotic ptip are required for segmentation and axis patterning during larval development. Loss of ptip results in a decrease in global levels of H3K4 methylation and an increase in the levels of H3K27 methylation. In cell culture, Drosophila ptip is required to activate homeotic gene expression in response to the derepression of Polycomb group genes. Activation of developmental genes is coincident with PTIP protein binding to promoter sequences and increased H3K4 trimethylation. These data suggest a highly conserved function for ptip in epigenetic control of development and differentiation (Fang, 2009).

The establishment and maintenance of gene expression patterns in development is regulated in part at the level of chromatin modification through the concerted actions of the Polycomb and trithorax family of genes (PcG/trxG). In Drosophila, Polycomb and Trithorax response elements (PRE/TREs) are cis-acting DNA sequences that bind to Trithorax or Polycomb protein complexes and maintain active or silent states, presumably in a heritable manner. In mammalian cells however, such PRE/TREs have not been conclusively identified. Polycomb and Trithorax gene products function by methylating specific histone lysine residues, yet how these complexes recognize individual loci in a temporal and tissue specific manner during development is unclear. Recently, a novel protein, PTIP (also known as PAXIP1), was identified that is part of a histone H3K4 methyltransferase complex and binds to the Pax family of DNA-binding proteins (Patel, 2007). PTIP is essential for assembly of the histone methyltransferase (HMT) complex at a Pax DNA-binding site. These data suggest that Pax proteins, and other similar DNA-binding proteins, can provide the locus and tissue specificity for HMT complexes during mammalian development (Fang, 2009).

In mammals, the PTIP protein is found within an HMT complex that includes the SET domain proteins ALR (GFER) and MLL3, and the accessory proteins WDR5, RBBP5 and ASH2. This PTIP containing complex can methylate lysine 4 (K4) of histone H3, a modification implicated in epigenetic activation and maintenance of gene expression patterns. Furthermore, conventional Ptip-/- mouse embryos and conditionally inactivated Ptip-/- neural stem cell derivatives show a marked decrease in the levels of global H3K4 methylation, suggesting that PTIP is required for some subset of H3K4 methylation events (Patel, 2007). The PTIP protein contains six BRCT (BRCA1 carboxy terminal) domains that can bind to phosphorylated serine residues. This is consistent with the observation that PAX2 is serine-phosphorylated in response to inductive signals. In mammals, PAX2 specifies a region of mesoderm fated to become urogenital epithelia at a time when the mesoderm becomes compartmentalized into axial, intermediate and lateral plate. These data suggest that PTIP provides a link between tissue specific DNA-binding proteins that specify cell lineages and the H3K4 methylation machinery (Fang, 2009).

To extend these finding to a non-mammalian organism and address the evolutionary conservation of Ptip, it was asked whether a Drosophila ptip homolog could be identified and if so, whether it is also an essential developmental regulator and part of the epigenetic machinery. The mammalian Ptip gene encodes a novel nuclear protein with two amino-terminal and four carboxy-terminal BRCT domains, flanking a glutamine-rich sequence. Based on the number and position of the BRCT domains and the glutamine-rich domain, the Drosophila genome contains a single ptip homolog. To understand the function of Drosophila ptip in development, a ptip mutant allele was characterized that contained a piggyBac transposon insertion between BRCT domains three and four. Maternal and zygotic ptip mutant embryos exhibited severe patterning defects and developmental arrest, whereas zygotic null mutants developed to the third instar larval stage but also exhibited anterior/posterior (A/P) patterning defects. In cell culture, depletion of Polycomb-mediated repression activates developmental regulatory genes, such as the homeotic gene Ultrabithorax (Ubx). This derepression is dependent on trxG activity and also requires PTIP. Microarray analyses in cell culture of Polycomb and polyhomeotic target genes indicate that many, but not all, require PTIP for activation once repression is removed. The activation of PcG target genes is coincident with PTIP binding to promoter sequences and increased H3K4 trimethylation. These data argue for a conserved role for PTIP in Trithorax-mediated epigenetic imprinting during development (Fang, 2009).

Embryonic development requires epigenetic imprinting of active and inactive chromatin in a spatially and temporally regulated manner, such that correct gene expression patterns are established and maintained. This study shows that Drosophila ptip is essential for early embryonic development. In larval development, ptip coordinately regulates the methylation of histone H3K4 and demethylation of H3K27, consistent with the reports that mammalian PTIP complexes with HMT proteins ALR and MLL3, and the histone demethylase UTX. In wing discs, ptip is required for appropriate A/P patterning by affecting morphogenesis determinant genes, such as en and ci. These data demonstrate in vivo that dynamic histone modifications play crucial roles in animal development and PTIP might be necessary for coherent histone coding. In addition, ptip is required for the activation of a broad array of PcG target genes in response to derepression in cultured fly cells. These data are consistent with a role for ptip in trxG-mediated activation of gene expression patterns (Fang, 2009).

Early development requires ptip for the appropriate expression of the pair rule genes eve and ftz. The characteristic seven-stripe eve expression pattern is regulated by separate enhancer sequences, which are not all equally affected by the loss of ptip. The complete absence of en expression at the extended germband stage also indicates the dramatic effect of ptip mutations on transcription. The characteristic 14 stripes of en expression depends on the correct expression of pair rule genes, which are clearly affected in ptip mutants. However, the maintenance of en expression at later stages and in imaginal discs is regulated by PREs and PcG proteins. If ptip functions as a trxG cofactor, then expression of en along the entire A/P axis in the imaginal discs of ptip mutants might be due to the absence of a repressor. This might explain the surprising presence of ectopic en in the anterior halves of imaginal discs from zygotic ptip mutants. This ectopic en expression is likely to result in suppression of ci through a PcG-mediated mechanism. Yet, it is not clear how en is normally repressed in the anterior half, nor which genes are responsible for derepression of en in the ptip mutant wing and leg discs (Fang, 2009).

The direct interaction of PTIP protein with developmental regulatory genes is supported by ChIP studies in cell culture. Given the structural and functional conservation of mouse and fly PTIP, mPTIP was expressed in fly cells; it can localize to the 5' regulatory regions of many PcG target genes that are activated upon loss of PC and PH activity. Consistent with the interpretation that a PTIP trxG complex is necessary for activation of repressed genes, mPTIP only bound to DNA upon loss of Pc and ph function. In the Kc cells, suppression of both Pc and ph results in the activation of many important developmental regulators, including homeotic genes. A recent report details the genome-wide binding of PcG complexes at different developmental stages in Drosophila and reveals hundreds of PREs located near transcription start sites. Strikingly, most of the genes found to be activated in the Kc cells after PcG knockdown also contain PRE elements near the transcription start site (Fang, 2009).

In vertebrates, PTIP interacts with the Trithorax homologs ALR/MLL3 to promote assembly of an H3K4 methyltransferase complex. The tissue and locus specificity for assembly may be mediated by DNA-binding proteins such as PAX2 (Patel, 2007) or SMAD2 (Shimizu, 2001), which regulate cell fate and cell lineages in response to positional information in the embryo. In flies, recruitment of PcG or trxG complexes to specific sites also can require DNA-binding proteins such as Zeste, DSP1, Pleiohomeotic and Pipsqueak. Whereas PcG complexes have been purified and described in detail, much less is known about the Drosophila trxG complexes. Purification of a trxG complex capable of histone acetylation (TAC1) revealed the proteins CBP and SBF1 in addition to TRX. By contrast, the mammalian MLL/ALL proteins are components of large multi-protein complexes capable of histone H3K4 methylation. Although the mutant analysis, the reduction of H3K4 methylation and the dsRNA knockdowns in Kc cells all suggest that Drosophila ptip has trxG-like activity and hence might be a suppressor of PcG proteins, a more definitve biochemical analysis awaits the generation of antibodies and the delineation of in vivo DNA-binding sites for PTIP and its associated proteins at specific target genes (Fang, 2009).

Mammalian PTIP is also thought to play a role in the DNA damage response based on its ability to bind to phosphorylated p53BP1. PTIP also binds preferentially to the P-SQ motif, which is a good substrate for the ATR/ATM cell cycle checkpoint regulating kinases. Several reports demonstrate that PTIP is part of a RAD50/p53BP1 DNA damage response complex, which can be separated from the MLL2 histone H3K4 methyltransferase complex. Both budding and fission yeast contain multiple BRCT domain proteins that are involved in the DNA damage response, including Esc4, Crb2, Rad9 and Cut5. All of these yeast proteins have mammalian counterparts. However, neither the fission nor budding yeast genomes encodes a protein with six BRCT domains and a glutamine-rich region between domains two and three, whereas such characteristic PTIP proteins are found in Drosophila, the honey bee, C. elegans and all vertebrate genomes. These comparative genome analyses suggest that ptip evolved in metazoans, consistent with an important role in development and differentiation (Fang, 2009).

In summary, Drosophila ptip is an essential gene for early embryonic development and pattern formation. Maternal ptip null embryos show early patterning defects including altered and reduced levels of pair rule gene expression prior to gastrulation. In cultured cells PTIP activity is required for the activation of Polycomb target genes upon derepression, suggesting an important role for the PTIP protein in trxG-mediated activation of developmental regulatory genes. The conservation of gene structure and function, from flies to mammals, suggests an essential epigenetic role for ptip in metazoans that has remained unchanged (Fang, 2009).

Accessibility of the Drosophila genome discriminates PcG repression, H4K16 acetylation and replication timing

Histone modifications are thought to regulate gene expression in part modulating DNA accessibility. This study measured genome-wide DNA accessibility in Drosophila melanogaster by combining M.SssI methylation footprinting with methylated DNA immunoprecipitation. Methylase accessibility demarcates differential distribution of active and repressive histone modifications as well as sites of transcription and replication initiation. DNA accessibility is increased at active promoters and chromosomal regions that are hyperacetylated at H4K16, particularly at the male X chromosome, suggesting that transcriptional dosage compensation is facilitated by permissive chromatin structure. Conversely, inactive chromosomal domains decorated with H3K27me3 are least accessible, supporting a model for Polycomb-mediated chromatin compaction. In addition, higher accessibility was detected at chromosomal regions that replicate early and at sites of replication initiation. Together, these findings indicate that differential histone-modification patterns and the organization of replication have distinct and measurable effects on the exposure of the DNA template (Bell, 2010).

Covalent modifications of histones, including acetylation and methylation, are important determinants of chromatin structure. These modifications are thought either to serve as platforms to recruit proteins to chromatin or to directly alter its physical properties, yet little is known about the exact molecular mechanisms involved. Chromatin structure is often separated into 'active chromatin' versus 'heterochromatin'. However, this bimodal classification does not take into account the degree of DNA accessibility or the multiple variables of chromatin in vivo. Active modifications such as histone H3 Lys4 methylation (H3K4me) and histone H4 hyperacetylation localize to promoters and the 5' ends of active genes, which also show low nucleosomal density and frequently harbor sites of DNase I hypersensitivity. Direct links have been established between histone H4 acetylation and higher-order chromatin folding. In vitro, acetylation of H4 at Lys16 (H4K16ac) can inhibit the formation of compact 30-nm fibers and impair the activity of ATP-dependent chromatin remodeling. As H4K16ac is critical for male dosage compensation in Drosophila, the effects observed in vitro may directly contribute to increase the accessibility of factors that promote transcriptional upregulation in vivo In contrast, heterochromatin has the property to remain condensed during interphase and is characterized by histone hypoacetylation and methylation at Lys9 of histone H3 (H3K9me2). H3K9 methylation is enriched at pericentric heterochromatin|, where it specifically interacts with heterochromatin protein 1 (HP1). HP1 binding to methylated Lys9 is proposed to serve as a scaffold to promote the formation of compact chromatin. Histone H3 lysine 27 methylation (H3K27me3), which is set by the Polycomb system, has also been implicated in the formation of repressive chromatin domains, as it inversely correlates with gene activity. Unlike the localized patterns of active marks, H3K27me3 spreads over larger regions harboring many target genes. The exact mechanism of repression remains unclear but may involve the recruitment of components of the Polycomb repressive complex 1 (PRC1). PRC1 core components have been shown to promote the compaction of nucleosomal arrays in vitro and to mediate long-range interactions in vivo, indicating that a change in chromatin organization could account for its repressive activity. Thus, it is generally assumed that repressive histone modifications form compact structures that reduce the accessibility for DNA-binding proteins such as transcription factors and the transcriptional machinery, whereas permissive marks would favor activator binding by exposing the DNA template. This study provide a genome-wide view of DNA accessibility in Drosophila using a modified DNA methylase accessibility assay (MeDIP footprint). Relating accessibility to a comprehensive analysis of histone modifications, different levels of chromatin structure were observed associated with distinct modification patterns. Gene-rich regions are embedded in generally accessible chromatin. Notably, DNA is highly exposed at active promoters, yet appreciable differences were detected in accessibility between transcribed and silent intragenic regions, suggesting similar degrees of chromatin compaction outside of cis-regulatory regions. However, it was not found that early-replicating regions, especially zones of replication initiation, are generally more accessible than late-replicating regions. This suggests that an open chromatin conformation that exposes DNA is not only a feature of active promoters but also of active origins of replication. In contrast, large chromosomal regions covered with H3K27me3 are least accessible for M.SssI methylation. This result supports a model of Polycomb-dependent chromatin compaction as a mode of repression of target genes. In contrast, regions marked with H3K9 methylation do not show reduced methylation footprinting, arguing for a mode of repression that, at the level of DNA accessibility, is distinct from that of the Polycomb pathway (Bell, 2010).

This genome-wide study provides a comprehensive view of DNA accessibility relative to a set of other chromosomal measures. The results suggest complex levels of chromatin organization delineated by distinct patterns of post-translational histone modifications and DNA replication (Bell, 2010).

Chromatin signatures of the Drosophila replication program

DNA replication initiates from thousands of start sites throughout the Drosophila genome and must be coordinated with other ongoing nuclear processes such as transcription to ensure genetic and epigenetic inheritance. Considerable progress has been made toward understanding how chromatin modifications regulate the transcription program; in contrast, relatively little is known about the role of the chromatin landscape in defining how start sites of DNA replication are selected and regulated. This study describes the Drosophila replication program in the context of the chromatin and transcription landscape for multiple cell lines using data generated by the modENCODE consortium. While the cell lines exhibit similar replication programs, there are numerous cell line-specific differences that correlate with changes in the chromatin architecture. Chromatin features were identified that are associated with replication timing, early origin usage, and ORC binding. Primary sequence, activating chromatin marks, and DNA-binding proteins (including chromatin remodelers) contribute in an additive manner to specify ORC-binding sites. Accurate and predictive models were generated from the chromatin data to describe origin usage and strength between cell lines. Multiple activating chromatin modifications contribute to the function and relative strength of replication origins, suggesting that the chromatin environment does not regulate origins of replication as a simple binary switch, but rather acts as a tunable rheostat to regulate replication initiation events (Eaton, 2011).

The chromatin landscape clearly impacts both the expression and the replication of the genome. For example, the transcriptionally active euchromatin typically replicates prior to the repressed heterochromatic sequences. Studies in yeast, Drosophila, and mammalian systems have shown that changes in histone acetylation are associated with changes in the replication program. However, a comprehensive view of the replication program in the context of chromatin modifications and DNA-binding proteins is lacking (Eaton, 2011).

The different modENCODE data types across multiple cell lines (The modENCODE Consortium 2010) allowed the definition of the chromatin and transcription landscape associated with features of the DNA replication program. For each replication data type (replication timing, early origins, and ORC binding), a 43 × 3 matrix was generated, with each column representing a specific cell line and each row representing the enrichment or correlations with chromatin marks, DNA-binding proteins, nucleosome density, histone variants, nucleosome turnover (CATCH-IT), and gene expression (RNA-seq). For the replication timing profiles where there are no discrete peak calls, the Spearman's correlation was calculated between each factor with the whole-genome replication timing profile. For early origins of replication and ORC-binding sites, the median log2 enrichment was calculated of each factor within all BrdU peaks and within 500 bp of ORC ChIP-seq peak centers, respectively (Eaton, 2011).

The selection and regulation of DNA replication origins was found to be associated with distinct sets of chromatin marks and DNA-binding proteins. Prior studies have associated early replication with active transcription and the presence of 'activating' chromatin modifications such as histone acetylation, whereas late replication is associated with 'repressive' chromatin marks such as those found in the heterochromatin. Indeed, this study found that gene expression is positively correlated with replication timing, as are generally euchromatic marks such as H3K4me1 and H3K18ac. In contrast, heterochromatic marks such as H3K27me3 and H3K9me2 are negatively correlated with replication timing. The sequences surrounding early origins were also enriched for activating chromatin marks as well as specific DNA-binding proteins, including chromatin remodeling factors (Eaton, 2011).

Because many of the ORC-binding sites colocalized with promoters of active genes, the ORC-binding sites were separated into those that are TSS proximal (within 1 kb of a TSS) and those that were not at a TSS (distal). Particular interest was placed on chromatin features that are shared between ORC-binding sites both proximal and distal to promoters. Additionally, marks that are specific to ORC sites distal from a promoter will be of interest, as these marks may be required for ORC binding or function in the absence of a promoter (Eaton, 2011).

ORC-binding sites proximal to TSSs were enriched for chromatin remodelers such as the NURF complex (NURF301 [also known as E(BX)], ISWI) as well as other DNA-binding proteins such as GAF, RNA Pol II, and CHRO. These TSS-associated ORC sites were also enriched for H3K9ac, H3K27ac, H3K4me2, and H3K4me3 -- marks frequently found at promoters. Interestingly, those ORC sites that did not overlap with a TSS (distal) were also enriched for chromatin remodelers ISWI and NURF301, as well as GAF, which has also been implicated in chromatin remodeling. Consistent with the idea of ORC localizing to dynamic and active chromatin, an enrichment was found for CATCH-IT and H3.3 at ORC sites both proximal and distal to TSSs, as well as a reduction in bulk nucleosome occupancy. ORC sites not located at promoters were enriched for many of the same histone marks as those at promoters, with a few notable exceptions. A decrease in H3K4me3 was found at ORC sites distal from a promoter, as well as an increase in H3K18ac and H3K4me1 (Eaton, 2011).

Chromatin features specific to transcription start sites such as RNA Pol II and H2Av were decreased at ORC-binding sites distal to promoter elements. A small amount of RNA Pol II signal remained in the TSS distal ORC-binding sites; however, in comparison to the local enrichment of ISWI and GAF, there was a clear reduction in local signal. The remaining signal may be due to unannotated transcription start sites (Eaton, 2011).

Chromatin marks that are associated with active transcription through gene bodies (e.g., H3K79me1, H3K36me1, and H3K36me3) were not found above background levels at any ORC-binding sites. However, H3K36me1 was found specifically flanking those ORC-binding sites that did not coincide with a TSS. ORC has been shown to facilitate the formation of heterochromatin and HP1 binding; however, ORC sites were depleted for heterochromatic histone modifications such as H3K27me3 and H3K9me2/3 and were only slightly enriched for HP1. This may be due, in part, to the inability to map distinct ORC-binding sites in repetitive sequences, a current limitation of high-throughput sequencing approaches (Eaton, 2011).

The chromatin signatures were examined of promoter elements with and without ORC associated to determine whether there were unique chromatin signatures specific for ORC associated promoters. Since those promoters with proximal ORC binding tend to be far more actively transcribed than those without ORC, the comparison was limited to active promoter elements only. It was found that ORC-associated promoters had modestly increased chromatin remodeling activities, decreased nucleosome occupancy, and greater evidence of nucleosome turn-over relative to other active promoters not associated with ORC. In summary, these results indicate that dynamic chromatin environments may contribute to ORC localization and the subsequent activation of replication origins (Eaton, 2011).

Histone H3K9 trimethylase Eggless controls germline stem cell maintenance and differentiation

Epigenetic regulation plays critical roles in the regulation of cell proliferation, fate determination, and survival. It has been shown to control self-renewal and lineage differentiation of embryonic stem cells. However, epigenetic regulation of adult stem cell function remains poorly defined. Drosophila ovarian germline stem cells (GSCs) are a productive adult stem cell system for revealing regulatory mechanisms controlling self-renewal and differentiation. This study shows that Eggless (Egg), a H3K9 methyltransferase in Drosophila, is required in GSCs for controlling self-renewal and in escort cells for regulating germ cell differentiation. egg mutant ovaries primarily exhibit germ cell differentiation defects in young females and gradually lose GSCs with time, indicating that Egg regulates both germ cell maintenance and differentiation. Marked mutant egg GSCs lack expression of trimethylated H3K9 (H3k9me3) and are rapidly lost from the niche, but their mutant progeny can still differentiate into 16-cell cysts, indicating that Egg is required intrinsically to control GSC self-renewal but not differentiation. Interestingly, BMP-mediated transcriptional repression of differentiation factor bam in marked egg mutant GSCs remains normal, indicating that Egg is dispensable for BMP signaling in GSCs. Normally, Bam and Bgcn interact with each other to promote GSC differentiation. Interestingly, marked double mutant egg bgcn GSCs are still lost, but their progeny are able to differentiate into 16-cell cysts though bgcn mutant GSCs normally do not differentiate, indicating that Egg intrinsically controls GSC self-renewal through repressing a Bam/Bgcn-independent pathway. Surprisingly, RNAi-mediated egg knockdown in escort cells leads to their gradual loss and a germ cell differentiation defect. The germ cell differentiation defect is at least in part attributed to an increase in BMP signaling in the germ cell differentiation niche. Therefore, this study has revealed the essential roles of histone H3K9 trimethylation in controlling stem cell maintenance and differentiation through distinct mechanisms (Wang, 2011).

Although the mouse H3K9 trimethylase SETDB1 was recently shown to be important for maintaining ESC self-renewal by repressing the expression of developmentally regulated genes (Bilodeau, 2009), its role in regulation of adult stem cells has not yet been established. In this study, it was shown that the Drosophila SETDB1 homolog, Egg, is required intrinsically for controlling GSC self-renewal and extrinsically for controlling GSC differentiation in the Drosophila ovary. The egg mutant ovaries exhibit both GSC loss and germ cell differentiation defects. It was further demonstrated that Egg controls GSC self-renewal by repressing a Bam/Bgcn-independent pathway. In addition, escort cell-specific RNAi-mediated knockdown of egg function leads to gradual escort cells (EC) loss and germ cell differentiation defects, indicating that Egg is required for EC maintenance and germ cell differentiation. Recently, it has been proposed that ECs function as a niche for promoting germ cell differentiation (Kirilly, 2011). Furthermore, Egg functions in ECs to control germ cell differentiation at least in part by preventing BMP signaling from spreading to the differentiation niche and regulating EC survival. Therefore, it is proposed that Egg is a key H3K9 trimethylase in the Drosophila ovary that is required intrinsically for controlling GSC self-renewal via repressing a Bam/Bgcn-independent differentiation pathway and in ECs for controlling germ cell differentiation by preventing BMP signaling spreading to the differentiation niche. The findings from this study have further supported the idea that ECs function as a germ cell differentiation niche. It will be of great interest to test if SETDB1 is also important for controlling adult stem cell self-renewal and differentiation in mammalian systems (Wang, 2011).

A previous study has shown Egg to be a primary H3K9 trimethylase in follicle progenitor cells for maintaining H3K9me3 and regulating their proliferation and survival. Egg and its co-factor Wde were also shown to be required for maintaining H3K9me3 in early germ cells and regulating their survival. This study has further demonstrated that Egg is required intrinsically for controlling GSC self-renewal and proliferation. H3K9me3 but not H3K9me2 is eliminated in marked egg mutant GSCs. In addition, marked egg mutant GSCs are lost rapidly from the niche in comparison with the marked control GSCs, further supporting the idea that Egg is required for GSC maintenance. Moreover, the marked egg mutant GSCs and mitotic cysts are negative for TUNEL-based ApopTag labeling, but the marked 16-cell cysts in the regions 2b and 3 of the germarium are observed to be positive, indicating that Egg is dispensable for the survival of GSCs and early mitotic cysts but is required for the survival of 16-cell cysts. Finally, marked egg mutant GSCs appear to proliferate slower than the control GSCs based on cyst production and BrdU labeling. RNAi-mediated knockdown was used to show that loss of Egg function from GSCs and their progeny leads to the accumulation of DNA damage, suggesting that Egg is required for maintaining genome integrity. The accumulated DNA damage could also explain retarded GSC proliferation and increased 16-cell cyst apoptosis. These results demonstrate that Egg is required intrinsically for GSC self-renewal and proliferation and for the survival of 16-cell cysts (Wang, 2011).

BMP signaling and E-cadherin-mediated cell adhesion are essential for maintaining GSCs in the Drosophila ovary. BMP signaling represses bam-GFP expression and activates Dad-lacZ expression in GSCs. H3K9me3 is thought to be a histone marker for heterochromatin formation and transcriptional repression. Surprisingly, in marked egg mutant GSCs, bam-GFP remains repressed as in wild-type GSCs, but Dad-lacZ expression fails to be activated, indicating that Egg, and presumably H3K9me3, is dispensable for BMP signaling-mediated transcriptional repression of bam. The requirement of Egg for transcriptional activation of Dad could be indirect, but the detailed mechanism awaits further investigation. It was further demonstrated functionally that Egg controls GSC self-renewal by repressing a Bam/Bgcn-independent pathway by showing that marked bgcn egg double mutant GSCs are still lost at a much faster rate than marked control GSCs. Previously, Pumilio and Pelota were proposed to control GSC self-renewal by repressing a Bam/Bgcn-independent differentiation pathway as mutations for either factor can drive differentiation of bam mutant germ cells. Interestingly, mutations in egg can also cause differentiation of bgcn mutant germ cells, further supporting the idea that Egg represses a Bam/Bgcn-independent differentiation pathway to maintain GSC self-renewal. There are two possible strategies for Egg to repress differentiation and thus maintain GSC self-renewal: Egg represses the expression of a gene(s) important for GSC differentiation or activates the expression of a gene(s) critical for repressing GSC differentiation. Unfortunately, it remains unclear how Egg represses GSC differentiation to maintain self-renewal. Therefore, the identification of Egg target genes in GSCs will help define the unknown GSC differentiation pathway along with the identification of target genes of Pumilio and Pelota in order to gain a deeper understanding of GSC self-renewing mechanisms (Wang, 2011).

During the revision of this manuscript, a study was published to propose that Egg is required for H3K9me3 and heterochromatin formation in CBs and differentiated cysts, and is required for expression of piRNA genes and thus repression of transposable elements (TEs) (Rangan, 2011). Loss of piRNAs in germ cells is known to cause the activation of transposable elements (TEs) and consequently an increase in DNA damage. Consistently, this study shows that loss of egg function in germ cells leads to the accumulation of DNA damage. The regulation of piRNA by Egg offers mechanistic insight into why Egg is required for GSC maintenance and proliferation (Rangan, 2011). However, the current study has two different findings. One is that H3K9me3 establishment begins from GSCs, but not from CBs as the published study proposed. The other is that Egg is also required intrinsically for GSC maintenance and proliferation, but not for CB differentiation. The published study showed that spectrosome-containing single germ cells accumulate following germline-specific egg knockdown. In the current study, germline-specific expression of eggRNAi-1 leads to GSC loss, which is consistent with the mutant clonal analysis results, whereas the expression of eggRNAi-2 results in swollen germaria containing a few more spectrosome-containing CBs and cysts than control. The accumulation of the few more single germ cells is likely due to DNA damage-caused slowdown of mitotic progression. The difference between the published study and the current study could be simply caused by different egg knockdown efficiencies (Wang, 2011).

egg homozygous ovaries accumulate more undifferentiated germ cells and gradually lose their GSCs, which appear to be paradoxical. The egg mutant GSC loss phenotype can be attributed to the intrinsic requirement for GSC self-renewal. Further genetic analysis has revealed the requirement of Egg in ECs for controlling GSC differentiation by EC-specific RNAi-mediated egg knockdown. In the absence of Egg function from ECs, GSC progeny fail to differentiate and continuously proliferate as single germ cells, indicative of differentiation defects. In addition, loss of Egg function in ECs also causes EC loss, and in the complete absence of ECs, the progeny that have been generated before GSC loss also accumulate as single germ cells, further supporting that ECs are required for CB differentiation. Some of the accumulated single germ cells appear to upregulate Dad-lacZ expression and repress bam-GFP expression, suggesting that BMP signaling spreads to the germ cell differentiation niche, thereby interfering with germ cell differentiation. These findings suggest that Egg is required in ECs to promote germ cell differentiation at least in part by preventing self-renewal-promoting BMP signaling from spreading to the germ cell differentiation niche (Wang, 2011).

EFGR signaling has been suggested to act in ECs to control germ cell differentiation by repressing expression of Dally, a protein important for facilitating BMP diffusion. Interestingly, in the egg knockdown ECs, the expression of pERK, an EGFR signaling indicator, still remains normal, indicating that Egg is not essential for EGFR signaling in ECs. However, dally knockdown in ECs can partially suppress the egg knockdown mutant germ cell tumor phenotype, indicating that upregulation of dally in egg knockdown ECs contributes to BMP upregulation in the differentiation niche and to germ cell differentiation defects. The regulation of dally in ECs by Egg could be direct or indirect. The newly published study on Egg has shown that loss of Egg function in ECs leads to defective piRNA production and germ cell differentiation defects (Rangan, 2011). Consistently, this study also confirmed that egg knockdown in ECs results in dramatically increased expression of transposable elements (TEs). The germ cell differentiation defect can be rescued by a mutation in one of the DNA damage checkpoint genes, suggesting that DNA damage in ECs affects their ability to regulate germ cell differentiation. It will be of great interest to investigate if the mutation in the checkpoint gene also rescues defective BMP signaling in differentiated cells. Based on the findings from this study, it is proposed that Egg functions downstream of or in parallel with EGFR signaling to repress dally expression in ECs, thereby preventing BMP signaling from spreading to the differentiation niche. Because the signal(s) from ECs to control germ cell differentiation has not been identified yet, it remains unclear whether Egg also regulates additional factors independent of BMP signaling in ECs to control germ cell differentiation (Wang, 2011).

In this study, it was also shown that the egg knockdown ECs are gradually lost, and that GSCs cannot be maintained in the complete absence of ECs. This is consistent with the recently published finding that disruption of Rho function in ECs also cause EC loss and thus GSC loss (Kirilly, 2011). Because 5 to 6 most anteriorly localized ECs directly contact cap cells and GSCs, it is proposed that these ECs function as a part of the GSC niche to promote self-renewal by directly providing signals or indirectly by regulating cap cells function. A previous study suggests that JAK-STAT signaling functions in ECs to control GSC maintenance indirectly. How these GSC-contacting ECs contribute to GSC regulation remains to be further investigated (Wang, 2011).

Panoramix enforces piRNA-dependent cotranscriptional silencing

The Piwi-interacting RNA (piRNA) pathway is a small RNA-based innate immune system that defends germ cell genomes against transposons. In Drosophila ovaries, the nuclear Piwi protein is required for transcriptional silencing of transposons, though the precise mechanisms by which this occurs are unknown. This study shows that the CG9754 protein is a component of Piwi complexes that functions downstream of Piwi and its binding partner, Asterix, in transcriptional silencing. Enforced tethering of CG9754 to nascent messenger RNA transcripts causes cotranscriptional silencing of the source locus and the deposition of repressive chromatin marks. CG9754 has been named 'Panoramix,' and it is proposed that this protein could act as an adaptor, scaffolding interactions between the piRNA pathway and the general silencing machinery that it recruits to enforce transcriptional repression (Yu, 2015).

The Piwi interacting RNA (piRNA) pathway controls transposons through a number of distinct, but likely interlinked, mechanisms. Whereas cytoplasmic Piwi proteins silence their targets posttranscriptionally through piRNA-directed cleavage and the ping-pong cycle, nuclear Piwi-piRNA complexes function at the transcriptional level. Piwi-directed repression of transcription is thought to be dependent on piRNA-guided recognition of nascent transposon transcripts. Transcriptional gene silencing (TGS) correlates with the presence of histone H3 lysine 9 trimethylation (H3K9me3) marks, yet the mechanism through which Piwi binding promotes the deposition of these marks remains enigmatic. With the exception of the zinc finger protein Asterix (also known as DmGTSF1), the components of Piwi effector complexes at target loci are largely unexplored (Yu, 2015).

This study systematically mined candidate genes from RNA interference (RNAi) screens for potential TGS effector proteins and identified CG9754 in three independently published screens as being critical in both the germ cells and follicle cells for transposon silencing. Loss of CG9754 had essentially no effect on the abundance or content of piRNA populations or on the nuclear localization of Piwi protein, suggesting that it is probably an effector component. CG9754 encodes a ~60-kD nuclear protein with no identifiable domains. The expression of CG9754 is restricted to the female gonads, as is seen for other core piRNA pathway components such as Asterix (Yu, 2015).

To examine global effects on transposon expression, RNA sequencing (RNA-seq) was used to measure steady-state RNA levels from ovaries with germline-specific knockdowns of either CG9754 or Piwi. Piwi knockdown caused a sharp rise in transposon transcripts, with minimal effects on protein-coding gene expression. Knockdown of CG9754 caused effects very similar to those of Piwi, with most transposon targets being shared. Changes in steady-state RNA levels could have resulted from alterations in either element transcription or the stability of transposon mRNAs. Global run-on sequencing (GRO-seq) was used to measure nascent RNA synthesis following gene knockdown. Loss of either CG9754 or Piwi produced very similar profiles, suggesting that CG9754 is specifically required for transcriptional silencing of transposons targeted by Piwi (Yu, 2015).

Piwi-mediated TGS correlates with the presence of H3K9me3 marks at silenced transposons. Depletion of either CG9754 or Piwi resulted in nearly identical losses of H3K9me3 over transposons. Four independent frameshift mutations of CG9754 generated via the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein system were isolated. A consistent global up-regulation of transposable elements and the corresponding loss of H3K9me3 marks in CG9754 mutant ovaries were observed without changes in piRNA levels. Similarly to other core piRNA pathway mutants, female flies lacking CG9754 were sterile. Moreover, flies double-mutant for CG9754 and Asterix showed transposon derepression comparable to that of flies with either single mutation, suggesting that both genes act in the same pathway. Thus, CG9754 functions along with Piwi and Asterix in the repression of transposon transcription (Yu, 2015).

Next it was asked whether the presence of CG9754 at a target locus might be sufficient to induce its silencing. Because piRNAs likely direct binding to nascent RNAs rather than to the DNA of their targets, CG9754 was delivered via protein-RNA interactions. A series of luciferase reporters was constructed with BoxB sites in their 3' untranslated regions (UTRs) and they were used to create transgenic reporter flies. BoxB sites are bound by the λN protein, which can also bring other components to the RNA as part of a fusion. Flies were also generated expressing λN proteins fused to CG9754, Asterix, nuclear Piwi, and, as a negative control, a cytoplasmic Piwi missing its nuclear localization signal (dN-Piwi). When coexpressed with the reporter, the dN-Piwi fusion failed to induce any change in luciferase expression. Of the remainder, only the CG9754 fusion considerably reduced luciferase activity. Silencing appeared to be dosage-dependent, as the degree of repression correlated with the number of BoxB binding sites inserted into the reporter mRNA. Consistent with a role for CG9754 in transcriptional silencing, the abundance of the reporter mRNAs was significantly reduced upon tethering (Yu, 2015).

Although CG9754-triggered repression appeared to be independent of chromatin context, integration of the reporter into genomic DNA appeared to be critical for repression. Transient cotransfection of reporter constructs into the OSS cell line, which contains an active piRNA pathway, resulted in little to no detectable silencing. In contrast, tethering Drosophila Ago1 (λN-dAgo1) to the luciferase reporter mRNA in OSS cells caused substantial repression of the same reporter. These results indicate that CG9754 can function properly only in the context of chromatin, likely acting at the transcriptional level, by interacting with nascent transcripts (Yu, 2015).

To test the hypothesis that λN-CG9754 acts on nascent transcripts, a reporter was generated for which the BoxB binding sites were located within the intron of the primary transcript. λN-CG9754 maintained the ability to repress this reporter but not a similar transcript carrying BoxB sites in the antisense orientation integrated into the same genomic locus. Because the spliced, mature reporter transcripts lack the BoxB sites, it was reason that λN-CG9754 must be able to exert its effects by binding to unspliced precursor mRNAs. Given that splicing occurs cotranscriptionally, this implies that CG9754 confers its effects by interaction with the nascent transcript (Yu, 2015).

If CG9754 mediates Piwi-dependent transcriptional silencing, delivery of CG9754 alone might recapitulate hallmarks of piRNA-directed repression. Tethering of CG9754 had a highly specific effect, changing levels of only the reporter mRNA. Repression occurred at the transcriptional level, as GRO-seq indicated a loss of nascent RNA from the integrated reporter. Repression by CG9754 also correlated with specific deposition of H3K9me3 marks over the reporter locus. Tethering of CG9754 failed to trigger piRNA production from the reporter, as has been seen previously for some loci that become targets of the piRNA pathway. Of note, spreading of H3K9me3 marks to other regions of the reporter gene was observed, as described previously for regions flanking piRNA-targeted transposon insertions. Thus, delivering CG9754 to the nascent RNA causes repression of a locus in a manner that mimics targeting by the piRNA pathway (Yu, 2015).

Whether CG9754 might be a component of Piwi complexes, as predicted by epistasis experiments, was tested. Functional GFP-Piwi fusion proteins copurified with hemagglutinin (HA)-tagged CG9754 from OSS cells, but not with a negative-control fusion (HA-mKate2). Conversely, Flag-tagged CG9754 was able to specifically precipitate endogenous Piwi proteins from OSS cell lysates, confirming the interaction between these two proteins. Given its properties, CG9754 was named 'Panoramix,' after the mentor who empowers the French comic book character Asterix to perform his feats of strength (Yu, 2015).

The identification of Panoramix as a key mediator of piRNA-directed TGS presented an opportunity to use the tethering assay to dissect the mechanism of transcriptional silencing. RNAi was used to deplete selected piRNA pathway genes in flies in which λN-Panoramix was tethered to the luciferase-BoxB reporter. Knockdown of Panoramix itself weakened the repression significantly, as compared with a control knockdown (mCherry). Silencing of factors required for piRNA biogenesis (Zuc and Armi) or those that are expected to act upstream of Panoramix (Piwi and Asterix) did not significantly affect repression. Depletion of dLSD1/Su(var)3-3 and its cofactor, CoREST, which normally form a complex that removes H3K4me2 marks from promoters, had significant effects on the ability of Panoramix to repress the reporter. Because H3K4me2 marks actively transcribed genes, it is possible that dLSD1-mediated removal of these marks is a key step in Panoramix-mediated transcriptional silencing. This raises a potential parallel with piRNA-directed silencing in mice, wherein engagement by DNMT3L, which is necessary for piRNA-induced DNA methylation, requires removal of such marks. Similarly, knockdown of HP1a caused derepression, in agreement with its role as a constitutive heterochromatin component required for transposon silencing and with the observation that the presence of Panoramix is correlated with the deposition of H3K9me3 marks at target loci. The H3K9 methyltransferase Eggless/dSETDB1 and its cofactor Windei appeared to be required specifically for Panoramix-mediated silencing, as knockdown of G9a, another H3K9 methyltransferase, showed no effect on the reporter. In eggless mutants, essentially complete derepression of the reporter was observed, despite Panoramix tethering. In contrast, the piRNA biogenesis mutant zuc showed little to no effect on the repression of the reporter, as also observed in zuc RNAi experiments. These data raise the possibility that Eggless could be one of the enzymes responsible for the deposition of H3K9me3 marks over silenced transposons in a Piwi-targeted fashion (Yu, 2015).

Panoramix functions downstream of Piwi and Asterix and is both necessary and sufficient to elicit transcriptional repression when bound to nascent transcripts. Panoramix represents an example in metazoans of a protein inducing cotranscriptional silencing when recruited to the nascent transcript from a locus. In fact, only cotranscriptional silencing can resolve the conundrum of a target being transcriptionally repressed while transcripts from that target locus are responsible for recruiting their own repressors. Orthologs of some of the general silencing factors that act with Panoramix to deposit and interpret repressive chromatin marks have also been implicated in mammalian transposon silencing, in which the pathway functions by causing heritable DNA methylation. Though one cannot identify a mammalian ortholog of Panoramix based on primary sequence alone, the overall conservation of the piRNA-mediated transcriptional machinery suggests that a protein with an equivalent function likely exists in mammals (Yu, 2015).

Dynamic acetylation of all lysine-4 trimethylated histone H3 is evolutionarily conserved and mediated by p300/CBP

Histone modifications are reported to show different behaviors, associations, and functions in different genomic niches and organisms. This study shows that rapid, continuous turnover of acetylation specifically targeted to all K4-trimethylated H3 tails (H3K4me3), but not to bulk histone H3 or H3 carrying other methylated lysines, is a common uniform characteristic of chromatin biology in higher eukaryotes, being precisely conserved in human, mouse, and Drosophila. Furthermore, dynamic acetylation targeted to H3K4me3 is mediated by the same lysine acetyltransferase, p300/cAMP response element binding (CREB)-binding protein (CBP), in both mouse and fly cells. RNA interference or chemical inhibition of p300/CBP using a newly discovered small molecule inhibitor, C646, blocks dynamic acetylation of H3K4me3 globally in mouse and fly cells, and locally across the promoter and start-site of inducible genes in the mouse, thereby disrupting RNA polymerase II association and the activation of these genes. Thus, rapid dynamic acetylation of all H3K4me3 mediated by p300/CBP is a general, evolutionarily conserved phenomenon playing an essential role in the induction of immediate-early (IE) genes. These studies indicate a more global function of p300/CBP in mediating rapid turnover of acetylation of all H3K4me3 across the nuclei of higher eukaryotes, rather than a tight promoter-restricted function targeted by complex formation with specific transcription factors (Crump, 2011).

Dynamic acetylation of all H3K4me3 mediated by CBP is evolutionarily conserved, observed before the divergence of the single Drosophila enzyme dCBP into the paralogs p300 and CBP in mammals. CBP was discovered as a transcriptional coactivator that binds to CREB and p300 complements this activity. Whereas p300 and CBP are often considered functionally redundant, some studies support unique roles. The majority of genes bound by one also show high levels of the other, suggesting common targeting. They interact with many transcription factors and coactivators, initially suggesting a structural role in promoter complexes; p300 and CBP have been localized to c-fos and c-jun by ChIP and through interactions with other proteins. A purely structural role was challenged following discovery of their acetyltransferase activity with the catalytic domain required for transcription from chromatinized promoter constructs in vitro and in vivo. Recent in vitro studies with reconstituted nucleosome arrays show a requirement for p300 KAT activity to allow decompaction of 30 nm chromatin, nucleosome remodelling, and transcription factor binding (Crump, 2011).

Consistent with previous mass spectrometry and biochemical work, this study shows that H3K4me3 tails are dynamically modified up to the pentaacetylated state, including at lysines 9, 14, and 18, This suggests that the enzyme responsible, p300/CBP, targets specific H3 tails but no specific lysine. In p300/CBP double-knockout mouse fibroblasts, forskolin-induced acetylation of lysine 5, 8, 12, and 16 of histone H4 at c-fos is inhibited, further suggesting no specific targeting of residues. A high level of acetylation is insufficient for efficient gene expression in vivo; treatment of cells with TSA enhances acetylation but interferes with c-fos and c-jun induction. Further, loss of gene expression and Pol II localization caused by p300/CBP inhibition cannot be relieved by preacetylating nucleosomes before inhibition. This indicates a more dynamic role for acetylation in gene expression, suggesting that turnover may be the important factor. Analyses of quiescent cells in which c-fos and c-jun are poised but inactive and inhibition of transcription with DRB both indicate that transcription is not required for dynamic acetylation (Crump, 2011).

The finding that dynamic acetylation and H3K4me3 colocalize on the same nucleosomes across the promoter and start site of c-fos and c-jun raises the question of their cotargeting. Even when the KAT-HDAC enzyme balance is drastically forced in favor of acetylation by HDAC inhibitors, strict targeting to H3K4me3 does not break down. Numerous ChIP studies have established the presence of H3K4me3, H3K9ac, p300/CBP, and HDACs at the promoter and 5' end of many genes, suggesting widespread colocalization (Crump, 2011).

There are two classes of model by which cotargeting of H3K4me3 and rapid dynamic acetylation may occur. The first involves independent targeting to the same loci and H3 tails, as previously shown for serine-10 phosphorylation and lysine-9 acetylation. This suggests that the relevant enzymes may be part of a common process, and cotargeting may arise from independent DNA sequence recognition or unique interactions with the machinery of signal transduction and transcriptional regulation; p300 and CBP have been isolated in complexes containing TATA-binding protein (TBP) and RNA polymerase II (Crump, 2011).

A second class of model is based on dependence of one modification on the other. For example, p300/CBP may mediate dynamic acetylation through direct or indirect recognition of trimethylated lysine 4, which may provide a binding platform or enhance KAT activity. In support of this mechanism WDR5 knockdown, which depletes H3K4 methylation, attenuates the TSA-induced increase in H3K9ac levels at promoters. Other KATs are known to be recruited to H3K4me3 to induce histone acetylation; yeast Yng1 and Yng2, which recognize H3K4me3 via their PHD fingers, form part of the NuA3 and NuA4 KAT complexes, respectively, and mammalian ING4 links HBO1 acetyltransferase activity to H3 lysine-4 trimethylated nucleosomes (Crump, 2011).

A sequential targeting mechanism is conceivable. Unmethylated CpG dinucleotides within CpG islands may primarily recruit CXXC motif-containing proteins, including the H3K4 methyltransferase MLL1 (56) and CGBP/Cfp1, which associates with H3K4 methyltransferases. DNA binding by Cfp1 has recently been shown to restrict Setd1A and H3K4me3 to euchromatic nonmethylated CpG regions. Similarly, ChIP-seq analysis has shown a tight association between Cfp1 and H3K4me3 at CpG islands, and Cfp1 knockdown depletes H3K4me3 levels at nonmethylated CpGs. This provides a plausible mechanism to target H3K4me3 to these regions, which could then recruit p300/CBP for dynamic histone acetylation (Crump, 2011).

Histone demethylase Utx and chromatin remodeler Brm bind directly to CBP and modulate acetylation of histone H3 lysine 27

Trithorax group (TrxG) proteins antagonize Polycomb silencing and are required for maintenance of transcriptionally active states. Previous studies have shown that the Drosophila acetyltransferase CREB-binding protein (CBP; Nejire) acetylates histone H3 lysine 27 (H3K27ac), thereby directly blocking its trimethylation (H3K27me3) by Polycomb repressive complex 2 (PRC2) in Polycomb target genes. This study shows that H3K27ac levels also depend on other TrxG proteins, including the histone H3K27-specific demethylase Utx and the chromatin-remodeling ATPase Brahma (Brm). Utx and Brm are physically associated with CBP in vivo, and Utx, Brm, and CBP colocalize genome-wide on Polycomb response elements (PREs) and on many active Polycomb target genes marked by H3K27ac. Utx and Brm bind directly to conserved zinc fingers of CBP, suggesting that their individual activities are functionally coupled in vivo. The bromodomain-containing C terminus of Brm binds to the CBP PHD finger, enhances PHD binding to histone H3, and enhances in vitro acetylation of H3K27 by recombinant CBP. brm mutations and knockdown of Utx by RNA interference (RNAi) reduce H3K27ac levels and increase H3K27me3 levels. It is proposed that direct binding of Utx and Brm to CBP and their modulation of H3K27ac play an important role in antagonizing Polycomb silencing (Tie, 2012).

Acetylation of histone H3K27, a strong predictor of active genes, has emerged as one of the central mechanisms for antagonizing/reversing Polycomb silencing since it directly prevents trimethylation of H3K27 by PRC2. Interestingly, H3K27ac is present in animals, plants, and fungi, but H3K27me3 and PRC2 homologs are present only in animals and plants, clearly indicating that H3K27ac has functions other than preventing H3K27 methylation. This study provides evidence that the Drosophila TrxG proteins Utx and Brm modulate H3K27 acetylation by CBP. Utx and Brm are physically associated with CBP in vivo and bind directly to ZF1 and the PHD finger of CBP. Genome-wide ChIP-chip analysis revealed that the chromatin binding sites of Utx, Brm, and CBP coincide on many genes and that strong peaks of all three are highly correlated with the presence of high levels of H3K27ac. Importantly, brm mutants and RNAi knockdown of Brm in vivo result in a decrease of H3K27ac and a concomitant increase of H3K27me3. Similarly, knockdown and overexpression of Utx with no change in the CBP level are sufficient to promote, respectively, a decrease and increase in the bulk H3K27ac level. This suggests that coupled H3K27 deacetylation/trimethylation and demethylation/acetylation are dynamically antagonistic. It further suggests that regulating the balance of these opposing activities is likely to play an important role in determining whether active and silent chromatin states will be maintained or switched (Tie, 2012).

This is the first report that Utx is physically associated with CBP. While functional collaboration between Utx and CBP is required to execute the sequential reactions required to switch Polycomb target genes from silent to active states, it was not obvious that this should require that they be physically associated. The fact that they are suggests that their two reactions are more efficiently coupled. It also suggests that despite the many histone and nonhistone substrates of CBP, H3K27 acetylation on Polycomb target genes to prevent their silencing is sufficiently critical to have evolved a Utx-CBP methyl-to-acetyl switching module, perhaps to counter the complementary coupling effect of the physical association of the H3K27 deacetylase RPD3 with PRC2 to create the antagonistic acetyl-to-methyl switch. Coupling of the Utx and CBP activities may increase the fidelity of maintenance of active chromatin states of Polycomb target genes by ensuring rapid reversal of H3K27ac deacetylation and methylation by RPD3 and PRC2 that may occur either adventitiously or as part of an ongoing dynamic balance between these antagonistic activities. Such coupling could also increase the efficiency of switching Polycomb target genes from a transcriptionally silent to an active state in response to developmentally programmed signals or other cellular signals, ensuring definitive establishment of the new active transcriptional state (Tie, 2012).

While Brm is well known for the chromatin remodeling activity associated with its highly conserved ATPase domain, the current findings identify another activity of Brm associated with its highly conserved BrD-containing C terminus [Brm(1417-1634)], which binds histone H3, enhances H3 binding to the CBP PHD finger, and enhances acetylation of H3K27 by CBP in vitro. The latter effect is most likely due to the simultaneous binding of Brm to H3 and the CBP PHD finger, thereby stabilizing the H3 interaction with the CBP HAT domain. However, it cannot be ruled out that it may also reflect a direct stimulatory effect of Brm on the intrinsic activity of the CBP HAT domain. In any case, this suggests that the physical association and genome-wide colocalization of CBP and Brm do not simply reflect a spatial and temporal coordination of their separate acetylation and chromatin remodeling activities but also reflect regulatory interactions. The reduced H3K27ac level in brm2 mutants and after RNAi knockdown is consistent with the observed enhancement of CBP HAT activity in vitro. However, at this time the possibility cannot be ruled out that the loss of Brm chromatin-remodeling activity in brm2 mutants may also contribute to their reduced H3K27ac level (Tie, 2012).

The physical association of Brm with CBP and with Utx reported in this study is consistent with recent reports that BRG1 can be coimmunoprecipitated with human CBP and p300 from tumor cells and with Utx/Jmjd3 in murine EL4 T cells. However, the region of human CBP reported to bind BRG1 differs from the current findings. It was reported that BRG1 interacts directly with the human CBP fragment containing ZF3, and the proline-rich region of BRG1 is required for this interaction. The current results indicate that only ZF1 and the PHD finger of Drosophila CBP bind directly to the BrD-containing C terminus of Brm. Since the proline-rich region of Drosophila Brm was not assayed due to its insolubility, the possibility cannot be ruled out that it mediates additional contact(s) with CBP (Tie, 2012).

CBP was not found in the previously purified Drosophila Brm-containing complexes, suggesting that only a portion of Brm is physically associated with CBP or that this association may be stabilized only on chromatin and/or may be regulated by other cellular signals. Consistent with this, there are some sites detected by ChIP-chip that are enriched for Brm and Utx without CBP and there are some column fractions containing Brm and Utx without CBP. It is possible that the CBP-Brm association may also serve to recruit Brm to some sites, e.g., recruitment of human Brm or BRG1 to the beta interferon (IFN-β) promoter depends on the prior presence of CBP and leads to subsequent nucleosome remodelin (Tie, 2012).

This study found that Utx, Brm, and CBP colocalize not only with H3K27ac at many regions, including promoters, transcribed regions, PREs, and other presumed cis-regulatory elements. They are also present, albeit at lower levels, on repressed genes marked by strong H3K27me3 domains (e.g., the ANT-C and BX-C). At such sites, the H3K27me3 may be protected from Utx-mediated demethylation by the binding of PC/PRC1. Alternatively, their lower levels at repressed genes may simply result in a dynamic balance of deacetylation/methylation and demethylation/acetylation that overwhelmingly favors the former. It is also possible that Utx and CBP may also be involved in Brm-dependent transcriptional repression at some sites. Recent evidence suggests that maintenance of steady-state levels of histone acetylation is highly dynamic, and the reciprocal changes in H3K27me3 and H3K27ac levels that occur upon altering Utx or E(Z) levels suggest that maintenance of histone methylation levels may also be highly dynamic. Much remains to be discovered about the factors that regulate the demethylase and acetyltransferase activities of Utx and CBP in different chromatin environments (Tie, 2012).

The bromodomains of yeast SWI2/SNF2 and human BRG1 bind specifically to H3K14ac, indicating that this binding specificity has been highly conserved during evolution. Brm(1417-1634) also binds specifically to H3K14ac, and the BrD of Brm is required for CBP binding and histone H3 binding. The BrD-containing C termini of human and plant Brm have been reported to be functionally important in vivo. Surprisingly, the BrD of Drosophila Brm has been reported to be dispensable for viability. A brm transgene containing a deletion of the central 72 residues of the BrD can rescue the late embryonic lethality of brm2 mutants, allowing them to develop into adults. Whether the reduced H3K27ac level of these brm2 mutants is also rescued has not been determined. This rescue could indicate that the Brm BrD is functionally redundant or at least not critical for achieving adequate expression of the genes responsible for the inviability of brm2 mutants. The brm2 mutation behaves genetically as a strong hypomorphic or null allele, but the sequence alteration responsible for its phenotype has not been determined and so the precise nature of its functional deficit is unknown (Tie, 2012).

In summary, this study has shown that Utx and Brm interact directly with CBP and modulate H3K27 acetylation. Utx presumably does so indirectly, at Polycomb target genes, by providing demethylated H3K27 substrate for acetylation by CBP. Their direct physical coupling could provide obvious gains in the efficiency of their two sequential reactions and their consequent H3K27ac yield. The interaction between Brm and CBP may similarly couple their activities, but this study also presents initial evidence to suggest that the binding of the Brm BrD-containing C terminus to H3 and the CBP PHD finger may also directly enhance H3K27 acetylation through its effect on H3 binding by the CBP HAT domain. It is expected that additional TrxG proteins will modulate H3K27 acetylation, including KIS and ASH1, which have recently been shown to affect H3K27me3 levels. The broad distributions of H3K27me3 over many Polycomb target genes is mirrored by similar broad distributions of H3K27ac when those genes are active, suggesting that in addition to its general genome-wide association with active genes, it may play a more specialized dual role at Polycomb target genes, where it also dynamically antagonizes the encroachment of Polycomb silencing (Tie, 2012).

Trithorax monomethylates histone H3K4 and interacts directly with CBP to promote H3K27 acetylation and antagonize Polycomb silencing

Trithorax (Trx) antagonizes epigenetic silencing by Polycomb group (PcG) proteins, stimulates enhancer-dependent transcription, and establishes a 'cellular memory' of active transcription of PcG-regulated genes. The mechanisms underlying these Trx functions remain largely unknown, but are presumed to involve its histone H3K4 methyltransferase activity. This study report that the SET domains of Trx and Trx-related (Trr) have robust histone H3K4 monomethyltransferase activity in vitro and that Tyr3701 of Trx and Tyr2404 of Trr prevent them from being trimethyltransferases. The trxZ11 missense mutation (G3601S), which abolishes H3K4 methyltransferase activity in vitro, reduces the H3 H3K4me1 but not the H3K4me3 level in vivo. trxZ11 also suppresses the impaired silencing phenotypes of the Pc3 mutant, suggesting that H3K4me1 is involved in antagonizing Polycomb silencing. Polycomb silencing is also antagonized by Trx-dependent H3K27 acetylation by CREB-binding protein (CBP). Perturbation of Polycomb silencing by Trx overexpression requires CBP. It was also shown that Trx and Trr are each physically associated with CBP in vivo, that Trx binds directly to the CBP KIX domain, and that the chromatin binding patterns of Trx and Trr are highly correlated with CBP and H3K4me1 genome-wide. In vitro acetylation of H3K27 by CBP is enhanced on K4me1-containing H3 substrates, and independently altering the H3K4me1 level in vivo, via the H3K4 demethylase LSD1, produces concordant changes in H3K27ac. These data indicate that the catalytic activities of Trx and CBP are physically coupled and suggest that both activities play roles in antagonizing Polycomb silencing, stimulating enhancer activity and cellular memory (Tie, 2014).

The major findings presented in this study are: (1) TRX and TRR are monomethyltransferases and together account for the bulk of the H3K4me1 in vivo; (2) the catalytic activities of both TRX and CBP are required to antagonize PcG silencing; (3) TRX and TRR are physically associated with CBP in vivo and TRX binds directly to the CBP KIX domain via a region that contains multiple KIX-binding motifs; (4) TRX and TRR colocalize genome-wide with H3K4me1 and CBP at PREs and enhancers; and (5) H3K4me1 enhances histone acetylation by CBP. Together, these data suggest that the primary target of TRX monomethyltransferase activity is not promoters but PREs and neighboring enhancers. They suggest a new model for how TRX antagonizes Polycomb silencing, stimulates active enhancers, and establishes a cellular memory of active transcription. This differs significantly from the previous view that TRX trimethylates H3K4 (Tie, 2014).

The evidence presented in this study indicates that the SET domains of TRX, TRR and their human orthologs possess intrinsic H3K4 monomethyltransferase activities and are prevented from being trimethyltransferases by the presence of the bulkier Tyr residue at their respective F/Y switch positions, as previously shown for MLL1. Although these data do not rule out the possibility that TRX and TRR complexes might have some H3K4 trimethylation activity in vivo in some chromatin contexts, the reduced H3K4me1 and apparently normal H3K4me3 levels in the catalytically inactive trxZ11 and trr3 mutants strongly suggest that H3K4 monomethylation is the predominant activity of TRX and TRR in vivo. Moreover, the absence of detectable H3K4me1 in trr3; trxZ11 double-mutant embryos suggests that they are the principal H3K4 monomethyltransferases in vivo, consistent with their genome-wide colocalization with H3K4me1 at PREs and enhancers. Suppression of Pc3 mutant phenotypes by trxZ11 further suggests that the monomethyltransferase activity of TRX plays a role in antagonizing Polycomb silencing (Tie, 2014).

This study found that TRX and TRR are physically associated with CBP in embryo extracts, confirming a previous report for TRX. The direct binding of TRX to the CBP KIX domain and the genome-wide correlation of H3K27ac with H3K4me1 on active genes suggests that their activities are coupled in vivo. Consistent with this, TRX-CBP complexes pulled down from embryo extracts have both H3K4 monomethyltransferase and H3K27 acetyltransferase activities. Moreover, the impaired Polycomb silencing caused by TRX overexpression in vivo (which elevates both H3K4me1 and H3K27ac levels) requires CBP and presumably the TRX-CBP interaction. Mutating the CID will be required to show this conclusively. No direct interaction between CBP and the TRR C-terminus was found, but it has been previously reported that CBP interacts directly with the H3K27 demethylase UTX, which is another subunit of the TRR complex. Together, these data suggest that these direct interactions are required for TRX- and TRR-dependent H3K27 acetylation and further suggest that TRX and TRR complexes function by fundamentally similar mechanisms (Tie, 2014).

The enhanced in vitro acetylation of H3K27 on K4me1-containing recombinant H3 substrates suggests that H3K4me1 might be a preferred CBP substrate in vivo. Consistent with this, altering the H3K4me1 level in vivo by manipulating LSD1 causes concordant changes in H3K27ac in adults. Moreover, a genome-wide analysis of hundreds of bona fide enhancers in purified mesodermal cells from Drosophila embryos revealed that H3K27ac is not present on enhancers without H3K4me1, whereas H3K4me1 is present without H3K27ac prior to enhancer 'activation'. This suggests that the presence of H3K4me1 might be a prerequisite for the deposition of H3K27ac at enhancers. Interestingly, some of the catalytically inactive trxZ11 mutants survive until the late pupal period and exhibit strong homeotic transformations. This suggests that TRX catalytic activity might be more important for stimulating enhancers that drive robust homeotic gene expression, whereas the physical association of TRX with CBP, which is intact in trxZ11, is more important for preventing silencing of normally active PcG-regulated genes in the embryo (Tie, 2014).

H3K4me1 and CBP are part of a conserved chromatin 'signature' of enhancers and H3K27ac marks 'active' enhancers. The data strongly suggest that TRX and TRR are responsible both for the H3K4me1 on enhancers and, via their physical association with CBP, for the H3K27ac on active enhancers. Determining which H3K4me1 is TRX dependent will require ChIP-seq analysis of trxZ11 mutant cells (Tie, 2014).

Like TRX, H3K4me1 and CBP are also present at PRE/TREs of both active and inactive genes, suggesting that PRE/TREs have a functional connection to enhancers. Functional analyses of the strong bxd PRE/TRE in vivo suggest that PRE/TREs are distinct from enhancers, do not possess enhancer activity, but can boost enhancer-dependent transcription in a TRX-dependent manner. A GAL4-TRX fusion protein tethered to a transgene reporter exhibits these same properties (Tie, 2014).

TRR was recently shown to occupy many presumed enhancers. This study has found that TRR binds more sites than TRX and also co-occupies most TRX binding sites genome-wide, including PRE/TREs. This raises the possibility that both TRX and TRR regulate many PcG-regulated genes, perhaps in different contexts or in response to different signals. The presence of UTX in the TRR complex suggests that TRR can facilitate switching of PcG-regulated genes from silent to active, whereas TRX might only be capable of maintaining the expression of genes activated prior to the onset of Polycomb silencing in the early embryo, or genes subsequently derepressed by the UTX activity associated with the TRR complex. This might explain the previously reported critical requirement for TRX in early embryogenesis (0-4 hours; i.e. prior to the onset of Polycomb silencing) for later robust expression of the homeotic genes in imaginal discs. Absence of TRX in 0- to 4-hour embryos cannot be compensated by its subsequent restoration. Further investigation will be required to determine whether and in what contexts there is functional collaboration or division of labor between TRX and TRR (Tie, 2014).

Although it is required continuously, the critical early requirement for TRX might provide an important clue to its function. This suggests that TRX and CBP, bound to PRE/TREs, might be required for de novo 'priming' of surrounding enhancers with H3K4me1 and H3K27ac in the early embryo, prior to the onset of Polycomb silencing and perhaps even prior to transcriptional activation of the zygotic genome (Tie, 2014).

There is little detectable H3K27me3 in 0- to 4-hour embryos, whereas the H3K27ac level is already high relative to later embryonic stages. H3K4me1 is already present during syncytial stages. It is speculated that before zygotic genome activation, TRX and CBP are constitutively bound to PRE/TREs and deposition of H3K4me1 and H3K27ac might initially be restricted to nucleosomes adjacent to PRE/TREs. Binding of activators to early-acting enhancers promotes spreading of H3K4me1 and H3K27ac from PRE/TREs across adjacent cis-regulatory regions to form broad domains, perhaps facilitated by interactions between activators and TRX/CBP complexes. Spreading of H3K27ac initially proceeds unchecked by H3K27me3, encompassing all surrounding enhancers, including those that will be 'activated' later (e.g. the imaginal disc enhancers) and protects them from subsequent deposition of H3K27me3 by PRC2 at the onset of Polycomb silencing. PcG-regulated genes that are not activated in the early embryo become subject to deposition/spreading of H3K27me3 in similar broad domains, blocking subsequent H3K27 acetylation. There might also be some active removal of pre-existing H3K27ac by PRC1/PRC2-associated RPD3. Subsequent activation requires removal of H3K27me3 by UTX, and thus might require TRR, which is also present at PRE/TREs and so is poised to respond to the binding of TRR-dependent activators, such as EcR (Tie, 2014).

Other functions of H3K4me1 and H3K27ac at PREs and enhancers are not yet understood, but they might (1) recruit H3K4me1 and H3K27ac 'readers' that further stimulate/maintain the active transcriptional state, (2) facilitate the targeting of enhancers to promoters and (3) perpetuate the broad domains of H3K4me1 and H3K27ac by enhancing their own deposition by TRX and CBP, as suggested by the enhancing effect of H3K4me1 on H3K27 acetylation in vitro. Perpetuation of the broad domains of H3K4me1 and possibly H3K27ac through replication and mitosis could also constitute the elusive cellular memory of past transcriptional activity (Tie, 2014).

Life span extension by targeting a link between metabolism and histone acetylation in Drosophila

Old age is associated with a progressive decline of mitochondrial function and changes in nuclear chromatin. However, little is known about how metabolic activity and epigenetic modifications change as organisms reach their midlife. This study assessed how cellular metabolism and protein acetylation change during early aging in Drosophila melanogaster. Contrary to common assumptions, it was found that flies increase oxygen consumption and become less sensitive to histone deacetylase inhibitors as they reach midlife. Further, midlife flies show changes in the metabolome, elevated acetyl-CoA levels, alterations in protein-notably histone-acetylation, as well as associated transcriptome changes. Based on these observations, the activity of the acetyl-CoA-synthesizing enzyme ATP citrate lyase (ATPCL) or the levels of the histone H4 K12-specific acetyltransferase Chameau were decreased. These targeted interventions both alleviate the observed aging-associated changes and promote longevity. These findings reveal a pathway that couples changes of intermediate metabolism during aging with the chromatin-mediated regulation of transcription and changes in the activity of associated enzymes that modulate organismal life span (Peleg, 2016).

The process of aging is characterized by a deterioration of multiple interconnected cellular pathways, which makes the identification of molecular mechanisms of phenotypic aging and death particularly difficult. Many molecular analyses have focused on the comparison of young and old organisms, which resulted in the formulation of nine hallmarks of aging ranging from telomere shortening and epigenetic alterations, to differences in nutrient sensing and stem cell depletion. While many of these experiments have identified valuable paths toward life span extension, such studies face the complication that old individuals suffer from the progressive deterioration of multiple cellular systems, which can make it challenging to distinguish primary from secondary effects. To identify changes involved in the onset of aging, this study compared D. melanogaster flies at young age and during midlife at the onset of a premortality plateau, when most individuals of a population are still alive (Peleg, 2016).

Surprisingly, heads from midlife flies consume more oxygen than the young ones. This is in apparent contradiction to the general observation of a reduced metabolism when animals age, which this study also observe in old flies. There are several possible explanations for this unexpected finding. In many studies, the oxygen consumption rate was extrapolated from measurements of isolated mitochondria, which may lack crucial extra-mitochondrial signals when investigated in isolation, whereas this study has measured activity in isolated fly heads. Alternatively, flies may change their feeding behavior when reaching midlife, or switch from an anaerobic to a more aerobic metabolism due to their decreased activity, which is consistent with higher levels of metabolites generated by oxidative processes in midlife flies. Finally, the metabolic changes may be due to a feed-forward activation of metabolic enzymes that become stimulated by hyper-acetylation. The observation that the treatment of isolated fly heads with lysine deacetylase (KDAC) inhibitors increases oxygen consumption rate (OCR) within minutes suggests that such a direct feed-forward mechanism might indeed exist. The finding that midlife flies have a higher ground state of acetylation and are less susceptible to a stimulation by KDAC inhibitors argues for similar acetylation events triggered by KDAC inhibitor treatment and aging (Peleg, 2016).

The increased level of acetyl-CoA in midlife flies correlates with a very specific change in the histone modification pattern as flies reach midlife. As it is not possible to distinguish between mitochondrial and cytosolic acetyl-CoA, the substrate for acetyltransferases, the observed correlation may not be causal. However, an increased activity of the main enzyme was also observed for the synthesis of cytosolic acetyl-CoA, ATPCL, in midlife flies, and therefore it was assumed that the cytosolic acetyl-CoA level is indeed higher when flies reach midlife. Interestingly, this increased activity is not caused by increased protein synthesis of ATPCL, but potentially by posttranslational mechanisms such as a hyper-acetylation. This is also supported by the observation that a fly strain heterozygous for an atpcl mutation shows only a 15% reduction in ATPCL activity, suggesting that there is a substantial degree of posttranscriptional regulation of this enzymatic activity. Such a regulation of ATPCL has also been proposed to stimulate lipid synthesis and tumor growth in rats. The current findings that a fly strain carrying a mutation in the ATPCL gene has an extended life span and a delayed onset of aging further confirm the importance of extra-mitochondrial acetyl-CoA for the regulation of aging. Interestingly, the reduction in ATPCL has a much stronger effect on the metabolism of midlife animals when compared to young animals. The effects observed analyzing head tissue of Drosophila melanogaster are in line with earlier reports that the targeted depletion of an unrelated acetyl-CoA synthase in fly neurons extends life span. It will be interesting to resolve the physiological effects of ATPCL mutation on the metabolome, the histone acetylation, and the transcriptome in isolated neurons (Peleg, 2016).

The ATPCL mutation results in a rather specific change in histone acetylation and does not affect all acetylation sites to the same degree. In midlife animals, the ATPCL mutation has the strongest effect on H4K12ac-an acetylation site that had been implicated in age-dependent memory impairment and transcriptional elongation and which is increased when flies reach their premortality plateau phase. This may be due to modulation of the enzymatic properties of Chameau or of a corresponding deacetylase. An increased activity in several deacetylases has been shown to extend life span in various organisms and higher concentrations of the sirtuin cofactor NAD+ have been shown to be beneficial for life span extension. However, the effect of sirtuins on life span continues to be debated and their effect has so far not been associated with a particular histone modification pattern. The quantitative analysis of specific histone modifications in this study has allowed identification of Chameau as an enzyme responsible for the increased modification in midlife flies (Feller, 2015). It is worth mentioning that the chm mutant allele is homozygous lethal and the beneficial effect on life span is more pronounced in males than in females, suggesting that Chameau has additional function, which are not yet fully understood. However, the fact that a reduction in the activity of the acetyltransferase Chameau robustly promotes longevity in male flies supports the hypothesis that this enzyme has an active role in modulating life span at least in Drosophila males (Peleg, 2016).

Previous studies demonstrated that old flies show an impaired transcriptome surveillance, as manifested in increased transcriptional noise and expression of aberrant or immature mRNAs. This study found substantial changes in the transcriptional profile as flies reach midlife, suggesting that the differential regulation of gene expression is one of the early hallmarks of the aging process. It remains to be explored how specific changes in gene expression integrate with regulatory modifications and metabolic activity. Chameau appears to promote the expression of a large number of genes particularly during the midlife period genes. Conceivably, the enhanced H4K12 acetylation leads to widespread chromatin opening, with positive effects for the transcription of specific genes. A side effect of this loosening of chromatin structure may be the increased transcriptional noise, which might compromise a variety of physiological functions. Considering the localization of H4K12ac at the gene body of highly expressed genes, it will be interesting to investigate whether the increased transcription during midlife is due to a higher rate of transcript elongation or a higher activity of cryptic promoters. It is hypothesized that the attenuation of this effect in chm mutant flies is the cause for their extended life span. A similar effect is also seen in ATPCL mutant flies, and the observation that life span is not further extended if the ATPCL and chm alleles are combined suggests that the two enzymes may act in the same pathway (Peleg, 2016).

These data provide an overview of the metabolic, proteomic, and transcriptomic changes that occur as flies reach the premortality plateau phase. Conceivably, metabolic processes are linked to changes in gene expression through differential protein acetylation, in general, and histone acetylation, in particular. Currently it cannot be unambiguously distinguish whether the shift in metabolic activity upon fly aging precedes the increases in protein/histone acetylation, or whether increases in protein/histone acetylation result in specific metabolic changes. Most likely, both principles affect each other in a complex network of feedback and feed-forward loops. Indeed, many mitochondrial enzymes that have been shown to be acetylated in response to metabolic changes either gain or lose enzymatic activity (Peleg, 2016).

Considering the high conservation of central metabolism, metabolic regulation, and epigenetics between flies and humans, these data raise the possibility that small molecule regulators of acetyl-CoA production or consumption, or changes in the activity of selective acetyltransferase functions, could prolong a healthy midlife also in humans. These model organism data reveal a potential alternative strategy that could extend midlife and delay aging-associated homeostatic decline in humans (Peleg, 2016).

The RING finger protein MSL2 in the MOF complex is an E3 ubiquitin ligase for H2B K34 and is involved in crosstalk with H3 K4 and K79 methylation

This study demonstrates that RING finger protein MSL2 in the MOF-MSL complex is a histone ubiquitin E3 ligase. MSL2, together with MSL1, has robust histone ubiquitylation activity that mainly targets nucleosomal H2B on lysine 34 (H2B K34ub), a site within a conserved basic patch on H2B tail. H2B K34ub by MSL1/2 directly regulates H3 K4 and K79 methylation through trans-tail crosstalk both in vitro and in cells. The significance of MSL1/2-mediated histone H2B ubiquitylation is underscored by the facts that MSL1/2 activity is important for transcription activation at HOXA9 and MEIS1 loci and that this activity is evolutionarily conserved in the Drosophila dosage compensation complex. Altogether, these results indicate that the MOF-MSL complex possesses two distinct chromatin-modifying activities (i.e., H4 K16 acetylation and H2B K34 ubiquitylation) through MOF and MSL2 subunits. They also shed light on how an intricate network of chromatin-modifying enzymes functions coordinately in gene activation (Wu, 2011).

This study describes a histone E3 ligase activity for the MOF-MSL complex. This activity mainly targets nucleosomal H2B K34, a site distinct from well-characterized H2B K120. Both MSL2 and MSL1, but not MOF and MSL3, are indispensable for the optimal activity. Importantly, H2B K34ub by MOF-MSL directly stimulates H3 K4 and K79 methylation through trans-tail regulation. It also affects H2B K120ub by regulating RNF20/40 chromatin association. The significance of MSL1/2-mediated H2B K34ub is underscored by data indicating that MSL1/2 activity is evolutionarily conserved in Drosophila DCC. Altogether, these results shed lights on the intricate network of chromatin-modifying enzymes that often function coordinately in gene activation (Wu, 2011).

Several early studies suggest that histone H2B can be ubiquitylated at sites other than K120 in vivo. For example, a proteomic study using a mouse brain sample identifies five ubiquitylation sites on histone H2B: K34, K46, K108, K116, and K120. Ubiquitylation of endogenous yeast H2B at multiple lysine residues in addition to K123 (K120 equivalent) has also been reported. Despite these observations, functions of these additional H2B ubiquitylation events as well as their respective Ub-conjugating enzymes and E3 ligases are largely unknown. Therefore, this study represents a step toward understanding the potential complexity of H2B ubiquitylation and their functions in transcription regulation beyond H2B K120ub. Comparing the activity of MSL1/2 to that of RNF20/40, several conclusions can be drawn: (1) MSL1/2 activity is lower than that of RNF20/40, contributing to low abundance of H2B K34ub in cells; (2) MSL1/2 has less strict substrate specificity, capable of weakly ubiquitylating H3 and H4 in vitro; and (3) MSL1/2 is mainly a monoubiquitin ligase, but it is able to add polyubiquitin chain to histones at lower efficiency. Given the distinct substrate specificity of MSL1/2 and RNF20/ 40, it is likely that similar to other histone modifications (e.g., methylation, acetylation), H2B ubiquitylation is catalyzed by a multitude of E3 ubiquitin ligases with distinct substrate preferences. Future studies on other RING finger-containing proteins will reveal yet-uncharacterized E3 enzymes and histone targets (Wu, 2011).

Like H2B K120ub, H2B K34ub plays important roles in regulating H3 K4/K79 methylation. It directly stimulates H3 K4 methylation by MLL and H3 K79 methylation by DOT1L in vitro. This result implies that trans-tail interactions between H2Bub and H3 methylation have certain plasticity in terms of H2B ubiquitylation site requirement. This is consistent with a previous report that disulfide mimics of ubH2B (uH2Bss) at K125 and K116 sites are able to stimulate DOT1L activity in vitro (Chatterjee, 2010). Given the demonstrated trans-tail regulation in this study, especially that H2B K34 resides between DNA gyres, it is important to further examine the basis for H2Bub-mediated stimulation of methyltransferase activity. It would be particularly interesting to test whether H2B K34ub stimulates H3 methylation by altering intrinsic nucleosome stability and/or nucleosome breathing mode (Böhm, 2011). In addition to a direct effect on H3 methylation, MSL1/2 also affects H3 methylation indirectly through regulating recruitment of RNF20/40 to chromatin and thus activity of RNF20/40. It was possible to differentiate these two effects by overexpression of RNF20/40 in MSL2 knockdown cells. Both immunoblot and ChIP assays support that MSL1/2 regulates H3 methylation through both direct and indirect mechanisms, further strengthening the case that MSL1/2 regulates an intricate network of histone modifications for transcription activation in cells (Wu, 2011).

Since it was not possible to detect any MOF-independent MSL1/2 heterodimer fraction on a size-exclusion column, it is likely that MSL1/2 always functions as an integral part of the MOF-MSL complex. Taking advantage of the fact that different histone modifying activities require different MOF-MSL components, it was possible to distinguish contributions of H2B K34ub and H4K16ac to transcription activation. MSL1 knockdown, which affects both H2B K34ub and H4 K16ac, has much more profound effects on HOX gene expression than MSL3 knockdown, which only affects H4 K16ac. It indicates functions of MOF-MSL in transcription regulation beyond HAT (Wu, 2011).

The finding that MOF-MSL regulates H2B K120ub and H3 K79me2, two important marks for transcription elongation, supports a role of MOF-MSL in later transcription events. Interestingly, the dMSL complex is implicated in the regulation of transcription elongation in Drosophila: (1) it is recruited to the bodies of X-linked genes, and (2) recruitment of the dMSL complex to ectopic loci with X chromosome enhancer sequences depends on active transcription, and (3) using global run-on sequencing (GRO-Seq), a recent study shows that the dMSL complex functions to facilitate progression of RNA Pol II across the bodies of active X-linked genes (Larschan, 2011). It would be interesting to test whether MOF-MSL in mammals regulates gene expression in the same manner and whether H2B K34ub (relative to H4 K16ac) contributes to the cascade of events associated with elongating Pol II. Future genome-wide analyses of H2B K34ub and MSL1/2 in mammalian cells will be very informative to further dissect the functions of different histone-modifying activities of MOF-MSL in transcription regulation (Wu, 2011).

Several remaining issues for H2B K34ub await future studies. First, how is H2B K34ub regulated in vivo? A series of elegant yeast genetic studies and in vitro biochemical studies show that H2B K120ub is extensively regulated by factors associated with Pol II transcription. They include the PAF complex, Pol II CTD, and Kin28 kinas. In addition, H2B K120ub is dynamically controlled by ubiquitin proteases such as Ubp8 and Ubp10. Given the robust activity of MSL1/2 in vitro and low H2B K34ub level in cells, it is likely that H2B K34ub is under extensive regulation. It is important to examine whether regulation of H2B K34ub and H2B K120ub is the same, or whether it involves different mechanisms despite their shared downstream effects on H3 methylation (Wu, 2011)

Second, what is the function of H2B K34ub in regulating chromatin structures? K34 resides in a highly conserved region in H2B from yeast to mammal. This region consists of a stretch of eight basic residues. Like the basic patch on the H4 N-terminal tail (14-20 aa), this H2B region is also proposed to play important roles in the formation of chromatin fibers by facilitating interactions with neighboring nucleosomes. This H2B basic patch overlaps with a conserved 'HBR' motif (30-37 aa) identified in S. cerevisiae, whose deletion leads to derepression of 8.6% of yeast genes. Given that both H2B K34 and H4 K16 are substrates of MOF-MSL and both reside within a basic patch, it is important to determine whether these two distinct histone modifications play any synergistic roles in regulating higher-order chromatin structures (Wu, 2011).

Finally, does dH2B K31ub play a role in Drosophila dosage compensation? Given the E3 ligase activity of the dMSL complex, it is important to test whether dH2B K31ub marks Drosophila male X chromosome and whether inactive dMSL2 leads to defects in expression of X-linked genes in male fly. It has been reported that dMSL1/2, but not other dMOF complex components, binds to large chromosome domains defined as high-affinity sites (HAS) or chromosome entry sites (CES) and initiates the spreading of H4 K16 acetylation mark along male X chromosome. It is important to examine whether the dMSL1/2 activity plays any role in setting up these HAS/CES sites in vivo. One common feature of HAS/CES is their relatively low nucleosome density compared to other chromatin regions. In light of the E3 activity of Drosophila dMSL1/2, it is important to test whether dMSL1/2 prefers to bind the nucleosome-free region or whether this is a result of nucleo- some destabilization caused by dH2B K31ub (Wu, 2011).

HP1a targets the Drosophila KDM4A demethylase to a subset of heterochromatic genes to regulate H3K36me3 levels

Recent discoveries of histone demethylases demonstrate that histone methylation is reversible. However, mechanisms governing the targeting and regulation of histone demethylation remain elusive. A Drosophila melanogaster JmjC domain-containing protein, dKDM4A (Histone demethylase 4A), is a histone H3K36 demethylase. dKDM4A specifically demethylates H3K36me2 and me3 both in vitro and in vivo. Affinity purification and mass spectrometry analysis revealed that Heterochromatin Protein 1a (HP1a) associates with dKDMA4A. The chromoshadow domain of HP1a and a HP1-interacting motif of dKDM4A are responsible for this interaction. HP1a stimulates the histone H3K36 demethylation activity of dKDM4A and this stimulation depends on the H3K9me binding motif of HP1a. Finally, in vivo evidence is provided suggesting that HP1a and dKDM4A interact with each other and loss of HP1a leads to increased level of histone H3K36me3. Collectively, these results suggest a function of HP1a in transcription facilitating H3K36 demethylation at transcribed and/or heterochromatin regions (Lin, 2012).

This study has identified one of the JmjC domain-containing KDM4 orthologs in Drosophila, dKDM4A. The in vitro demethylation assay shows that dKDM4A demethylates histone H3K36me3 and me2 using an oxidative demethylation mechanism which requires Fe (II) and α-ketoglutarate as cofactors. Overexpression of dKDM4A in Drosophila S2 cells reduces the level of histone H3K36me3, whereas knockdown of endogenous dKDM4A increases the level of histone H3K36me3 and me2. These results together demonstrate that dKDM4A is a bona fide histone H3K36 demethylase in vivo (Lin, 2012).

Through multidimensional protein identification technology (MudPIT) analysis of the affinity-purified native dKDM4A complex, it was found that HP1a associates with dKDM4A. More importantly, it was demonstrated that HP1a stimulates the demethylation activity of dKDM4A, while the HP1a CD mutant V26M, that cannot bind methyl-K9 histone H3, fails to stimulate dKDM4 activity. In addition, dKDM4A directly binds to the HP1 CSD and this binding requires an intact HP1 CSD dimer interface. A consensus HP1 binding PxVxL motif of dKDM4A is responsible for its interaction with CSD of HP1a. Interestingly, overexpression of dKDM4A causes the spread of HP1a to euchromatin regions, presumably through this specific interaction, and dKDM4A-V423A, which does not bind to HP1a, failed to localize HP1a to euchromatin. These data suggest HP1a-dKDM4A is a euchromatic H3K36 demethylase complex (Lin, 2012).

Set2 mediated histone H3K36 methylation is an important mark on chromatin during transcription elongation . In fungi, such as S. cerevisiae, S. pombe, and N. crassa, a sole histone lysine-methyltransferase Set2 is responsible for all three methylation states of H3K36. In Drosophila, histone H3K36 methylation is catalyzed by two enzymes, dSet2 and dMes-4. Although yeast Set2 is the only histone methyltransferase that catalyzes methylation of histone H3K36, two histone H3K36 demethylases, Jhd1 and Rph1, are responsible for demethylation of histone H3K36 at different modification states in budding yeast. In Drosophila, there are three histone demethylases that govern demethylation of histone H3K36. dKDM2 has been identified as a histone H3K36me2 demethylase (Lagarou, 2008). This study demonstrates that dKDM4A is a histone H3K36me3 and me2 demethylase, and dKDM4B has demethylation activity on both histone H3K9 and K36me3/me2 in vitro. Therefore, histone H3K36 methylation in Drosophila is likely regulated by highly specific enzymes in both directions. Since both modification and de-modification enzymes possess high modification state specificity, histone H3K36 may be subjected to much more sophisticated regulation in higher eukaryotes than in yeast (Lin, 2012).

Purification of the dKDM4A complex from S2 cells revealed a specific association of HP1a with dKDM4A. Three of the HP1-like chromatin proteins (HP1a, HP1b, HP1c) in Drosophila share high amino acid sequence similarity. Both HP1a and HP1b localize to euchromatin and heterochromatin, while HP1c is found only in euchromatin. It is unclear whether these HP1-like chromatin proteins have specific or redundant functions in transcription regulation. However, this study demonstrates that dKDM4A specifically interacts with HP1a, but not HP1b and HP1c. Furthermore, HP1b and HP1c cannot stimulate dKDM4A demethylation activity in vitro. A previous study showed that the yeast homolog of KDM4, Rph1 (ScKDM4), did not stably associate with any other protein. It was speculated that the C-terminal ZF domain of Rph1, which can potentially bind to DNA, allows Rph1 to function without associated factors. Unlike other proteins in the KDM4 family which commonly contain PHD, tudor or ZF domains, dKDM4A only has JmjN and JmjC domains. This study found that HP1a stably associates with dKDM4A and stimulates its demethylation activity. Since the H3K9 binding motif is required for this stimulation, it is proposed that the CD of HP1a might contribute to target dKDM4A to specific loci, particularly to H3K9me enriched regions, to regulate gene expression (Lin, 2012).

In S. pombe, the HP1 homolog recruits a JmjC domain-containing protein Epe1 to heterochromatin loci where they function together to counteract repressive chromatin. This study shows that HP1a directly interacts with dKDM4A through a consensus binding motif PxVxL. Most importantly, the presence of HP1a stimulates histone demethylation activity of dKDM4A in vitro, and HP1a is required for maintaining normal level of H3K36me3 in vivo as well. Since Epe1 on its own seems to have no histone demethylation activity, it would be interesting to see whether a similar scenario also occurs in S. pombe, in which Swi6 may stimulate enzymatic activity of Epe1 towards other non-histone substrates (Lin, 2012).

HP1 has been reported to associate with actively transcribed euchromatin regions. Mammalian HP1γ and histone H3K9 methylation are enriched at the coding region of active genes, implying that they may play a role during transcription elongation. In yeast, histone H3K36me3 appears to be a repressive mark at coding region of actively transcribed genes. In higher eukaryotes, histone H3K9 methylation, which is absent in the budding yeast, might replace the role of K36 methylation in the coding regions of transcribed genes. However, the mechanism by which HP1 functions in active transcription is largely unknown. The current findings suggest a possible role of HP1a in recruitment of the histone H3K36me3/me2 demethylase dKDM4A to transcribed regions to remove histone H3K36 methylation. The formation of the HP1a-dKDM4A complex may help to release HP1a from heterochromatin regions, thus targeting it to specific gene loci. It is also possible that dKDM4A, which targets histone modification marks within the 3' ORF of actively transcribed genes, recruits HP1a to euchromatic regions. A model is favored in which HP1a facilitates recruitment of dKDM4A, because the HP1a CD mutant, V26M, fails to stimulate dKDM4A activity. This result suggests that HP1a binding to histone H3 is required for the enhancement of dKDM4A demethylation activity. It is speculated that HP1a-mediated histone demethylation may serve as a regulatory mechanism to control chromatin states during active transcription elongation. Alternatively, a similar mechanism might also apply to maintaining silenced states of heterochromatin (Lin, 2012).

Histone recognition and nuclear receptor co-activator functions of Drosophila Cara Mitad, a homolog of the N-terminal portion of mammalian MLL2 and MLL3

MLL2 and MLL3 histone lysine methyltransferases are conserved components of COMPASS-like co-activator complexes. In vertebrates, the paralogous MLL2 and MLL3 contain multiple domains required for epigenetic reading and writing of the histone code involved in hormone-stimulated gene programming, including receptor-binding motifs, SET methyltransferase, HMG and PHD domains. The genes encoding MLL2 and MLL3 arose from a common ancestor. Phylogenetic analyses reveal that the ancestral gene underwent a fission event in some Brachycera dipterans, including Drosophila species, creating two independent genes corresponding to the N- and C-terminal portions. In Drosophila, the C-terminal SET domain is encoded by trithorax-related (trr), which is required for hormone-dependent gene activation. This study identified the cara mitad (cmi) gene (FlyBase name: Lost PHDs of trr), which encodes the previously undiscovered N-terminal region consisting of PHD and HMG domains and receptor-binding motifs. The cmi gene is essential and its functions are dosage sensitive. CMI associates with TRR, as well as the EcR-USP receptor, and is required for hormone-dependent transcription. Unexpectedly, although the CMI and MLL2 PHDf3 domains could bind histone H3, neither showed preference for trimethylated lysine 4. Genetic tests reveal that cmi is required for proper global trimethylation of H3K4 and that hormone-stimulated transcription requires chromatin binding by CMI, methylation of H3K4 by TRR and demethylation of H3K27 by the demethylase UTX. The evolutionary split of MLL2 into two distinct genes in Drosophila provides important insight into distinct epigenetic functions of conserved readers and writers of the histone code (Chauhan, 2012).

Nuclear receptors (NRs) function as transcription factors that respond to cellular signals to initiate new gene expression programs and have essential roles in embryonic development, growth and differentiation. NRs collaborate with greater than 300 co-factors that provide important enzymatic and regulatory functions. Co-factors can be activators or repressors and are typically recruited to gene promoters through associations with receptors. Some co-factors direct changes in the epigenetic environment of target genes by direct covalent chromatin modification or nucleosome remodeling. Co-activators are recruited in a ligand-dependent manner, whereas unliganded receptors often associate with co-repressors. Co-activators exist in large complexes required for the transcription of genes that are regulated by at least 48 vertebrate NRs, including retinoic acid receptor (RAR), liver-X-receptor (LXR), farnesoid-X-receptor (FXR), as well as a co-activator for p53. Disruptions of both NRs and their co-regulators have been linked to many cancers and developmental disorders (Chauhan, 2012).

Hormone signaling pathways in Drosophila melanogaster rely on two primary hormones, the steroid hormone 20-hydroxyecdysone (20HE) and sesquiterpenoid juvenile hormone (JH), and 18 receptors representing all major conserved nuclear receptor subfamilies. Drosophila Ecdysone Receptor (EcR) is an FXR/LXR ortholog, whereas its heterodimeric partner Ultraspiracle (USP) is an RXR ortholog (Chauhan, 2012).

Drosophila Trithorax-related (TRR) is a co-activator of EcR-USP. TRR is a histone lysine methyltransferase (HMT) that trimethylates histone 3 on lysine 4 (H3K4me3) and TRR functions are essential for activating ecdysone-regulated genes. TRR is closely related to another Drosophila protein, Trithorax (TRX), which regulates homeotic (Hox) gene expression through similar methyltransferase activity. The mammalian counterparts of TRR are MLL2 (also known as ALR or MLL4) and MLL3 (also known as HALR). MLL2 and MLL3 are enormous (5537 aa and 4911 aa, respectively), with multiple conserved domains, including histone methyltransferase (SET domain), five plant homeodomain (PHD) zinc fingers, an HMG-I binding motif, LXXLL NR binding motifs and FY-rich regions. Through the SET domain, both MLL2 and MLL3 directly methylate histone H3 to mediate transcription activation (Chauhan, 2012).

MLL2 and MLL3 are components of large SET1/COMPASS-like co-activator complexes that are required for NR-directed gene regulation. These complexes have important human disease connections, including developmental disorders and cancers. MLL2 and MLL3 are mutated in many Kabuki syndrome patients. MLL2 is frequently mutated in childhood medulloblastomas (14%), follicular lymphoma (89%) and diffuse large B-cell lymphoma (32%) (the two most common forms of non-Hodgkin lymphoma), suggesting that MLL2 and MLL3 COMPASS-like complex activities have important epigenetic gene regulatory roles that normally function to inhibit cancer progression (Chauhan, 2012).

Proteins that co-purify with the MLL2 include ASH2, RBBP5 (RBQ3), DPY30, WDR5, adaptor protein ASC2, PTIP, PA1 and histone demethylase UTX. Recently, TRR was found in Drosophila COMPASS-like complexes (Mohan, 2011). Despite functional similarities, TRR is much smaller than MLL2 or MLL3 with homology limited to the C-terminal SET domain portion. TRR lacks the N-terminal PHD and HMG domains that might contribute to chromatin binding. MLL2-related family members are always encoded by large single genes in species other than Brachycera dipterans. To further studies on epigenetic regulation of ecdysone target genes, Drosophila genes were sought that could encode a protein highly related to the N-terminal half of MLL2, and a single open reading frame (CG5591) was identified. The gene was named cara mitad (cmi; translated as 'dear half'). Although cmi is unlinked to trr in the genome, genetic studies using null mutants, in vivo depletion and overexpression revealed functions for cmi as a nuclear receptor co-factor necessary for hormone-regulated gene expression. Unexpectedly, the CMI type 3 PHD finger (PHDf3) was found to accommodate non-methylated, mono- and dimethylated H3K4, rather than trimethylated H3K4. Moreover, CMI-dependent activation also required demethylation functions of UTX, suggesting that NR-stimulated transcription involved at least three steps: binding of H3K4me1/2 by CMI, trimethylation of H3K4 by TRR and demethylation of H3K27 by UTX. The intriguing possibility that COMPASS-like functions in NR-directed transcription are associated with two independent proteins in flies suggests that recognition and binding to modified histones is a distinct step, separate from the epigenetic modification associated with other enzymes in the complex. This presents a unique opportunity to examine functions of histone recognition/binding and covalent histone tail lysine modifications as separate and essential features of NR-directed activation (Chauhan, 2012).

Although the precise roles of proteins directly participating in nuclear receptor signaling remain largely speculative, many are thought to regulate transcription through effects on chromatin. The MLL2 and MLL3 co-activators function to epigenetically decode or modify histone lysine residues and provide activation functions for NR signaling at target genes. In Drosophila, CMI and TRR together have a single MLL family homolog. This is the first example of an evolutionary 'splitting' of an epigenetic regulator involved in nuclear receptor signaling, whereby the essential gene regulatory functions of one protein have been parsed into two distinct proteins. CMI forms complexes with TRR, associates directly with hormone receptors and interacts with other putative COMPASS-like components, suggesting that Drosophila contains a functional counterpart to the mammalian ASCOM-MLL2 nuclear receptor co-activator complex (Chauhan, 2012).

The MLL histone lysine methyltransferases (KMTs) can be divided into two conserved groups, the MLL1-MLL4(2) and MLL2(4)/ALR-MLL3/HALR subfamilies. Each MLL member is capable of forming related discrete complexes with several common components. The MLL-based complexes activate transcription in part through methyltransferase activity on histone H3 Lys4 residues within promoter-associated nucleosomes. There might be partial functional overlap between MLL2 and MLL3; however, they are not redundant with the MLL1-MLL4 subfamily. The SET-domain methyltransferase activity of the MLL proteins is essential for transcription activation through histone lysine methylation, but the precise biological role of PHD fingers remains somewhat elusive. Closely related PHDf3 fingers bind H3K4me3/2, the product of the methyltransferase activity. Within the context of a single protein, such as MLL1, the PHDf3 recognition and binding of H3K4me3 is required for transcription activation of target genes (Chauhan, 2012).

The findings that CMI and TRR function coordinately in a COMPASS-like complex suggest that cmi and trr probably split from a common ancestor. Gene-protein fusions are four times more common than fissions, perhaps reflecting a simpler genetic event. In cases in which fissions occur, it has been suggested that many involve subunits of multimeric complexes in which the two independent proteins interact physically. The process of splitting into two independent genes might involve gene duplication with subsequent partial degeneration, as has been observed in the monkey king (mkg) gene family in Drosophila (Chauhan, 2012).

The notion that a large protein contains domains that function both together and independently is not without precedent. TRX and MLL1 are cleaved by a specific protease, taspase-1. The two 'halves' interact with each other in a functional complex, but there is evidence that the N-terminal TRX peptide (TRX-N) binds chromatin without its TRX-C partner in transcribed regions of Hox genes. Transcription factor TFIIA and herpes simplex virus host cell factor (HCF1) are cleaved during maturation, with both halves necessary for a functional product. There is presently no evidence that MLL2 or MLL3 are cleaved or processed (Chauhan, 2012).

An important question is whether both the chromatin-binding and methyltransferase functions of the MLL family are required for transcription activation. The data indicate that depletion of trr can suppress the effects of overexpressing cmi, suggesting that the activation potential of CMI depends on TRR methyltransferase activity. Similarly, simultaneous depletion of cmi and trr produces stronger phenotypes than depletion of either alone, indicative of cooperation on similar gene targets. Moreover, in vivo depletion of cmi results in reduced global H3 trimethylation, despite a functional trr gene (Chauhan, 2012).

Phenotypes associated with changes in CMI levels reveal important functions in hormone-regulated development. The larval defects in molting, morphogenetic furrow progression and necrosis associated with a cmi null allele, similar to trr, are consistent with impaired hormone signaling. Similarly, depletion of MLL2 in HeLa cells using siRNA led to reduced expression of genes known to be important for development and trimethylation of H3K4 was reduced at some promoters. Knockdown of MLL2 in MCF-7 cells impaired estrogen receptor (ERα) transcription activity and inhibited estrogen-dependent growth. Inactivation of the murine Mll3 resulted in stunted growth and reduced PPARgamma-dependent adipogenesis with increased insulin sensitivity. Perhaps reflecting synonymous functions in Drosophila, cmi/CG5591 was found to be important for regulating muscle triglyceride levels, suggesting conserved adipogenic functions. Furthermore, CG5591 (cmi) is involved in phagocytosis and regulation of caspase functions in response to cellular stress, implicating cmi in immune-cell regulation. The increased hemocyte number associated with elevated CMI suggests functions in hemocyte development, perhaps as an effector of chromatin remodeling or signaling (JAK/STAT, Hedgehog, Notch) pathways. It was previously shown that trr was important for Hedgehog (HH)-dependent signaling during eye development and cmi overexpression and depletion data are consistent with that possibility. However, the dosage-dependent cmi wing phenotypes are not consistent with changes in HH signaling, raising the possibility that cmi and trr are important for other growth and signaling pathways in wing development, including Decapentaplegic (DPP/TGFβ) and Wingless (WG/WNT) pathways (Chauhan, 2012).

Several steps are involved in activation of hormone-responsive target genes, including methylation of H3K4 by the MLL2-MLL3 COMPASS-like complex and displacement of demethylases. Reduced cmi function resulted in lower hormone-responsive enhancer activation and genetic interactions between cmi, trr and Utx revealed that chromatin binding by CMI was important for gene activation in vivo. Furthermore, RNAi depletion of Utx suppressed HA-cmi overexpression wing phenotypes, suggesting that demethylation of H3K27 is a pre-requisite for activation of some hormone target genes. This is supported by genetic evidence from C. elegans that indicated both histone H3K4 methylation by SET-16 (MLL2/MLL3 ortholog) and H3K27 demethylation by UTX-1 were required for attenuation of RAS signaling in the vulva and MLL2-MLL3 complex-related components were required for proper germ line development. Genetic epistasis data reveals that Utx, trr and cmi functions are all required for activation in Drosophila (Chauhan, 2012).

Unexpectedly, the CMI PHDf3.b showed binding to mono- and dimethylated H3K4, rather than trimethylated H3K4. Although CMI contains two PHDf3 domains in two clusters similar to MLL3, MLL2 contains one PHDf3 most closely related to the CMI and MLL3 PHDf3.b domains. The second cluster appears in all isoforms of MLL3, whereas the N-terminal 'a' cluster is optional. Additionally, the 'b' cluster is more closely related to the PHD cluster found in other MLL family proteins. PHD modules are thought to bind histones and present tail residues to the modifying enzyme subunits or stabilize those enzymes with their substrates. Recently, RNAi knockdown of trr in S2 cells was shown to affect H3K4 mono-, di-, and trimethylation, revealing widespread functions in regulating methylation in vivo and suggesting that loss of TRR might destabilize the co-activator complex leading to de-protection of H3K4 methylation. One possibility is that CMI binds mono- and dimethylated H3K4 to prevent demethylation and stabilize TRR to allow for hormone-stimulated methylation and gene activation. CMI might disengage to allow for removal of methylation marks as hormone levels decrease and gene transcription is reduced. In contrast to MLL1-TRX function in maintenance of active gene transcription, CMI and TRR might be required for NR-targeted gene activation in response to temporally restricted hormone-dependent genome reprogramming (Chauhan, 2012).

Developmental regulation of N-terminal H2B methylation in Drosophila melanogaster

Histone post-translational modifications play an important role in regulating chromatin structure and gene expression in vivo. Extensive studies investigated the post-translational modifications of the core histones H3 and H4 or the linker histone H1. Much less is known on the regulation of H2A and H2B modifications. This study shows that a major modification of H2B in Drosophila is the methylation of the N-terminal proline, which increases during fly development. Experiments performed in cultured cells revealed higher levels of H2B methylation when cells are dense, regardless of their cell cycle distribution. dNTMT was identified as the enzyme responsible for H2B methylation. It was also found that the level of N-terminal methylation is regulated by dART8, an arginine methyltransferase that physically interacts with dNTMT and asymmetrically methylates Histone Histone H3 arginine 2 (H3R2). These results demonstrate the existence of a complex containing two methyltransferases enzymes that negatively influence each other's activity (Villar-Garea, 2012).

In the eukaryotic nucleus DNA is packaged into chromatin by its association with the basic core histones. The folding of chromatin fibers has a major impact on many aspects of nuclear function and is regulated by the post-translational modification of the histone tail domains. In multicellular organisms malfunction of the enzymatic machinery that establishes these modifications leads to abnormalities such as failures in embryonic development, cancer and other diseases. Human H2B is phosphorylated at S14 by the caspase cleaved mammalian Mst-1 kinase. H2A is phosphorylated at S1 where an ordered appearance and removal of distinct modifications is required for proper chromatin assembly. The modifications on the histone N-terminal tails are recognized by specialized proteins that selectively bind modified histones. The specific binding to particular modifications can then either lead to structural changes of chromatin or recruit enzymatic activities to specific loci, which in turn can either stimulate or inhibit a subsequent modification. Examples of this phenomenon are the stimulation of the acetylation of H3K14 by a phosphorylation of H3S10 and methylated at the N-terminal proline. Although the methylation of the terminal α-amino group has been found in H2B of a variety of organisms and a number of proteins other than histones. The two modifications that influence each other do not have to reside on the same molecule as it has been demonstrated that the ubiquitination of H2B by Rad6 facilitates the methylation of H3K4, suggesting a crosstalk of the two histone tails. Another example for such a crosstalk is the phosphorylation of H3S10 by the Pim1 kinase, which stimulates the acetylation (Villar-Garea, 2012).

Most of the global histone modification analyses done so far were performed on the two core histones H3 and H4 whereas the post-translational modifications of canonical H2A and H2B have been less well studied in metazoa. Only the ubiquitination of H2A and H2B has been suggested to have a specific function such as the silencing of genes or the stimulation of H3K4 and H3K79 methylation, respectively. Human H2B is phosphorylated at S14 by the caspase cleaved mammalian Mst-1 kinase. This phosphorylation has been proposed to mediate chromatin condensation during apoptosis, which is counteracted by the acetylation of the adjacent K15. H2A is phosphorylated at S1 but so far this has not been shown to be regulated. The analysis of H2A and H2B methylation and acetylation in higher eukaryotes by mass spectrometry (MS) has been severely hampered by the multitude of different isoforms with similar molecular masses making it difficult to distinguish post-translational modifications from sequence variants. Drosophila melanogaster, in contrast to most higher organisms, has just a single H2B variant, which greatly facilitates the analysis of H2B PTMs (Villar-Garea, 2012).

Drosophila H2B is ubiquitinated at K120, phosphorylated at S33 and methylated at the N-terminal proline. Although the methylation of the terminal α-amino group has been found in H2B of a variety of organisms and a number of proteins other than histones, its biological significance is largely unknown. Drosophila tissue culture cells show an increased proline methylation in response to heat shock and arsenite treatment. Also, the N-terminal methylation of mammalian Regulator of Chromosome Condensation 1 (RCC1) has been shown to be crucial for its binding to chromatin and for proper mitotic chromosomal segregation. A pair of ortholog enzymes that perform N-terminal methylation of proteins in humans and yeast, NRMT (METTL11A) and YBR261C/Tae1, respectively, has been also isolated (WVillar-Garea, 2012).

This study shows that N-terminal methylation of H2B in D. melanogaster is not only regulated by cellular stress such as heat shock but also changes during development. In tissue culture cells the proportion of methylated histone depends on cell density, but not on the cell cycle distribution. Knockdown experiments and in vitro assays demonstrate that Drosophila's ortholog of NRMT, dNTMT, mono- and di-methylates the N-terminus of H2B. Interestingly, dNTMT forms a complex with an arginine-specific H3 methyltransferase dART8, which targets H3R2. Although the interaction has no effects on the activity of the enzymes in vitro, modulation of dART8 levels in cells has a substantial effect on H2B N-terminal methylation in vivo. A knockdown of dART8 results in an increase of H2B methylation whereas over-expression leads to a reduction, suggesting a repressive effect of dART8 levels on the modification of the H2B N-terminus (Villar-Garea, 2012).

While studying the post-translational modifications of H2B in D. melanogaster this study found the methylation of its N-terminus as the major modification of this histone. In contrast to many other known N-terminal modifications, the methylation is highly regulated. The enzyme responsible for establishing this modification (dNTMT/CG1675) was identified and it was also confirmed that it physically interacts with another histone methyltransferase (dART8) specific for H3R2, thereby constituting a bifunctional methyltransferase complex. The two enzymes not only interact with each other physically but also have opposing functions as the reduction of dART8 protein levels results in an increase of N-terminal methylation and the overexpression in a strong decrease. This effect of the modulation of dART8 concentration on H2B methylation may be due to a negative crosstalk between the methylation of H3R2 and the N-terminal methylation of H2B or due to a competition of the two enzymes for the common cofactor SAM (Villar-Garea, 2012).

N-terminal methylation is a rare modification in eukaryotic proteins and has only recently been addressed functionally. The methylation of the N-terminus of human RCC1 has been shown to be important for its stable interaction with chromatin and its disturbance leads to mitotic defects in vivo. This regulation is in accordance with the structure of the nucleosome bound RCC1 molecule, where a N-terminal loop has been suggested to interact with the nucleosomal DNA. Interestingly, despite the strong conservation of RCC1 function in metazoans, the N-terminal methylation site of RCC1 is not conserved in the Drosophila ortholog of RCC1, suggesting that the stabilization of RCC1 on chromatin is either accomplished by a different mechanism or not required in Drosophila (Villar-Garea, 2012).

Eight proteins out of 36 that carry a recognition site for a presumptive N-terminal methyltransferases are conserved between humans and fruit flies. Those are three ribosomal subunits, two proteins associated with stress response and/or growth arrest, two with an unknown function and one member of the respiratory chain. Considering the conservation of the enzyme responsible for this modification, the low degree of overlap is very surprising. However, it is striking that frequently the human orthologous of proteins that carry a recognition site only in Drosophila can be found associated with putative targets in humans. So is for example RCC1 (methylated in humans but not in Drosophila) in a complex with H2B (methylated in Drosophila but not in humans) and many factors that are predicted to become N-terminally methylated are expressed in a testis specific manner in humans as well as in Drosophila. This suggests that the N-terminal methylation exerts its function on a protein complex as long as one subunit carries the modification (Villar-Garea, 2012).

What may be the function of H2B methylation in Drosophila? The observation that it increases during temperature stress as well as during differentiation points towards a role of H2B methylation in stabilizing chromatin. In both circumstances (heat shock and aging) the overall transcription is reduced and becomes restricted to a limited number of active genes. At the same time, only low levels of methylation are detected at early stages of embryonic development, where chromatin has been reported to be hyper-dynamic in other systems. In general, histones have been shown to have a higher turnover in dynamically transcribed regions compared to non-transcribed domains therefore lowering this turnover may have a repressive effect on general transcriptional activity. As N-terminal modifications have been shown to regulate protein turnover, H2B methylation might similarly stabilize the protein and contribute to a reduced overall transcription in differentiated or stressed cells. Alternatively, H2B methylation could also be a consequence of low transcriptional activity, which is the supported by the observation that it also increases when cells are treated with transcriptional inhibitors or with inhibitors of TopoII (Villar-Garea, 2012).

N-terminal modification of histones is not restricted to H2B in flies but has also been detected in H4, which is N-acetylated in virtually all eukaryotes, H2A, which is N-acetylated in human tissue culture cells and H2B from yeast, which is also acetylated at its N-terminal residue. None of the N-acetylations have been associated with a particular function and are thought to be constitutive modifications following histone synthesis. This description of a developmentally and stress-induced regulated N-terminal modification of H2B sheds new light on the potential function of this modification in chromatin metabolism (Villar-Garea, 2012).

The relative expression levels of dNTMT during different developmental stages correlate very well with the relative levels of H2B methylation. This suggests that the proportion of H2B methylation is regulated by the amount of enzyme present in the cell. However, in SL2 cells, it was also observed that the level of dART8 regulates H2B methylation, which suggests a second layer of control for H2B methylation. Although the two proteins interact physically, they do not methylate each other and the interaction does not lead to an alteration of dNTMTs activity. The observed interference of dART8 expression with H2B methylation can therefore not be explained by a direct effect mediated by the simple interaction of the two polypeptides. Expression studies show that the dNTMT expression is not downregulated by dART8 expression or upregulated in cells that lack dART8 (Villar-Garea, 2012).

Recently local SAM concentrations within the nucleus have been suggested to play an important role in regulating the activity of histone methyltransferases. The regulation of H2B methylation this study has observed in vivo by modulating dART8 levels may therefore be due to a competition of the two enzymes residing in the same complex for the limiting cofactor SAM. Several nuclear complexes have been shown to contain multiple methyltransferases activities that could potential be regulated by a similar mechanism. Alternatively, as dART8 methylates R2 at H3, a crosstalk between the two histone-tails where methylation of H3R2 inhibits H2B N-terminal methylation could also be an explanation for the reciprocal activities of the two enzymes. Based on these data, future studies are necessary that distinguish the possible mechanisms of how dART8 modulates dNTMTs activity within a single protein complex and analyze the role of the striking increase in H2B methylation during fly development (Villar-Garea, 2012).

Evolutionary adaptation of the fly Pygo PHD finger toward recognizing histone H3 tail methylated at Arginine 2

Pygo proteins promote Armadillo- and beta-catenin-dependent transcription, by relieving Groucho-dependent repression of Wnt targets. Their PHD fingers bind histone H3 tail methylated at lysine 4, and to the HD1 domain of their Legless/BCL9 cofactors, linking Pygo to Armadillo/beta-catenin. Intriguingly, fly Pygo orthologs exhibit a tryptophan > phenylalanine substitution in their histone pocket-divider which reduces their affinity for histones. This study used X-ray crystallography and NMR to discover a conspicuous groove bordering this phenylalanine in the Drosophila PHD-HD1 complex-a semi-aromatic cage recognizing asymmetrically methylated arginine 2 (R2me2a), a chromatin mark of silenced genes. A structural model of the ternary complex reveals a distinct mode of dimethylarginine recognition, involving a polar interaction between R2me2a and its groove, the structural integrity of which is crucial for normal tissue patterning. Notably, humanized fly Pygo derepresses Notch targets, implying an inherent Notch-related function of classical Pygo orthologs, disabled in fly Pygo, which thus appears dedicated to Wnt signaling (Miller, 2013).

Drosophila CTCF tandemly aligns with other insulator proteins at the borders of H3K27me3 domains

Several multiprotein DNA complexes capable of insulator activity have been identified in Drosophila melanogaster, yet only CTCF, a highly conserved zinc finger protein, and the transcription factor TFIIIC have been shown to function in mammals. CTCF is involved in diverse nuclear activities, and recent studies suggest that the proteins with which it associates and the DNA sequences that it targets may underlie these various roles. This study shows that the Drosophila homolog of CTCF (dCTCF) aligns in the genome with other Drosophila insulator proteins such as Suppressor of Hairy wing (SU(HW)) and Boundary Element Associated Factor of 32 kDa (BEAF-32) at the borders of H3K27me3 domains, which are also enriched for associated insulator proteins and additional cofactors. RNAi depletion of dCTCF and combinatorial knockdown of gene expression for other Drosophila insulator proteins leads to a reduction in H3K27me3 levels within repressed domains, suggesting that insulators are important for the maintenance of appropriate repressive chromatin structure in Polycomb (Pc) domains. These results shed new insights into the roles of insulators in chromatin domain organization and support recent models suggesting that insulators underlie interactions important for Pc-mediated repression. This study reveals important relationship between dCTCF and other Drosophila insulator proteins and speculates that vertebrate CTCF may also align with other nuclear proteins to accomplish similar functions (Van Bortle, 2012).

Improvements in genomic strategies for mapping genome-wide interactions have allowed recent studies to probe basic genome folding principles as well as insulator-mediated chromatin interactions. Results consistently support current models proposing roles for insulator proteins in chromosome organization and challenge the basic barrier and enhancer-blocking activities that classically defined these proteins. Instead, the ability of insulators to block the spread of heterochromatin and impede enhancer-promoter interactions may simply be consequences of a more paramount role in chromosome organization. New findings in Drosophila also suggest that insulators are required to mediate long-range interactions important for Polycomb (Pc) repression, and the recent identification of CTCF in transcription factories suggests that insulators may direct the localization of specific genomic loci to discrete nuclear subcompartments for gene regulation. Nevertheless, the finding that heterochromatin does not spread into flanking chromatin domains in response to insulator knockdown is surprising based on numerous examples of insulator mediated barrier function. Though individual insulator elements may indeed serve to prevent the spread of silencing chromatin, disruption of total insulator protein levels instead significantly affected the levels of H3K27me3 within rather than outside of repressive chromatin domains. Insulator knockdown had no effect on the expression of E(z) or total H3K27me3 levels. Therefore, the loss of H3K27me3 within Pc domains genome-wide suggests insulators play a critical role necessary for the maintenance of appropriate chromatin architecture at these specific loci. Given the requirement for insulators in long-range Pc interactions, it is speculated that long-range interactions mediated by dCTCF and other Drosophila insulator proteins are ultimately disrupted by insulator knockdown, and that H3K27me3 depletion likely reflects a defect in Pc mediated compaction and maintenance of H3K27me3 at developmental loci (Van Bortle, 2012).

Interestingly, however, expression of genes within repressive H3K27me3 domains was not significantly affected, suggesting Pc mediated gene silencing was not abrogated, or that additional steps are required to activate these developmental genes. Future studies investigating the role of insulators in Pc-mediated repression, and the effects of insulator knockdown in nuclear organization, will provide valuable insight into the relationship between insulator proteins and chromatin architecture (Van Bortle, 2012).

The diverse activities of CTCF in gene expression and chromatin organization require exploration of the proteins with which it functions and the target sequences associated with specific functions. By combining the resolution conferred by high throughput sequencing (ChIPseq), with mapping of core target sequences, this study provides a stringent but exhaustive map of direct binding sites for Drosophila insulators, and extends previous analyses of dCTCF, SU(HW), BEAF-32, and CP190 to include the insulator protein MOD(MDG4). It was shown that dCTCF aligns with both the SU(HW) and BEAF-32 insulators, where dCTCF becomes enriched for additional insulator and insulator-associated proteins. The presence of aligned dCTCF sites at the borders of H3K27me3 domains provides an excellent system to query the importance of insulator proteins at the boundaries of discrete chromatin domains. Recently identified correlations for insulator proteins at the boundaries of physical domains mapped in Drosophila melanogaster (Sexton, 2012) provide evidence for why only a subset of aligned dCTCF localize to H3K27me3 domain borders, and clearly demonstrate that insulators are also involved in the organization of other, distinct chromatin domains. Whereas Pc-repressed domains are relatively easily identifiable in the form of H3K27me3 signatures, future characterization of discrete physical domains and domain boundaries will require genome-wide interrogation of chromosome interactions in individual cell-types of interest. Nearly 40% of aligned dCTCF sites (~355) localize to physical domain boundaries mapped in late embryos by Sexton (2012), suggesting physical domains and insulator localization may be conserved at many loci across cell-types (Van Bortle, 2012).

Interestingly, dCTCF appears to target three different sequences in D. melanogaster, including the highly conserved core motif for which dCTCF has been described as binding in both Drosophila and mammals. The secondary motif appears highly similar to the conserved core consensus (AGGNGGC) with an insertion between the first pair of guanines (AGTGTGGC), and average dCTCF levels suggest this represents a low occupancy and potentially lower affinity binding site. These novel dCTCF sites are highly enriched for insulator protein CP190 when compared to its primary target sequence. This finding, combined with previous data indicating CP190 is essential for dCTCF binding to a subset of its target sites, suggests that CP190 might facilitate dCTCF binding to these secondary sites. The absence of CP190 in vertebrates may explain why these sequences have not been identified as mammalian target sequences, raising the possibility that these binding sites are a Drosophila specific phenomenon (Van Bortle, 2012).

Analysis of dCTCF insulator alignment at the eve locus and genome-wide uncovers a tight association with BEAF-32 and SU(HW), which may provide dCTCF with numerous advantages for effectively establishing a functional insulator. First, alignment of multiple insulator DNA elements may increase the likelihood of sequence accessibility at important loci, as insulator binding sites have been characterized by reduced nucleosome density. For example, an insulator-binding protein may access its cognate sequence, thereby creating an accessible landscape for other, potentially different insulator proteins to bind their respective targets. Second, by aligning in close proximity, recruitment of essential insulator proteins [i.e., CP190 and MOD(MDG4)] by one insulator-binding protein may facilitate recruitment by others, given that CP190 and MOD(MDG4) may be recruited as multimers. Third, given that dCTCF binds secondary sites that potentially require CP190, recruitment of CP190 by a neighboring insulator (i.e., SU(HW) or BEAF-32) may preclude dCTCF binding, thereby providing a regulatory step in dCTCF recruitment to DNA. Finally, by aligning with SU(HW) and BEAF-32, dCTCF establishes a unique identity compared to independent dCTCF sites, where it becomes enriched for additional cofactors, including L(3)MBT and Chromator (Van Bortle, 2012).

Though the data shed new and valuable insight into what appears to be cooperative insulator function in Drosophila melanogaster, many questions remain. Given current models that insulators function via intra- and inter-chromosomal interactions, it is plausible that aligned dCTCF sites and their enrichment for CP190 and MOD(MDG4) allow for stable chromosomal interactions. Current locus- and genome-wide interaction assays may effectively answer this question in the near future. While BEAF-32 has been defined as lineage specific, and SU(HW) appears to lack a counterpart in mammals, the current results suggest that mammalian CTCF may align with other, unique DNA-binding proteins important for appropriate insulator function at the boundaries of Pc domains (Van Bortle, 2012).

In vivo Polycomb kinetics and mitotic chromatin binding distinguish stem cells from differentiated cells: Phosphorylation of histone H3 at Ser 28 was identified as a potential mechanism governing the extent and rate of mitotic PC dissociation in different lineages

Epigenetic memory mediated by Polycomb group (PcG) proteins must be maintained during cell division, but must also be flexible to allow cell fate transitions. This study quantified dynamic chromatin-binding properties of PH::GFP and PC::GFP in living Drosophila in two cell types that undergo defined differentiation and mitosis events. Quantitative fluorescence recovery after photobleaching (FRAP) analysis demonstrates that PcG binding has a higher plasticity in stem cells than in more determined cells and identifies a fraction of PcG proteins that binds mitotic chromatin with up to 300-fold longer residence times than in interphase. Mathematical modeling examines which parameters best distinguish stem cells from differentiated cells. Phosphorylation of histone H3 at Ser 28 was identified as a potential mechanism governing the extent and rate of mitotic PC dissociation in different lineages. It is proposed that regulation of the kinetic properties of PcG-chromatin binding is an essential factor in the choice between stability and flexibility in the establishment of cell identities (Fonseca, 2012).

This study used a combination of quantitative live imaging and mathematical modeling to investigate changes in the dynamic behavior of PcG proteins upon mitosis and cell fate transitions in living Drosophila, giving quantitative insight into the properties of the PcG system. For the PH::GFP fusion protein, the use of limited tissue-specific expression strategies was necessary to avoid cell death associated with PH overexpression. This, in turn, precluded the quantification of endogenous PH molecule numbers, since protocols for the isolation of GFP-marked SOPs and neuroblasts are not currently available. A goal of future studies will be to isolate the PH::GFP-expressing cell types of interest in order to enable relative quantification of PH::GFP and endogenous PH. For the PC::GFP fusion protein, the transgene was expressed under the endogenous Pc promoter, enabling quantification of relative amounts of transgenic and endogenous protein from whole tissues. It is important to consider to what extent the partial rescue of Pc mutants by the PC::GFP transgene will affect the quantitative conclusions draw in this study. By quantitative comparison with PH::GFP behavior, it has been proposed that the PC::GFP fusion is less favored by fourfold to fivefold in the PRC1 complex than the endogenous protein. Previous studies have concluded that the population of PRC1 is marked with PC::GFP, but the bound fraction of PC::GFP may be an underestimation of the bound fraction of endogenous PC. This effect may lead to the lower bound fraction that was measure for PC::GFP in comparison with PH::GFP. It also follows from this that second-order kinetic processes (on rates) will be prone to inaccuracies, but first-order processes (off rates and therefore residence times) will be unaffected. It is noted that the accurate determination of the true on rate (kon) from the pseudo-first-order association rate (k*on), extracted from FRAP experiments such as these, is also limited by the unknown quantity of free binding sites; thus, at best, one can extract relative kon values that allow comparisons between different cell types. This in itself allows meaningful comparisons. In summary, it is concluded that the PC::GFP fusion protein is a useful reporter of specific aspects of endogenous protein behavior: It enables the accurate determination of residence times, absolute protein quantities (which do not rely on protein activity), and relative differences between on rates in different cell types and at different cell cycle stages (Fonseca, 2012).

Comparison of PC::GFP and PH::GFP revealed twofold to ninefold longer residence times for PH::GFP than PC::GFP at interphase in all cell types. This result suggests that PC and PH do not solely operate as part of the PRC1 complex. The longer residence times observed for PH::GFP may reflect multimerization of PH via the SAM domain, which has been shown to be required for PH-mediated gene silencing (Robinson, 2012). The cell type-specific differences that were observe in the kinetic behavior of PH::GFP raise the intriguing possibility that some of these may be due to regulation of sterile a motif (SAM) domain polymerization and thus PH silencing properties (Fonseca, 2012).

The estimation of the number of endogenous PC molecules bound to chromatin in interphase (~2500-7500 depending on cell type) allows comparison with numbers of PcG target genes estimated from profiling studies (between 400 and 2000). It is noted that the interphase residence times for both proteins measured in this study (0.5-10 sec) are shorter than those previously reported for the same fusion proteins in other tissues (2-6 min). These differences may arise from the different cell types examined or from the different FRAP analysis models used. Indeed, the residence times measured in this study are consistent with those measured for several transcription factors using similar FRAP models. These findings suggest that in interphase, several PC molecules are bound to a given target gene and exchange within a matter of seconds on a time scale similar to transcription factor-binding events. The fact that shorter residence times were measured in neuroblasts than in SOPs suggests that the mode of PcG binding, and thus the extent of silencing, may be differently regulated in stem cells and differentiated cells (Fonseca, 2012).

The analysis of different cell lineages and of interphase-to- mitotic transitions led to two key findings. First, a progressive reduction was documented in mobility of both PC::GFP and PH::GFP upon lineage commitment both between cell types and within a single lineage, consistent with and extending previous studies showing reduced mobility of these proteins at later developmental stages (Ficz, 2005) and a general loss of chromatin plasticity upon embryonic stem (ES) cell differentiation. Interestingly, a recent study of TFIIH binding in developing mammalian tissues, performed in living mice, revealed a differentiation-driven reduction in TFIIH mobility, revealing long-lasting but reversible immobilization in post-mitotic cells. It will be of great interest in the future to examine PC, PH, and other PcG and TrxG (Trithorax group) proteins in other cell lineages to determine the extent to which residence times are modulatable upon changes in cell identity. In particular, it will be interesting to examine the kinetics of theDNA-binding proteins that recruit the PcG and TrxG proteins to their sites of action (Fonseca, 2012).

Second, a fraction of PcG molecules was identified that remain strongly bound to mitotic chromatin in both neuroblasts and SOPs. The long residence times (up to several minutes) of this bound fraction raise the important question of whether these molecules are carriers of mitotic memory. Thus, how the mitotic chromatin-binding properties of the PcG are differently regulated in SOPs and neuroblasts will be a key question for future studies. Does a strongly bound subpopulation exist in interphase? In the mathematical model for PC dissociation, all PC molecules are treated as belonging to a single population whose properties change upon entry into mitosis. It is noted that a model in which a subpopulation with long residence time exists during interphase would also be compatible with the observed data, but such a subpopulation was not discernible from the FRAP recovery data (Fonseca, 2012).

The determination of molecule numbers, concentrations, and kinetic constants gives insight into the absolute quantities and mobilities of free and bound PC molecules in specific cell types in the endogenous situation, thus providing in vivo quantitation of an epigenetic system. These in vivo measurements will be essential for interpretation of models based on in vitro findings. Furthermore, this analysis enabled use of quantitative mathematical modeling to examine the predicted behavior of the system over time during an entire cell cycle. The most important insight provided by the model is the requirement for accelerated PC displacement in SOPs and the prediction that this may be provided by a reduction in association rate during prophase. It was demonstrated that H3S28 phosphorylation is a good candidate mechanism for PC displacement during prophase and metaphase, in addition to its documented role in PcG displacement during interphase (Gehani, 2010; Lau, 2011). The increased residence time that was observed for PC::GFP upon RNAi-mediated knockdown of JIL-1 is consistent with a role of H3S28P in ejecting PC from H3K27me3 sites on chromatin. The observation of accumulation of this double mark in prophase and metaphase is consistent with observations of mitotic accumulation of H3K9me3/S10p but is in contrast to a study that report only slight changes in levels of H3K27me3/S28p from interphase to metaphase in human fibroblasts. This discrepancy strongly suggests that the extent of mitotic S28 phosphorylation on K27-methylated H3 tails is cell type-specific, consistent with a potential role for this mark in distinguishing the mitotic behavior of PC in SOPs and neuroblasts (Fonseca, 2012).

Since H3K27me3/S28p is associated with ejection of PC from chromatin, and the double mark is highly enriched on mitotic chromatin, additional mechanisms must contribute to the increased residence times of the small bound fraction of PC::GFP that was observed in mitosis. These may include post-translational modifications of PC and PH proteins themselves, a switch of binding platform (e.g., from histone tails to DNA or RNA), and modification of recruiting or competing molecules. Whether these proposed mechanisms contribute to mitotic PcG displacement and retention and whether they are regulated differently in different lineages will be key questions for future studies (Fonseca, 2012).

In summary, this study demonstrates that the properties of the PcG proteins are not only different in different lineages, but also profoundly altered at mitosis. It is proposed that this regulation of PcG properties may be essential to both the stability of determined cell identities and the flexibility of the stem cell state. The combination of absolute quantification with analysis in living animals that was used in this study offers three key advances to the study of epigenetic regulation: First, single, defined, genetically marked cell lineages were examined as they go through mitosis and differentiation or self-renewal. Only in a living animal can we observe a defined mitotic event and its differentiated or self-renewed daughter cells. Second, only by quantifying absolute numbers of chromatin-bound endogenous molecules in real volumes can the biological meaning of observed differences in terms of cellular concentrations and protein abundance be understood. Third, these quantitative measurements enable not only the comparison of dynamic transitions in different cell types, but also meaningful mathematical models, identifying which parameters of the system can best explain the observed changes in the plasticity of PcG-chromatin binding upon mitosis and differentiation in stem cells and in more determined lineages. In summary, the combined use of live imaging and mathematical modeling in genetically tractable, dynamically changing in vivo experiments provides quantitative insight into how a system whose components are in constant flux can ensure both stability and flexibility (Fonseca, 2012).

Progressive polycomb assembly on H3K27me3 compartments generates polycomb bodies with developmentally regulated motion

Polycomb group (PcG) proteins are conserved chromatin factors that maintain silencing of key developmental genes outside of their expression domains. Recent genome-wide analyses showed a Polycomb (PC) distribution with binding to discrete PcG response elements (PREs). Within the cell nucleus, PcG proteins localize in structures called PC bodies that contain PcG-silenced genes, and it has been recently shown that PREs form local and long-range spatial networks. The nuclear distribution of two PcG proteins, PC and Polyhomeotic (PH) was examined in this study. Thanks to a combination of immunostaining, immuno-FISH, and live imaging of GFP fusion proteins, it was possible to analyze the formation and the mobility of PC bodies during fly embryogenesis as well as compare their behavior to that of the condensed fraction of euchromatin. Immuno-FISH experiments show that PC bodies mainly correspond to 3D structural counterparts of the linear genomic domains identified in genome-wide studies. During early embryogenesis, PC and PH progressively accumulate within PC bodies, which form nuclear structures localized on distinct euchromatin domains containing histone H3 tri-methylated on K27. Time-lapse analysis indicates that two types of motion influence the displacement of PC bodies and chromatin domains containing H2Av-GFP. First, chromatin domains and PC bodies coordinately undergo long-range motions that may correspond to the movement of whole chromosome territories. Second, each PC body and chromatin domain has its own fast and highly constrained motion. In this motion regime, PC bodies move within volumes slightly larger than those of condensed chromatin domains. Moreover, both types of domains move within volumes much smaller than chromosome territories, strongly restricting their possibility of interaction with other nuclear structures. The fast motion of PC bodies and chromatin domains observed during early embryogenesis strongly decreases in late developmental stages, indicating a possible contribution of chromatin dynamics in the maintenance of stable gene silencing (Cheutin, 2012).

This study showed that PC bodies co-localize with H3K27me3 and form small nuclear domains of heterogeneous intensity. Surprisingly, PC bodies are found in DAPI poor regions, often adjacent to DAPI and histone-dense euchromatic regions. This result thus indicates that PC bodies are not among the most condensed chromatin portions of the euchromatic part of the genome. This localization of PC bodies is consistent with a previous study with electron microscopy, which has shown that PC is concentrated in the perichromatin compartment of the mammalian nucleus. In contrast, these data are in apparent contrast with a series of papers reporting PcG protein-dependent chromatin condensation. PcG complexes have been shown to compact chromatin in vitro and reduce DNA accessibility in vivo. Moreover, recent works show that PcG proteins are required to maintain compaction of Hox loci in mammalian embryonic stem cells and of the mouse Kcnq1 imprinted cluster. In those studies, condensation has been addressed by measuring either the compaction of nucleosomal fibers in electron microscopy, or the distance between close genomic loci by FISH. It is difficult to relate in vitro data to the current in vivo analysis. In particular, FISH analyses do not directly distinguish between a truly dense 3D organization and other types of conformations, such as a multi-looped architecture that would not necessarily induce an increase in chromatin density. Therefore, PcG target chromatin is probably organized in higher-order 3D structures that involve nucleosome-nucleosome and protein-protein interactions, but the net density of DNA (as seen by DAPI) or histones (as seen by tagged-histone microscopy) is not particularly high in these structures (Cheutin, 2012).

Earlier studies indicated that PcG proteins rapidly exchange between the nucleoplasm and PC bodies, suggesting that PC bodies consist of a local transient accumulation of PcG proteins in the cell nucleus. Earlier studies have detected the same number of PC bodies inside the nucleus as the number of bands observed on polytene chromosomes, suggesting that PC bodies are formed by PcG proteins binding to their target chromatin. The observed colocalization of PcG target genes with PC bodies in diploid cells confirms this view. An alternative scenario posits that PC bodies could form nucleation sites onto which PcG-target genes move to become silenced. Two lines of evidence from this work suggest the first scenario to be closer to reality. Firstly, it was found that the amount of PC within a PC body depends on the linear size of the genomic region coated by PC and H3K27me3. Secondly, the higher enrichment of PC in PC bodies after homologous chromosome pairing strongly suggests that PC bodies are the nuclear counterparts of linear genomic domains identified in genome-wide studies rather than nuclear structures to which Polycomb target genes have to be localized for their silencing (Cheutin, 2012).

In the head of embryos, where the Antp and Abd-B genes are silenced, they localize in large PC bodies in all cell nuclei. In contrast, loci where PC coating is restricted to smaller genomic regions do not always localize within PC bodies in interphase cell nuclei. Interestingly, time-lapse imaging shows that large PC bodies are stable structures that can be visualized in all frames of time series, whereas small PC bodies are apparently less stable because they are not visible in all of the frames. One possible explanation for the lack of colocalization between PC target genes and PC bodies is that small genomic regions may not be coated by PC in every cell. Alternatively, the amount of PC within the PC body in which small genomic regions localize might be too small to be directly observed, and only become visible when several small PC bodies interact together. For instance a previous study showed that a transgene containing only two copies of a PRE could be detected in about 50% of cell nuclei (Cheutin, 2012).

Intense PC bodies can be visualized during entire time-lapse experiments, allowing the study of their motion. The interpretation of these time-lapse experiments is not straightforward because the MSD of PC bodies only weakly correlates with the MSC. Interestingly, tracks of PC bodies are mainly composed of narrow angles. The analysis of the motion of chromatin domains containing H2Av-GFP gave similar results, but gave unambiguous evidence for the coordinated motion of several chromatin domains. By using the Lac repressor/lac operator system, two components of chromatin motion in early G2 Drosophila spermatocyte nuclei have been reported: a short range motion which occurs in approximately 0.5 µm radius domains, and long-range motion confined to a large, chromosome-sized domain. Another study has also identified a two-regime motion of a chromatin locus inside mammalian nucleus by using a two-photon microscope, which provides high spatial and temporal resolution. This work indicated that chromatin loci undergo apparent constrained diffusion during long periods, interrupted by jumps of 150 nm lasting less than 2 s. However, none of these previous works reported any coordinated motion of adjacent chromatin domains, and therefore they both described the motion of chromatin as being consistent with a random walk (Cheutin, 2012).

In tracking experiments, it was realized that the fast regime of motion is tightly constrained within volumes much smaller than chromosome territories. This suggests that any given locus will normally explore a restricted three-dimensional environment in the cell nucleus. Since this applies generally to chromatin at all developmental stages, one can deduce that each genomic locus is likely to locate in the vicinity of neighboring loci in the three-dimensional nuclear space. The prediction is thus that each locus should most frequently contact other loci that are in its linear neighborhood along the chromosome. This behavior matches the results observed in chromosome conformation capture on chip (4C) experiments, where each 4C bait had most contacts within few hundred kb to a few Mb of surrounding chromatin. Thus, the current results provide a possible scenario for the explanation of these results obtained from large cell populations. Recent studies showed that homeotic gene clusters form an extensive network of contacts with other PcG target loci. This is consistent with the observation of multiple PC body collisions that can be stable for prolonged times in the nucleus. In contrast, the fact that PC intensity correlates with the linear extension of genomic PC and H3K27me3 domains suggests that PC-mediated associations are relatively rare, at least during embryogenesis (Cheutin, 2012).

The slower regime of long-range motion depends on coordinated large-scale chromatin movements that were not documented before. This may depend on the tools used in previous studies. Time-lapse experiments performed by using the Lac repressor/lac operator system only follow one or a few points inside the cell nucleus, limiting the probability to observe coordinated motions, especially in species containing many chromosomes. In contrast, this study followed many chromatin domains inside Drosophila nuclei and long-range coordinated motions were easily identified when at least two distinct nuclear structures moved simultaneously with a similar trajectory. This motion is directional and chromatin domains and PC bodies can cover up to 1 µm in 10 sec. Different objects having coordinated motion probably belong to the same structure, which suggests that the ensemble of chromatin domains and PC bodies displaying a similar coordinated motion forms a single higher-order nuclear structure. This kind of motion is perfectly consistent with the observation of a chromosome territory, which implies that chromosomes form distinct nuclear structures in interphase cells. A displacement of an entire chromosome, or of a chromosome arm, or a large part thereof, would induce the coordinated motion of all chromatin domains and PC bodies associated to the corresponding chromosome portion (Cheutin, 2012).

The few association and dissociation events of PC bodies observed during this work are related to long-range coordinated motion events that affect both chromatin domains and PC bodies. Therefore, gene kissing depending on PcG proteins could rely on large scale chromatin movements which lead to transient fusion of PC bodies, and may be in turn specifically stabilized by interactions among PcG proteins. Moreover, the association and dissociation of PC bodies seems to be developmentally regulated, because dynamic associations and dissociations were observed during early embryogenesis, but are strongly reduced later in development (Cheutin, 2012).

Condensed chromatin domains and PC bodies move in confined volumes much smaller than chromosome territories. This highly constrained motion prevents chromatin domains from dispersing inside the cell nucleus and can explain why chromosomes form chromosome territories in interphase cells. This movement within highly confined volumes implies that some forces prevent chromatin from diffusing within entire chromosome territories. Interestingly, it was shown before that chromatin loci localized in peri-nucleolar areas or within heterochromatin move less than the ones included in euchromatin, and it was concluded that association of chromatin loci with different nuclear compartments induces specific constraints on their motion. Another time-lapse experiment performed on one Drosophila locus flanking a large block of heterochromatin showed that random association of this locus with pericentric heterochromatin is quite stable and decreases its motion. The motion of larger chromatin structures such as heterochromatin or euchromatin domains cannot be addressed by tracking single loci. By analyzing structures larger than individual chromatin loci, the motion of both bulk chromatin domains and of PC bodies seems to be influenced by their respective local enrichment of histone and PC proteins. Therefore, one key determinant of the motion constraint is an inner property of these structures, which is coherent with the concept of self-organization (Cheutin, 2012).

The most dramatic change of PC body motion occurs during embryogenesis when nuclear volumes strongly decrease, concomitant with a decrease in bulk chromatin motion. Comparison of chromatin motion between early and late G2 Drosophila spermatocytes or between undifferentiated and differentiated cells of eye imaginal discs indicated that the volume in which chromatin loci move decreases during differentiation. However, because of the particularly rapid motion of chromatin domains and PC bodies during early embryogenesis, the slowdown of chromatin motion occurring during embryogenesis is higher than the ones previously described during differentiation. Interestingly, the reduction of the volume of constraint during developmental progression suggests a correlation between the flexibility of chromatin structures and the potential for cell differentiation (Cheutin, 2012).

It is interesting to note that the motion of PC bodies appears less sensitive to temperature than chromatin domains in late embryos, suggesting that Polycomb proteins may specifically buffer environmental effects such as temperature change. This buffering may be an important determinant of the stability of Polycomb-dependent gene silencing during development. During this work, no other fundamental difference was observed between the motion of condensed chromatin domains and of PC bodies. This apparent absence in specificity is coherent with data implying that PC bodies form molecularly specialized chromatin regions, but suggests that the molecular identity of these structures is not the main determinant of their motion. Interestingly, a previous study has shown that the artificial Mx1-YFP nuclear body exhibits a very similar mobility compared with Promyelocytic leukemia and Cajal bodies. Although being molecularly different, no specific motion of these nuclear bodies was observed, indicating that the motion of nuclear bodies mainly depends on structural issues such as their size and the nuclear volume. During fly embryogenesis, PC bodies and condensed chromatin domains move similarly, but PC bodies move in a larger volume than chromatin domains. To explain this difference, one might argue that condensed chromatin domains would form much larger structures than PC bodies. This is difficult to ascertain until the identity of these DAPI- and histone-dense regions is better understood. Genome-wide analysis of chromatin components has recently identified five different types of chromatin in Drosophila cells, among which three contained silent genes (Filion, 2010). In addition to heterochromatin and Polycomb-repressed chromatin, a third type of silent chromatin was uncovered, which is composed of very large genomic domains encompassing half of the genomic euchromatin. It is proposed that this silent chromatin portion of the genome is physically manifested as the DAPI- and histone-dense chromatin that this study has identified to be distinct from PC bodies (Cheutin, 2012).

H3K27 modifications define segmental regulatory domains in the Drosophila bithorax complex

The bithorax complex (BX-C) in Drosophila melanogaster is a cluster of homeotic genes that determine body segment identity. Expression of these genes is governed by cis-regulatory domains, one for each parasegment. Stable repression of these domains depends on Polycomb Group (PcG) functions, which include trimethylation of lysine 27 of histone H3 (H3K27me3). To search for parasegment-specific signatures that reflect PcG function, chromatin from single parasegments was isolated and profiled. The H3K27me3 profiles across the BX-C in successive parasegments showed a 'stairstep' pattern that revealed sharp boundaries of the BX-C regulatory domains. Acetylated H3K27 was broadly enriched across active domains, in a pattern complementary to H3K27me3. The CCCTC-binding protein (CTCF) bound the borders between H3K27 modification domains; it was retained even in parasegments where adjacent domains lack H3K27me3. These findings provide a molecular definition of the homeotic domains, and implicate precisely positioned H3K27 modifications as a central determinant of segment identity (Bowman, 2014).

The Polycomb Group repression system is often described as a cellular memory mechanism, which can impose lifelong silencing of a gene in response to a transitory signal. That view seems valid, but the concept of a PcG regulatory domain is much richer. In the PS6 domain of the BX-C, for example, there are many enhancers to drive Ubx expression in specific cells at specific developmental times, all of which are blocked in parasegments one through five, but active in parasegments 6 through 12. Individual enhancers need not include a segmental address that is specified, for example, by gap and pair-rule DNA-binding factors; their function is segmentally restricted by the domain architecture. Indeed, these enhancers will drive expression in a different parasegment when inserted into a different domain (as in the Cbx transposition). Each domain has a distinctive collection of enhancers; the UBX pattern in PS5 is quite different from that in PS6. Thus, there are two developmental programs for Ubx, one in each of these parasegments, without the need for a duplication of the Ubx gene. Other loci with broad regions of H3K27 methylation may likewise be parsed into multiple domains, once histone marks are examined in specific cell types (Bowman, 2014).

The all-or-nothing H3K27me3 coverage of the BX-C parasegmental domains validates and refines the domain model. In particular, K27me3 is uniformly removed across the PS5 and PS7 domains in PS5 and PS7, even though the activated genes in those parasegments (Ubx and abd-A, respectively) are only transcribed in a subset of cells. It is interesting that both PRC1 and PRC2 components have binding patterns that do not fully reflect function (repression and K27 methylation, respectively), indicating the possibility that function of these complexes is regulated separately from binding. The challenges now are to understand how PcG regulated domains are established, differently in different parasegments, and to describe the molecular mechanisms, including changes in chromosome structure, that block gene activity in H3K27 trimethylated domains (Bowman, 2014).

Chromatin signatures at Notch-regulated enhancers reveal large-scale changes in H3K56ac upon activation

The conserved Notch pathway functions in diverse developmental and disease-related processes, requiring mechanisms to ensure appropriate target selection and gene activation in each context. To investigate the influence of chromatin organisation and dynamics on the response to Notch signalling, this study partitioned Drosophila chromatin using histone modifications and established the preferred chromatin conditions for binding of Su(H), the Notch pathway transcription factor. Manipulating activity of a co-operating factor, Lozenge/Runx, showed that it can help facilitate these conditions. While many histone modifications were unchanged by Su(H) binding or Notch activation, rapid changes were detected in acetylation of H3K56 at Notch-regulated enhancers. This modification extended over large regions, required the histone acetyl-transferase CBP and was independent of transcription. Such rapid changes in H3K56 acetylation appear to be a conserved indicator of enhancer activation as they also occurred at the mammalian Notch-regulated Hey1 gene and at Drosophila ecdysone-regulated genes. This intriguing example of a core histone modification increasing over short timescales may therefore underpin changes in chromatin accessibility needed to promote transcription following signalling activation (Skalska, 2015).

Signalling pathways such as Notch have diverse functions depending on the context in which they are activated and on the specific subsets of genes that are regulated in each context. This specificity necessitates mechanisms that enable Su(H) to recognise and bind to appropriate enhancers and effect relevant gene expression changes. By utilising the comprehensive collection of chromatin modifications gathered by the modENCODE project, this study has generated maps of chromatin states (see The full list of signal tracks) in two Drosophila cell types and related those to the loci that are bound by Su(H). In doing so, the profile of H3K56ac across the genome was also analysed, and that this core histone modification was found to be present at enhancers, and at transcription start sites, similar to the reported distribution in mammalian ES cells. Significantly, the inclusion of H3K56ac-binding data in the computational model helped to discriminate the active enhancers. Even more striking was the robust increase in this core nucleosome modification in response to Notch activation. Such changes were also detected in mammalian cells and at ecdysone-regulated genes in Drosophila, arguing that H3K56ac is likely to be a widespread modification associated with enhancer activation (Skalska, 2015).

Unlike the modifications to exposed histone tails, which primarily provide docking sites for further chromatin modifying proteins, H3K56ac can directly alter nucleosomal DNA accessibility by increasing DNA breathing and unwrapping rate. As a consequence, this modification can influence transcription factor (TF) occupancy within the nucleosome and it has been argued that H3K56ac drives chromatin towards the disassembled state during transcriptional activation. As the increase in H3K56ac appears to precede transcription elongation, it fits with the latter model. Furthermore, as mammalian CSL has been found to bind preferentially to motifs at the nucleosome exit point\, H3K56ac may enhance recruitment, giving a feed-forward benefit that could potentially explain the increase in occupancy following Notch activation. In addition, H3K56ac facilitates divergent transcription by promoting rapid nucleosome turnover and also promotes small RNA production in neurospora, which is consistent with the detection of intergenic enhancer-templated RNAs in the modified regions following Notch activation (Skalska, 2015).

The increase in H3K56ac appears to require CBP-HAT activity, which is also essential for catalysing this modification on free histones. It is plausible therefore that the increase in H3K56ac could occur through the incorporation of pre-modified nucleosomes. The modification of histone dimers requires interaction with the chaperones CAF1 and ASF1, and while genetic evidence that the chaperone subunit dCAF-1-p105 can help promote Notch signalling favours such a model, the current results suggest this is less likely. First, it was found that CBP is required at the time of activation, making it improbable that the increase in H3K56ac is a consequence of loading pre-modified histones. Second, an inhibitor of the CBP bromodomain, which plays an important role in enabling H3K56ac on histone dimers via its interaction with chaperones, had no effect on the increase in H3K56ac. Thus, it seems more likely that the modification occurs at the time of enhancer activation, although it may nevertheless involve nucleosome exchange. For example, SWI/SNF nucleosome remodellers have been found to act in combination with H3K56ac to promote nucleosome turnover and gene activity in yeasts. At several loci where changes were detected in H3K56ac, the modification extended broadly from the site of Su(H)/NICD binding, correlating with domains that already possessed H3K4me1. Along with data from other studies of enhancer activation, and the observation that levels of H3K56ac are affected by mutation of H3K4, this suggests that H3K4me1 is likely to be one of the earliest modifications, prefiguring sites of active enhancer. It may also facilitate the spread of H3K56ac across the regulated regions (Skalska, 2015).

Analysis of the relationship between chromatin states and regions occupied by Su(H) suggests that the pre-existing chromatin environment is likely to make an important contribution to recruitment. First, Su(H)-occupied motifs were almost exclusively located in highly accessible chromatin, with modifications such as H3K4me1 characteristic of enhancer states. Second, expression of the cooperating transcription factor Lz converted enhancers towards this preferred chromatin state where additional Su(H) was recruited. By having a preference for a particular chromatin signature, the vast majority (>91%) of potential Su(H) binding motifs will be masked by unfavourable chromatin. Indeed, the small fraction of sites that do not fit with this pattern may reflect false positives in the ChIP data or in chromatin assignment. The greater paradox is that only 7%-10% of CSL motifs within the favourable Enh chromatin were bound. Furthermore, many of the positions that were differentially bound in two cell types existed in Enh chromatin in both cell types examined. These observations suggest that additional factors restrict CSL binding to a subset of sites located within favourable chromatin. Such factors might include currently unknown histone modifications, protein-protein interactions, 3D organisation and/or DNA sequence properties around the CSL motif (Skalska, 2015).

Once bound, Su(H) itself also helps to shape the local chromatin environment. Depleting cells of Su(H) resulted in an increase in local histone acetylation (H3K27ac, H3K56ac), suggesting that, in the absence of NICD, Su(H) helps to suppress enhancer activity through its association with co-repressors. Thus, a model emerges in which Su(H) is recruited to regions that have already acquired regulatory competence and that it keeps these in a transitional state with low levels of H3K56ac. As there is considerable variability between enhancers, this suggests that each attains an activity that reflects the balance between the transcription factors promoting enhancer activity and those, such as Su(H), that can antagonise it. In those instances where Su(H)-corepressor complexes win out, then the enhancer is suppressed until the complimentary activity of NICD converts it from a transitional to an active state, a conversion that is associated with a large-scale increase in H3K56ac (Skalska, 2015).

The extent that the principles observed in this study will be of general relevance for other signalling pathways remains to be established, although it seems likely that their target gene specificity will be similarly dependant on the pre-existing chromatin substrate. However, it is possible that the inferred transitional enhancer states may be particularly relevant for those pathways/contexts where there is a fine-scale switch between repression and activation, as occurs for Notch and ecdysone signalling. Nevertheless, the correlation of H3K56ac with H3K4me1 suggests that H3K56ac is likely to be of widespread importance in enhancer activation. Whether this will be mediated through its direct effects on DNA-histone core interactions or through intermediate bromodomain containing proteins that link to the core transcription machinery, such as Brd4, remains to be determined (Skalska, 2015).

Loss of Drosophila Ataxin-7, a SAGA subunit, reduces H2B ubiquitination and leads to neural and retinal degeneration

The Spt-Ada-Gcn5-acetyltransferase (SAGA) chromatin-modifying complex possesses acetyltransferase and deubiquitinase activities. Within this modular complex, Ataxin-7 anchors the deubiquitinase activity to the larger complex. This study identified and characterized Drosophila Ataxin-7 (CG9866) and found that reduction of Ataxin-7 protein results in loss of components from the SAGA complex. In contrast to yeast, where loss of Ataxin-7 inactivates the deubiquitinase and results in increased H2B ubiquitination, loss of Ataxin-7 results in decreased H2B ubiquitination and H3K9 acetylation without affecting other histone marks. Interestingly, the effect on ubiquitination was conserved in human cells, suggesting a novel mechanism regulating histone deubiquitination in higher organisms. Consistent with this mechanism in vivo, this study found that a recombinant deubiquitinase module is active in the absence of Ataxin-7 in vitro. When the consequences of reduced Ataxin-7 were examined in vivo, it was found that flies exhibited pronounced neural and retinal degeneration, impaired movement, and early lethality (Mohan, 2014).

Back to Regulation of H3: Part 1

Histone H3: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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