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).
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; full text of article).
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).
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).
A novel gene, grappa (gpp) is the Drosophila ortholog of the Saccharomyces cerevisiae gene Dot1, a histone methyltransferase that modifies the lysine (K)79 residue of histone H3. gpp is an essential gene identified in a genetic screen for dominant suppressors of pairing-dependent silencing, a Polycomb-group (Pc-G)-mediated silencing mechanism necessary for the maintenance phase of Bithorax complex (BX-C) expression. Surprisingly, gpp mutants not only exhibit Pc-G phenotypes, but also display phenotypes characteristic of trithorax-group mutants. Mutations in gpp also disrupt telomeric silencing but do not affect centric heterochromatin. These apparent contradictory phenotypes may result from loss of gpp activity in mutants at sites of both active and inactive chromatin domains. Unlike the early histone H3 K4 and K9 methylation patterns, the appearance of methylated K79 during embryogenesis coincides with the maintenance phase of BX-C expression, suggesting that there is a unique role for this chromatin modification in development (Shanower, 2005).
Recent studies on telomeric silencing in S. cerevisiae have led to the identification of a histone methlylase, DOT1, which has a number of unusual properties. First, unlike the previously identified histone methylases, DOT1 does not have a canonical SET domain. Instead, the DOT1 protein resembles a family of S-adenosyl methione methyltransferases that modify arginine residues. DOT1 methylates histone H3 at lysine 79 only when it is assembled into nucleosomes and methylation strongly depends upon prior Rad6 dependent ubiquitination of histone H2B at K123. Second, in yeast, deletion or overexpression of Dot1 disrupts TPE and also silencing of the mating-type loci. In contrast, silencing in the yeast ribosomal gene cluster is disrupted only when DOT1 is overexpressed. Third, both telomeric and mating-type silencing are disrupted by mutations in the lysine 79 residue of histone H3. Fourth, methylation of K79 appears to influence the recruitment of the SIR silencing proteins to the telomeres. The SIR silencing proteins appear to preferentially associate with chromatin that is deficient in K79 methylation, while the proteins are generally not associated with chromatin in which there is an enrichment for K79 methylated H3. Fifth, there is evidence that K79 methylation is coordinated with polymerase transcription via the COMPASS complex. Consistent with the idea that K79 methylation might be coordinated with transcription, H3meK79 is enriched in transcribed sequences in yeast and mammals. Interestingly, the distribution of H3meK79 in the β-globin locus differs from H3meK4 in that it is not found at the locus control region. These findings have led to a model in which H3meK79 serves as a marker for transcribed sequences where it functions to block the association of chromatin proteins that mediate transcriptional silencing (Shanower, 2005).
While Dot1 homologs have been identified in higher eukaryotes, little is known about their biological functions. This report characterized the Drosophila Dot1 ortholog gpp. The gpp transcription unit is >40 kb in length and it encodes a complex array of alternatively spliced transcripts that range in size from 6.5 to >9 kb and are expressed at different developmental stages. Consistent with the assignment of the gpp gene, P-element and X-ray mutations disrupt this large transcription unit and in at least one case lead to the production of truncated mRNAs. The gpp transcripts are predicted to encode 170- to 232-kD polypeptides that share a common N-terminal domain that corresponds to about two-thirds of the protein but have different C-terminal domains. The common N-terminal domain contains the Dot1 homology region including the MT methyltransferase fold required for methylation of histone H3. Mutation of conserved glycine residues in the active site of both yeast and human DOT1 protein inactivates the enzyme. GPP also contains domains that are not present in DOT1 including a coiled-coil motif also found in the human, C. elegans, D. pseudoobscura, and A. gambia DOT1-like proteins. In yeast, K79 is mono-, di-, and trimethylated and Dot1 is responsible for all three modifications. The different methylated states of H3 at K79 suggest that multiple regulatory activities are conferred on these modified nucleosomes. However, in fly tissue culture cells, the mono- and di- but not the trimethylated form is observed. Since database searches indicate that gpp is the only fly Dot1 homolog, it should also be the sole fly protein in this class that methylates histone H3 on K79. Consistent with this suggestion, discs and other tissues isolated from gpp mutant larvae have little if any H3 mono- or dimethyl K79 (Shanower, 2005).
Like its yeast counterpart, gpp is required for the silencing of reporter transgenes inserted into telomeric heterochromatin. However suppression of silencing associated with pericentric heterochromatin is unaffected by mutations in gpp. While these observations point to a role of gpp in silencing specific for telomeric heterochromatin, antibody staining experiments indicate that there is a paucity of H3dmeK79 at telomeres in polytene chromosomes compared to many other chromosomal DNA segments. In this respect it is interesting that both telomeric and mating-type chromatin in yeast are hypomethylated on K79 compared to 'bulk' chromatin even though DOT1 is required for SIR silencing in each case. It has been suggested that the meK79 modification in euchromatic nucleosomes blocks SIR protein association and that silencing is lost in the absence of DOT1 because the SIR proteins spread into euchromatin. In contrast, in flies, since many euchromatic domains in wild-type polytene chromosomes have only little H3meK79, it is difficult to see how telomeric silencing proteins would be restricted to telomeres by this modification even when gpp is fully active (Shanower, 2005).
gpp also has functions in flies besides telomeric silencing. Unlike Dot1, gpp is essential for viability. Although the underlying cause of lethality remains to be established, gpp mutant larvae grow more slowly than wild type and this potentially implicates gpp in pathways that control growth rates and size in flies. In addition, gpp mutants display defects that are characteristic of both Pc-G and trx-G genes. The first gpp alleles were recovered as dominant suppressors of mini-white silencing by two BX-C PREs. Consistent with a role in Pc-G silencing, gpp mutants enhance the segmentation defects of several Pc-G genes. In this context, it is interesting to note that several Pc-G genes have recently been shown to play a role not only in the repression of genes in the homeotic complexes but also in telomeric silencing. Thus, it is possible that gpp activity in telomeric silencing may be linked in some manner to its role in Pc-G silencing (Shanower, 2005).
gpp mutants also exhibit transformations in segment identity and genetic interactions with Abd-B that are characteristic of trx-G mutations. This would point to a role in promoting rather than repressing gene expression. Some function in transcription would be consistent with studies in other systems as well as with the enrichment of meK79 seen in many polytene interbands and puffs. However, this correlation is not complete. Thus, there are many puffs and interbands that have only little H3dmeK79. Conversely, H3dmeK79 is sometimes enriched in bands. These findings would argue that in Drosophila, meK79 is not a ubiquitous marker for transcriptionally active chromatin, but rather may have functions that are specific to particular chromatin domains. In this case, the disruptions in homeotic gene expression seen in gpp mutants could reflect a special requirement for H3meK79 in the transcription of these particular genes. Domain-specific requirements for gpp activity in transcription could also potentially account for the effects of gpp mutations on Pc-G and telomeric silencing. In this model, Pc-G and telomeric silencing would be disrupted in gpp mutants because the expression of one or more Pc-G (and/or telomeric heterochromatin) genes is downregulated when gpp activity is compromised (Shanower, 2005).
The developmental profile of H3dmeK79 also suggests that this modification cannot be a ubiquitous marker for either transcriptionally active or silenced chromatin. High levels of Pol II transcription in somatic nuclei begin in the precellular blastoderm stage around nuclear cycle 11/12. Concomitant with the activation of transcription, H3meK4 can be first be detected at this stage, and the level of meK4 then increases through cellularization. By contrast, little if any H3 mono- or dimethyl K79 is in either the transcriptionally active somatic nuclei or the transcriptionally quiescent pole cell nuclei. H3meK79 can first be readily detected only later in development in germband extended embryos. However, at this stage accumulation is restricted primarily to a subset of cells in the embryo, most of which seem to be in the process of cell division. High levels of H3meK79 are not observed until stages 13-15, long after the initial upregulation of transcription in the early zygote. This result also suggests that the homeotic transformations seen in gpp mutants are unlikely to be due to defects in the initial establishment of parasegment-specific patterns of homeotic gene expression by the gap and pair-rule genes. Rather, these transformations probably reflect a requirement for gpp activity later in development during the maintenance phase of homeotic gene regulation -- a phase that is dependent upon Pc-G and trx-G genes. In this respect it is curious that homeotic transformations are not observed in gpp embryos when they hatch as first instar larvae. Maternally derived gpp activity in homozygous mutant embryos maybe sufficient to maintain specific parasegmental patterns of homeotic gene expression through the end of embryogenesis. Alternatively, there may not be absolute requirement for H3meK79 in maintaining appropriate parasegmental patterns of homeotic expression during embryogenesis (Shanower, 2005).
The developmental profile of H3meK79 indicates that this modification is present at low levels in specific developmental stages and tissues (CNS) undergoing active cell division. In contrast, the highest levels of H3meK79 are observed in epidermal cells that have exited the cell cycle and are undergoing differentiation. Thus, it seems possible that this modification may be activated when specific chromatin configurations, active or inactive, need to be maintained for extended periods of time in the absence of de novo DNA synthesis/chromatin assembly. In this respect it is interesting that it has been reported that the highest levels of meK79 are found in a histone H3 variant, H3.3, which is assembled into chromatin by a replication-independent mechanism. Further studies of gpp in Drosophila will be required to understand the mechanisms governing the temporal and tissue-specific regulation of the K79 modification and how this relates to the functions of this particular histone modification during development. Understanding this aspect of the histone code in a multicellular organism such as Drosophila will lead to a better understanding of chromatin regulatory mechanisms during development (Shanower, 2005).
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).
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 (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).
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).
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 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).
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, 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).
Mutant alleles of Drosophila were generated in which expression of the linker histone H1 could be be down-regulated over a wide range by RNAi. When the H1 protein level is reduced to ~20% of the level in wild-type larvae, lethality occured in the late larval - pupal stages of development. This study shows that H1 has an important function in gene regulation within or near heterochromatin. It is a strong dominant suppressor of position effect variegation (PEV). Similar to other suppressors of PEV, H1 is simultaneously involved in both the repression of euchromatic genes brought to the vicinity of pericentric heterochromatin and the activation of heterochromatic genes that depend on their pericentric localization for maximal transcriptional activity. Studies of H1-depleted salivary gland polytene chromosomes show that H1 participates in several fundamental aspects of chromosome structure and function. First, H1 is required for heterochromatin structural integrity and the deposition or maintenance of major pericentric heterochromatin-associated histone marks, including H3K9Me2 and H4K20Me2. Second, H1 also plays an unexpected role in the alignment of endoreplicated sister chromatids. Finally, H1 is essential for organization of pericentric regions of all polytene chromosomes into a single chromocenter. Thus, linker histone H1 is essential in Drosophila and plays a fundamental role in the architecture and activity of chromosomes in vivo (Lu, 2009).
This work provides evidence that maintaining the level of histone H1 expression is essential for proper Drosophila development. In vivo transcription of an H1-specific dsRNA 'hairpin' was used to induce post-transcriptional gene silencing in Drosophila. Lethality caused by abrogation of histone H1 synthesis is temperature-dependent. In this system, the transcription of the H1-specific hairpin RNA is activated ubiquitously by the yeast transactivator protein GAL4, which is known to exert stronger effects at elevated temperatures. Indeed, the depletion of H1 protein and penetrance of the RNAi-induced lethality in transgenic strains both directly correlated with the temperature . Thus, the temperature dependence of GAL4 transcriptional activity allows temporal control over the post-transcriptional silencing of H1; that is, by transferring developing animals from the permissive (18oC) to the restrictive (29oC) temperatures, or vice versa, one can target the RNAi effect to a specific developmental time period. For instance, it was found that activating the synthesis of the H1-specific RNAi during late stages of Drosophila development (in pupae and adults) did not cause an appreciable effect on viability, in contrast to H1 abrogation in embryos and larvae. Thus, there may be a less stringent requirement for maintaining H1 expression after metamorphosis. Alternatively, the endogenous H1 protein that accumulates in larvae prior to metamorphosis may be sufficient for proper cell function throughout the rest of the life cycle in Drosophila (Lu, 2009).
Previous studies with single and compound H1 subtype-specific knockout mice also revealed a direct correlation between the levels of H1 expression and survival. Mice lacking only one or two H1 subtypes, but containing a normal H1 to nucleosome ratio, survive and appear normal. On the other hand, mice lacking five H1 alleles, with a reduction from 20% to up to 50% in the H1-to-nucleosome ratios in different tissues, were small and born at a significantly lower rate than the single and double H1 knockout mice. Embryos lacking six alleles (three H1 subtypes) and containing approximately half of the normal H1 levels developed multiple abnormalities and died in midgestation, an indication that a minimum threshold level of H1 protein is required for normal mammalian embryonic development. These data in Drosophila parallel these findings, since at subpermissive temperatures (26°C or lower), intermediate reduction of H1 expression to ~70% of the wild-type larval level resulted in partial survival of affected animals. Thus, in contrast to simpler eukaryotes, in which the linker histone is not essential, metazoans require maintenance of a certain level of H1 expression for normal development (Lu, 2009).
Pericentric heterochromatin has been implicated in gene silencing that occurs when euchromatic genes are placed adjacent to heterochromatin by chromosome rearrangement or transposition—a phenomenon that was initially described in Drosophila as PEV. Through genetic screening, many important chromatin regulators have been identified, which, when mutated, act as modifiers (suppressors or enhancers) of PEV. Thus, PEV in Drosophila represents a valuable assay for identification and molecular study of evolutionarily conserved functions controlling epigenetic programming in eukaryotes. This study observed that the linker histone H1 stimulates silencing in pericentric heterochromatin. Although it was not feasible to make a classical mutant of the H1 genes, dose reduction of H1 by ~15% resulted in PEV suppression. In that respect, H1 resembles other dominant suppressors of PEV, such as Su(var)2-5, which encodes HP1. Dose reduction of HP1 in Su(var)2-5 heterozygotes results in strong PEV suppression. The data indicate that H1 is an essential structural component of pericentric heterochromatin, or it is necessary for recruitment of another such essential biochemical component(s) to heterochromatin. In fact, it was found that the level of H1 does affect the localization of two major markers of pericentric heterochromatin, HP1 and H3K9Me2 (Lu, 2009).
HP1 is an abundant nonhistone chromosomal protein first discovered in Drosophila because of its association with heterochromatin. HP1 is conserved in many eukaryotes, including fission yeast, insects, and mammals; involved in gene silencing; and consistently associated with pericentric heterochromatin and telomeres. In Drosophila polytene chromosomes, HP1 is diagnostic of heterochromatin, and the vast majority of HP1 protein concentrates at the chromocenter. Indirect immunofluorescence staining of polytene chromosomes indicates that histone H1 is abundant in pericentric heterochromatin. Furthermore, the chromocenter is severely disrupted in polytene chromosomes of salivary gland cells with depleted H1, and H1 abrogation also results in a delocalization of HP1. The dispersion of the chromocenter is not produced by mechanical stress during squashing, since it is similarly observed in whole-mount salivary gland cells. Thus, H1 plays important roles in the establishment and/or maintenance of the structure as well as in the biochemical composition of proximal heterochromatin in Drosophila larvae. It remains to be seen whether H1 is directly required for faithful deposition/recruitment of HP1 to its cognate loci in pericentric heterochromatin, or mislocalization of HP1 in chromosomes of H1-depleted cells is a secondary effect mediated by disruption of other nuclear processes that are regulated by the abundance of H1 (e.g., transcription). The former explanation is certainly possible since there are several reports that HP1 interacts directly with H1 (Lu, 2009).
Methylation of histone H3 Lys 9 (H3K9) has a well-established role in heterochromatin formation in metazoans, and H3K9Me3 (H3K9Me2 in Drosophila) is highly enriched in condensed heterochromatin. The chromodomain of HP1 specifically recognizes methylated H3K9, which facilitates its recruitment and leads to an overlapping distribution of HP1 and the H3K9 methylation mark in the genome. Upon H1 abrogation, however, very little or no H3K9Me2 is detected in the loci where HP1 remains present. It is concluded that in polytene chromosomes of H1-depleted larvae, HP1 is deposited by a mechanism that does not require histone H3 dimethylation. The persistence of HP1 in proximal heterochromatin in the absence of dimethylated H3K9 is consistent with reports indicating that HP1 can bind nonspecifically to nucleosome core particles and even to naked DNA. It is also consistent with findings that used a tethering system to recruit HP1 to euchromatic sites: these showed that HP1-mediated silencing can operate in a Su(var)3-9-independent manner. The current findings strengthen the view that, whereas HP1 may normally cooperate with Su(var)3-9 and K9-methylated H3 in heterochromatin formation and gene silencing at pericentric chromosome sites, it can be deposited in these regions independently of these other components, and even without the presence of H1 (Lu, 2009).
The Su(var)3-9-null mutants, although also lacking an appreciable level of H3K9Me2 signal in immunofluorescence-stained polytene chromosomes, do not exhibit the same spectrum of phenotypes as H1-depleted animals. For instance, the single polytene chromocenter is not disrupted in Su(var)3-9-null mutants. Thus, the observed phenotypes and defects in chromatin structure upon abrogation of H1 cannot be explained exclusively by the loss of H3K9 dimethylation, and H1 is therefore predicted to play a separate and unique role in the establishment and/or maintenance of pericentric heterochromatin. In the future, it will be interesting to see whether in addition to the reversal of heterochromatic silencing, similar to other suppressors of variegation, H1 depletion also affects other properties of heterochromatin, such as the reduced rates of meiotic recombination normally observed in these regions (Lu, 2009).
It is an intriguing observation that H3K9Me2 is not detectable in chromatin of H1-depleted salivary glands by indirect immunofluorescence, although total protein levels in cell lysates are elevated rather than reduced. Thus, H1 may be required for H3K9Me2 deposition in chromatin. Alternatively, if histone H3 Lys 9 is dimethylated by Su(var)3-9 predominantly in the context of a nucleosome, H1 depletion may result in specific expulsion of the K9-dimethylated form of H3 from pericentric regions and potentially other H3K9Me2-enriched loci. The presence of other repressive, heterochromatin-specific histone marks, such as H4K20Me2, H3K9Me1, and H3K9Me3, was examined in polytene chromosomes of H1 knockdown larvae by IF microscopy. It was discovered that they were all largely absent in pericentric heterochromatin. In contrast, there was no substantial effect on the active H3K4Me2 mark, which remained widely distributed in polytene chromosomes. Thus, H1 appears to be required for global maintenance of repressive marks in heterochromatin, rather than stimulation of particular programs/enzymes that affect specific histone modification states. This function of H1 might be linked to its role in the transcriptional activity of heterochromatin. Indeed, studies of heterochromatic gene expression in H1-depleted larvae showed that low levels of H1 cause altered transcriptional activity in heterochromatin. Further studies of the dynamics of formation and maintenance of H3K9Me2 and other repressive marks in H1-depleted chromatin may lead to a better understanding of this relationship (Lu, 2009).
H1 depletion has a dramatic effect on the distribution of H3K9Me2-containing nucleosomes in the genome. It is possible that H1 is similarly involved in maintenance of other repressive histone marks in Drosophila. However, it is unlikely that H1 is involved in Polycomb silencing, since no homeiotic phenotypes were observed in adult escapers that survive partial H1 depletion (at 26oC and below) (Lu, 2009).
Previous work with H1-depleted mouse ES cells, as well as studies in other species, suggested that H1 may participate in both transcriptional activation as well as repression in vivo. Likewise, studies with H1-depleted Drosophila larvae support dual roles for H1 in transcriptional regulation. Similar to other suppressors of PEV, H1 stimulates silencing of genes that are brought into juxtaposition with heterochromatin. In contrast, certain Drosophila genes that are embedded in heterochromatin (e.g., 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).
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).
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).
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