Histone H3


Distribution of methylated H3 on polytene chromosomes

If the H3 methylation activity associated with E(z) serves as a chromatin mark for PcG silencing, correspondingly methylated H3 should be found at PcG sites on polytene chromosomes. When chromosome spreads were double stained with an antibody directed against dimethylated H3 lysine 9 (anti-me2K9) together with anti-Psc to label PcG sites, no correspondence between the two was found. The anti-me2K9 antibody stains the chromocenter very strongly, as well as a very few euchromatic sites and telomeres. Telomeres are in fact the only places where Psc and me2K9 H3 are found together, consistent with reports that heterochromatin protein Hp1 and Psc coexist at subtelomeric sites. In contrast, when anti-me3K9 was used to stain polytene chromosomes, almost perfect colocalization was obtained of me3K9 and Psc at euchromatic sites. The relative intensity of the two signals varies from site to site, but with very few exceptions the two signals colocalize. In particular, both the BX-C locus and ANT-C locus, prime sites of action of PcG proteins, stain strongly with both antibodies. Clear exceptions are the chromocenter and most of chromosome 4, where the anti-me3K9 antibody stains powerfully while Psc is not found; a few rare euchromatic sites and some telomeres, where the Psc signal is not always present. Conversely, a few sites give a strong Psc signal but only a weak me3K9 signal. Two such sites are 2D, near the tip of the X chromosome, and 49F, on chromosome 2R. The first is the locus of polyhomeotic (ph); the second contains the divergently transcribed Psc and Su(z)2 genes. All three genes are components of PcG complexes, and ph and Psc have been shown to be themselves targets of PcG regulation (Czermin, 2002).

Since the me2K9 and me3K9 antibodies have nonoverlapping specificities at least in vitro for the me2K9 and me3K9 peptides, respectively, it is concluded that the cytological sites typically considered heterochromatin-like (chromocenter, chromosome 4, and telomeric regions) contain both me2K9 and me3K9 H3. While the me3K9 antibody may recognize weakly the methylated K27 peptide, the contribution of this interaction to the polytene staining cannot be evaluated. To test whether the methylation detected by the anti-me3K9 antibody is dependent on E(z) function, polytene chromosomes were prepared from larvae homozygous for the E(z)S2 temperature-sensitive mutation and raised at 29°C after hatching of the larvae. Inactivation of the E(z)S2 product causes loss of binding of PcG proteins from most but not all sites, compared to wild-type chromosomes. Inactivation appears to occur to different extents in different larvae or even in different nuclei from the same gland, judging from the distribution of anti-me3K9 or anti-Psc antibody staining. In general, the euchromatic bands of anti-me3K9 staining are lost well before the Psc staining. A few Psc sites remain strong in the absence of detectable me3K9 staining. Methylation at a few euchromatic sites persists; one in particular is region 31 of chromosome 2L, containing multiple bands that bind the heterochromatin protein Hp1. Staining of the chromocenter and of chromosome 4 also persists, but it is difficult to conclude unambiguously whether it is affected by the E(z)S2 mutation since the loss of function is often incomplete. Heterochromatic staining is lost in some nuclei but whether it is artifactual in these dying larvae is uncertain. Interestingly, staining at telomeres is also lost, often well before the corresponding staining with anti-Psc antibody. These results confirm the conclusion that most of the euchromatic methylation detected by the anti-me3K9 antibody is in fact dependent on E(z) (Czermin, 2002).

To distinguish the histone H3 methylation due to Su(var)3-9 and that due to E(z), chromosomes from larvae homozygous for the Su(var)3-906 mutation were stained. Larvae and flies homozygous for this mutation are viable and lack most but not all of the anti-me3K9 staining at the chromocenter while staining of chromosome 4, of the base of chromosome 2R, and of telomeres, as well as euchromatic sites, persists unaffected. Staining with anti-Psc is also normal in these chromosomes (Czermin, 2002).

The histone variant H3.3 marks active chromatin by replication-independent nucleosome assembly

Two very similar H3 histones -- differing at only four amino acid positions -- are produced in Drosophila cells. A mechanism of chromatin regulation is described whereby the variant H3.3 histone is deposited at particular loci, including active rDNA arrays. While the major H3 is incorporated strictly during DNA replication, amino acid changes toward H3.3 allow replication-independent (RI) deposition. In contrast to replication-coupled (RC) deposition, RI deposition does not require the N-terminal tail. H3.3 is the exclusive substrate for RI deposition, and its counterpart is the only substrate retained in yeast. RI substitution of H3.3 provides a mechanism for the immediate activation of genes that are silenced by histone modification. Inheritance of newly deposited nucleosomes may then mark sites as active loci (Ahmad, 2002).

To monitor histone dynamics in vivo, fusion genes encoding various histones and the green fluorescent protein (GFP) were constructed under the control of heat shock-inducible promoters. These constructs were transfected into exponentially growing Kc cells and induce. The deposition of histone H3-GFP in the nucleus parallels that of nucleotide analog incorporation into DNA. Localization of histone H3-GFP is completely blocked by pretreatment of cells with the DNA replication inhibitor aphidicolin, demonstrating that the deposition of histone H3 is strictly replication dependent. Detection of a component of the DNA replication machinery, PCNA, also confirms that deposition of histone H3-GFP is coupled to DNA replication: PCNA, BrdU, and H3-GFP give similar labeling patterns both in early S phase (when euchromatic DNA is replicating) and in late S phase (when heterochromatic DNA is replicating). BrdU and H3-GFP closely overlap because both are present for the entire 2 hr labeling period. PCNA labeling does not precisely overlap, since it provides a 'snapshot' of replication only at the time of fixation. In subsequent labeling experiments, PCNA as used to indicate the cell cycle stage (Ahmad, 2002).

Since histone H3 deposition is strictly replication dependent, it was reasoned that replication-independent deposition of histone H4 might be accompanied by the deposition of H3 variants to form variant nucleosomes. Centromeric histones are thought to be included in nucleosomes at centromeres, and it has been demonstrated that the Drosophila centromeric H3 variant Cid localizes to centromeres by a replication-independent pathway. Thus, it was expected that some sites showing H4 replication-independent deposition would be centromeres. Detection of centromeres in H4GFP-transfected cells demonstrates that four to six of the H4 replication-independent foci were indeed centromeres, consistent with the assembly of nucleosomes containing Cid and H4 at these sites. It was reasoned that the remaining H4 sites must be incorporating the final histone H3 variant, H3.3. Indeed, expression of H3.3-GFP in cells demonstrated that this variant does undergo both replication-coupled and replication-independent deposition. None of the H3.3-GFP foci coincided with centromeres, showing that centromeres use the Cid histone exclusively (Ahmad, 2002).

It was confirmed the H3.3-GFP is tightly bound to chromatin by extracting cells with 1.5 M salt before fixation. After this treatment, nuclei retain 48% of the H3.3-GFP but only 22% of the H2B-GFP. Such differential extraction is expected from the biochemical properties of these histones, and the proper behavior of GFP-tagged histones has been extensively documented (Ahmad, 2002).

To map the locations of the sites in the nucleus where replication-independent deposition of histone H3.3 and H4 occurs, mitotic figures were examined from cells transfected with histone-GFP constructs. The G2 phase in Kc cells is 4-6 hr long; thus, mitotic figures with H3-GFP labeling first appear 4-6 hr after heat-shock induction and show patterns consistent with histone-GFP production in late S phase, when heterochromatin is replicating. In contrast, labeled mitotic figures with H3.3-GFP and H4-GFP appear within 2 hr of induction. H4-GFP showed prominent labeling at a single extended site near the middle of an X chromosome. The pattern of H3.3-GFP is very similar to that of H4-GFP, showing the greatest labeling over an extended site on the X chromosome and at low levels specifically in euchromatin. These cells must have been in the G2 phase of the cell cycle when histone-GFP was produced. This was confirmed by the presence of H3.3 labeling on mitotic chromosomes that showed no incorporation of pulse-labeled nucleotides and by observing mitotic figures from aphidicolin-treated cultures that nevertheless displayed H3.3-GFP labeling. Thus, these mitotic labeling patterns with H3.3-GFP and H4-GFP must have resulted from replication-independent deposition (Ahmad, 2002).

The extended appearance and proximal location of the prominent H3.3 and H4 site on the labeled X chromosome suggested that it coincides with the large rDNA gene repeat array on this chromosome. In situ hybridization with probes to the 28S rDNA gene confirmed that this is so. Quantitative measurements of GFP signal over the rDNA array and over all of the chromosomes indicate that ~40% of all histone H3.3 in the cell is deposited at the rDNA locus. In Tetrahymena, a histone H3 replacement variant is enriched in the transcriptionally active macronucleus, suggesting that this Tetrahymena variant potentiates active chromatin (Allis, 1984). It is presumed that the high intensity of histone H3.3-GFP staining at the rDNA locus in Drosophila is due to the combination of its densely repeated genes with high transcriptional activity (Ahmad, 2002).

Notably, labeling with H3.3-GFP and H4-GFP was often observed of only one X chromosome. This is not due to absence of rDNA from other X chromosomes in these cells because the detection of 28S rDNA by in situ hybridization confirmed that rDNA arrays are present on each of the three X chromosomes. Other studies have pointed out that many Drosophila cell lines (including Kc) carry two distinguishable kinds of X chromosomes: a short one (XS) that resembles the normal X of flies, and a longer X (XL). The origin of XL has been attributed to an expansion of the rDNA locus on this chromosome, presumably as these cells adapted to culture conditions. It was observed that the rDNA array on XL is always labeled by H3.3-GFP, consistent with this locus being active in all cells. However, in some experiments, variable numbers of cells had additional labeling on XS chromosomes. To test whether some of this variability between experiments was due to differences in growth conditions, cells were transfected with the histone H3.3-GFP construct and then expression was induced in samples of this culture 16 or 24 hr later. It was found that many cells from exponentially growing cultures show replication-independent labeling on both XL and XS chromosomes, while metaphase spreads from the later time point, when culture growth had slowed, showed labeling on only the one XL. This change in frequency suggests that the smaller rDNA arrays on XS chromosomes are maintained in a transcriptionally silent state but can be activated (Ahmad, 2002).

The silencing of XS rDNA arrays might be due to heterochromatin-mediated silencing. Indeed, staining of metaphase spreads from cells expressing histone H3.3-GFP for the heterochromatin marker H3di-MethylK9 (H3Me) revealed that rDNA arrays labeled by replication-independent deposition of H3.3-GFP are depleted for H3Me, in spite of being flanked on both sides by heterochromatin. In every XS chromosome where the proximal region was labeled with H3.3-GFP, a corresponding gap in the H3Me pattern was found. That sites heavily labeled with H3.3-GFP are largely unlabeled with H3Me was confirmed in interphase nuclei. It is concluded that the chromatin state of rDNA arrays can be reversed in response to changes in growth conditions, and H3.3 accumulates de novo at activated genes (Ahmad, 2002).

Alternate interpretations of the phylogenetic history of the histone H3 family have been proposed. One analysis suggested that a replacement histone H3 variant was the common ancestor, but other interpretations have proposed that replacement histones have multiple independent origins. The presence of paralogous histone H3 genes in many organisms may preclude delineation of which sequence is ancestral. However, the findings of this study suggest that a replication-independent nucleosome assembly pathway is essential in all cells. This implies that, functionally, a replacement histone H3 has always been extant. In organisms that encode only one kind of canonical histone H3 protein that is used throughout chromatin, it is expected that this H3 variant must undergo both replication-coupled and replication-independent deposition. Fungal lineages are particularly intriguing in this regard because all ascomycetes, including laboratory yeasts and molds, carry only one canonical histone H3. Each of these is identical to animal H3.3 at positions 89 and 90, and often identical at position 31. Thus, by this criterion, it is proposed that the solitary histone H3 proteins in ascomycetes are equivalent to histone H3.3. Indeed, nucleosome assembly activity in the cell cycle gap phases has been detected in Saccharomyces. These fungi appear to have lost their ancestral H3, since genomes from the Basidiomycota sister clade have both H3 and H3.3. Histone H2A in Saccharomyces may have an analogous evolutionary history, since it now performs the functions of the H2A and the H2A.X variants in other organisms. Thus, both histone H3 and H2A in Saccharomyces appear to be evolutionary derivatives of replacement genes (Ahmad, 2002).

The lack of an H3 counterpart in yeasts and molds may provide insight into differences between simple fungi and complex multicellular eukaryotes in maintaining silent chromatin. Much of the Saccharomyces genome is continually in a transcriptionally competent state similar to H3.3-containing regions in complex genomes. Perhaps this relative lack of silent chromatin allowed the loss of the strictly replication-coupled histone substrate. Heterochromatic silencing in yeast may be needed only at special sites, such as silent mating type loci and telomeres, where SIR-based silencing has evolved. In multicellular eukaryotes, the need for maintaining most of the genome in a continuously silent state in differentiated cells may favor maintaining two distinct H3 histones (Ahmad, 2002).

Functional anatomy of polycomb and trithorax chromatin landscapes in Drosophila embryos

Polycomb group (PcG) and trithorax group (trxG) proteins are conserved chromatin factors that regulate key developmental genes throughout development. In Drosophila, PcG and trxG factors bind to regulatory DNA elements called PcG and trxG response elements (PREs and TREs). Several DNA binding proteins have been suggested to recruit PcG proteins to PREs, but the DNA sequences necessary and sufficient to define PREs are largely unknown. This study used chromatin immunoprecipitation (ChIP) on chip assays to map the chromosomal distribution of Drosophila PcG proteins, the N- and C-terminal fragments of the Trithorax (TRX) protein and four candidate DNA-binding factors for PcG recruitment. In addition, histone modifications associated with PcG-dependent silencing and TRX-mediated activation were mapped. PcG proteins colocalize in large regions that may be defined as polycomb domains and colocalize with recruiters to form several hundreds of putative PREs. Strikingly, the majority of PcG recruiter binding sites are associated with H3K4me3 and not with PcG binding, suggesting that recruiter proteins have a dual function in activation as well as silencing. One major discriminant between activation and silencing is the strong binding of Pleiohomeotic (PHO) to silenced regions, whereas its homolog Pleiohomeotic-like (PHOL) binds preferentially to active promoters. In addition, the C-terminal fragment of TRX (TRX-C) showed high affinity to PcG binding sites, whereas the N-terminal fragment (TRX-N) bound mainly to active promoter regions trimethylated on H3K4. The results indicate that DNA binding proteins serve as platforms to assist PcG and trxG binding. Furthermore, several DNA sequence features discriminate between PcG- and TRX-N-bound regions, indicating that underlying DNA sequence contains critical information to drive PREs and TREs towards silencing or activation (Schuettengruber, 2008; tull text of article).

The genome-wide mapping of PcG factors, TRX, their associated histone marks, and potential PcG recruiter proteins in Drosophila embryos revealed several important features. First, similar to the PcG distribution in Drosophila cell lines, PcG proteins strongly colocalize and form large domains containing multiple binding sites. Second, the N-terminal and C-terminal fragments of TRX show different binding affinities to repressed and active chromatin. The N-terminal fragment of TRX has low affinity to PcG binding sites but is strongly bound to thousands of active promoter regions that are trimethylated on H3K4, whereas the C-terminal fragment of TRX only showed high binding affinity to PcG binding sites. Third, the majority of PcG recruiter binding sites are associated with H3K4me3 and TRX-N foci and not with PH binding. The binding ratio between the PHO protein and its homolog PHOL is a major predictive feature of PcG versus TRX recruitment. Finally, supervised and unsupervised sequence analysis methods led to the identification of sequence motifs that discriminate between most of the PcG and TRX binding sites, but these motifs are likely to be working jointly, and none of them seems to drive recruitment by itself (Schuettengruber, 2008).

To date, PREs have been only characterized in Drosophila. These elements are not defined by a conserved sequence, but include several conserved motifs, which are recognized by known DNA binding proteins like GAGA factor (GAF), Pipsqueak (PSQ), Pleiohomeotic and Pleiohomeotic-(like) (PHO and PHOL), dorsal switch protein (DSP1), Zeste, Grainyhead (GH), and SP1/KLF. The genomic profiles provide a comprehensive view on the potential role of these factors in the establishment of PcG domains (Schuettengruber, 2008).

The presence of PHO at all PREs indicates that PHO is a crucial determinant of PcG-mediated silencing, consistent with earlier analysis on one particular PRE. On the other hand, PHOL and Zeste were bound at a small subset of PREs. Zeste was previously shown to be necessary for maintaining active chromatin states at the Fab-7 (Frontabdominal-7) PRE/TRE. Therefore, Zeste and PHOL may primarily assist transcription rather than PcG-mediated silencing. GAF and DSP1 resemble PHO as they bind to many (albeit less than PHO) PREs as well as to active promoters. Supervised DNA motif analysis indicated a higher density of GAF, DSP1, and PHO binding sites at PREs as compared to other bound regions at non-PH sites. This suggests that cooperative binding of these proteins may provide a platform for PcG protein binding. Moreover, GAF may act by inducing chromatin remodeling to remove nucleosomes, since the regions bound by PcG proteins show a characteristic dip in H3K27me3 signal that has been attributed to the absence of nucleosomes in those regions. These nucleosome depletion sites are the places wherein histone H3 to H3.3 replacement takes place. Indeed, several of the Zeste-bound regions and GAGA binding sequences were shown to localize to peaks of H3.3, suggesting the possibility that GAF may recruit PcG components to PHO-site-containing PREs as well as recruit TRX to promoters via nucleosome disruption (Schuettengruber, 2008).

In addition to an increased density of motifs for GAF, PHO, and PHOL, unsupervised spatial cluster analysis identified specific motifs that distinguish the PH sites from the K4me3 cluster. Although the identity of the factors binding to these motifs is unknown, this suggests that the DNA sequence of PREs contains much of the information needed to recruit PcG proteins and to define silent or active chromatin states. With this distinction, it may be possible to develop an algorithm to faithfully predict the genomic location of PREs. Earlier attempts to predict PREs in the fly genome have made progress toward this goal, but they are still far from reaching the required sensitivity and specificity. The use of a sequence analysis pipeline that is not dependent on prior knowledge was demonstrated here to generate new discriminative motifs with a potential predictive power. The unique genomic organization of PcG domains may suggest that the genome is using, not only local sequence (high-affinity transcription factor binding sites located at the binding peaks) information to determine PREs, but also integration of regional sequence information (stronger affinity on 5 kb surrounding PREs). Using such regional information to predict PREs may break the current specificity and sensitivity barriers (Schuettengruber, 2008).

ChIP on chip data showed that PHO binding comes in two distinct flavors. In one class of target sites, PHO binding coincides with PH sites within PC domains, whereas outside these domains, it is largely colocalized with PHOL, TRX-N, and H3K4me3 . PHOL binding was weaker at PH sites and was mainly present along with marks associated with gene activation. Quantitative ChIP assays revealed that PH, PHO, and PHOL were bound in PREs/TSS of their target genes in both ON and OFF states, but the ON state was marked by a decrease in PH binding and a corresponding increase in PHOL levels, whereas the OFF state was characterized by an increase in both PH and PHO binding levels (Schuettengruber, 2008).

Chromatin at the Ubx TSS, the bx PRE, and the bxd PRE (the same primers were used in the current study) by comparing haltere/third leg imaginal discs (ON state) with wing imaginal discs (OFF state). A 50% reduction was found of PH binding levels at the bx PRE, a minor decrease at bxd, and no change in the Ubx TSS. ChIP experiments demonstrated a 50% decrease in PH levels at bx PRE and at the Ubx TSS and a minor decrease at bxd PRE when comparing haltere/third leg imaginal discs to eye imaginal discs. A slight decrease was observed in the levels of PHO in haltere/third leg disc (ON state) as compared to eye imaginal discs (OFF state) at the bx and bxd PRE, whereas another study did not see differences in the levels of PHO. The most likely explanation for these discrepancies is that the peripodal membrane cells of the wing imaginal discs express Ubx, whereas all cells silence this gene in eye imaginal discs (Schuettengruber, 2008). pho1 mutant eye discs, the absence of PHO causes derepression of the homeotic genes Ubx and Antp. However, the expression levels in pho1 mutants are still much weaker compared to tissues where these genes are normally expressed. This low degree of activation could be explained by compensatory binding of PHOL to the PHO sites in order to maintain PcG-mediated silencing, even if the PHOL-dependent rescue function is incomplete as pho1 mutants die as pharate adults. PHO and PHOL have indeed been described as redundant in their role in PcG-mediated silencing since they bind to the same DNA sequence motif in vitro. However, out of the 1,757 places wherein both PHO and PHOL were significantly bound, only 807 shared the same local maxima. Another 559 (32%) peaks were within 250 bp of each other. This suggests that, in vivo, these two proteins prefer slightly different sequences, with PHO more strongly attracted to PREs, whereas PHOL binds better to promoters. Moreover, PHO interacts directly with PC and PH, as well as with the PRC2 components E(z) and Esc, whereas PHOL only interacts with Esc in yeast two-hybrid assays. Stronger interactions between PHO and PcG components may stabilize PHO binding at PREs, favoring it over the binding of PHOL. It is thus possible that the primary function of PHOL is as a transcription cofactor, and that its recruitment to PREs is subsidiary to PHO (Schuettengruber, 2008).

This study reports the genome-wide distribution of TRX. This protein has been proposed to counteract PcG-mediated silencing. It has been demonstrated that TRX colocalizes with Polymerase II and elongation factors in Drosophila polytene chromosomes. They it was showm that PcG and TRX proteins bind to a PRE mutually exclusively in salivary gland chromosomes. In contrast, other studies found binding of TRX at discrete sites at PREs and promoter regions of HOX genes, and suggested that TRX coexists with PRC1 components at silent genes. This study postulated that these differences might be explained by the use of different TRX antibodies, one against the N-terminal domain and one against the C-terminal domain of TRX. Notably, the TRX protein is proteolytically cleaved into an N-terminal and a C-terminal domain, but the fate of the two moieties after cleavage has never been addressed in vivo (Schuettengruber, 2008).

Genome-wide mapping studies using the same antibody against the N-terminal fragment (TRX-N) as used previously, showed that the binding affinity of the N-terminal fragment to PREs is rather weak, whereas TRX-N binds thousands of promoter regions trimethylated on H3K4, indicating a general role of TRX-N in gene activation. In contrast, ChIP on chip profiling using an antibody against the C-terminal TRX fragment showed high binding levels at PRE/TREs, whereas binding to promoter regions (where the TRX N-terminal fragment is strongly bound) is rather weak. The strong quantitative correlation between the binding intensities of PH and TRX-C suggests that TRX-C can indeed bind to silent PcG target genes. These data are confirmed by the colocalization of PH and TRX-C at inactive Hox genes in salivary gland polytene chromosomes and in diploid cell nuclei (as seen in a combination of DNA fluorescent in situ hybridization (FISH) and immunostaining; unpublished data). Thus, PcG silencing may involve locking the C-terminal portion of TRX in an inactive state that perturbs transcription activation events. The fact that TRX is recognized by two different antibodies that recognize PREs (H3K4me3-depleted regions) or TSSs suggests that these antibodies reflect the activity state of the protein and thus represent a powerful tool to study the switching of genes between silencing and activation (Schuettengruber, 2008).

Similar to mapping studies in Drosophila cell lines, H3K27me3 also forms large domains in Drosophila embryos. These large PcG domains could provide the basis of a robust epigenetic memory to maintain gene expression states during mitosis. As previously suggested, stably bound PcG complexes at PREs may loop out and form transient contacts with neighboring chromatin, which become trimethylated on H3K27. H3K27me3 might then attract the chromodomain of the PC protein, which may be occasionally trapped at these remote sites by cross-linking mediated by the chromodomain of PC. Alternatively, PcG subcomplexes missing some of the subunits might spread from the PRE into flanking genomic regions containing H3K27me3 histones (Schuettengruber, 2008).

Although genome-wide PcG profiles in Drosophila embryos correlate well with profiles from Drosophila cell lines, it has recently been shown that PcG protein binding profiles are partially remodeled during development. Comparison of PcG target genes showed that 40% of the targets are unique. The fact that a consistent number of targets are only found in one or two of the samples indicates tissue specific PcG occupancy. Thus, although PcG proteins have been often invoked as epigenetic gatekeepers of cellular memory processes, they may be involved as well in dynamic gene regulation during fly development, similar to their function in mammalian cells (Schuettengruber, 2008).

Sequential changes at differentiation gene promoters as they become active in a stem cell lineage

Transcriptional silencing of terminal differentiation genes by the Polycomb group (PcG) machinery is emerging as a key feature of precursor cells in stem cell lineages. How, then, is this epigenetic silencing reversed for proper cellular differentiation? This study investigate how the developmental program reverses local PcG action to allow expression of terminal differentiation genes in the Drosophila male germline stem cell (GSC) lineage. It was found that the silenced state, set up in precursor cells, is relieved through developmentally regulated sequential events at promoters once cells commit to spermatocyte differentiation. The programmed events include global downregulation of Polycomb repressive complex 2 (PRC2) components, recruitment of hypophosphorylated RNA polymerase II (Pol II) to promoters, as well as the expression and action of testis-specific homologs of TATA-binding protein-associated factors (tTAFs). In addition, action of the testis-specific meiotic arrest complex (tMAC; Drosophila RB, E2F and Myb), a tissue-specific version of the mammalian MIP/dREAM complex, is required both for recruitment of tTAFs to target differentiation genes and for proper cell type-specific localization of PRC1 components and tTAFs within the spermatocyte nucleolus. Together, the action of the tMAC and tTAF cell type-specific chromatin and transcription machinery leads to loss of Polycomb and release of stalled Pol II from the terminal differentiation gene promoters, allowing robust transcription (Chen, 2011).

The results suggest a stepwise series of developmentally programmed events as terminal differentiation genes convert from a transcriptionally silent state in precursor cells to full expression in differentiating spermatocytes. In precursor cells, differentiation genes are repressed and associated with background levels of hypophosphorylated Pol II and H3K4me3. These genes also display elevated levels of H3K27me3 and Polycomb at the promoter region, suggesting that they are acted upon by the PcG transcriptional silencing machinery. Notably, the differentiation genes studied in precursor cells in this study did not show the hallmark bivalent chromatin domains enriched for both the repressive H3K27me3 mark and the active H3K4me3 mark that have been characterized for a cohort of differentiation genes in mammalian ESCs (Chen, 2011).

The cell fate switch from proliferating spermatogonia to the spermatocyte differentiation program initiates both global and local changes in the transcriptional regulatory landscape, starting a cell type-specific gene expression cascade that eventually leads to robust transcription of the terminal differentiation genes. Globally, soon after the switch from spermatogonia to spermatocytes, core subunits of the PRC2 complex are downregulated, including E(z), the enzyme that generates the H3K27me3 mark. Locally, after male germ cells become spermatocytes, Pol II accumulates at the terminal differentiation gene promoters, although these genes still remain transcriptionally silent, with low H3K4me3 and high Polycomb protein levels near their promoters (Chen, 2011).

The next step awaits the expression of spermatocyte-specific forms of core transcription machinery and chromatin-associated regulators, including homologs of subunits of both the general transcription factor TFIID (tTAFs) and the MIP/dREAM complex (Aly and other testis-specific components of tMAC). The tMAC complex acts either locally or globally, perhaps at the level of chromatin or directly through interaction with tTAFs, to allow recruitment of tTAFs to promoters of target terminal differentiation genes. The action of tTAFs then allows full and robust transcription of the terminal differentiation genes, partly by displacing Polycomb from their promoters (Chen, 2011).

Strikingly, the two major PcG protein complexes appear to be regulated differently by the germ cell developmental program: whereas the PRC2 components E(z) and Su(z)12 are downregulated, the PRC1 components Polycomb, Polyhomeotic and dRing continue to be expressed in spermatocytes. The global downregulation of the epigenetic 'writer' E(z) in spermatocytes might facilitate displacement of the epigenetic 'reader', the PRC1 complex, from the differentiation genes, with the local action of tTAFs at promoters serving to select which genes are relieved of PRC1. In addition, the tTAFs act at a second level to regulate Polycomb by recruiting and accompanying Polycomb and several other PRC1 components to a particular subnucleolar domain in spermatocytes. It is not yet known whether sequestering of PRC1 to the nucleolus by tTAFs plays a role in the activation of terminal differentiation genes, perhaps by lowering the level of PRC1 that is available to exchange back on to differentiation gene promoters. Conversely, recruitment of PRC1 to the nucleolar region might have a separate function, such as in chromatin silencing in the XY body as observed in mammalian spermatocytes (Chen, 2011).

The findings indicate that, upon the switch from spermatogonia to spermatocytes, the terminal differentiation genes go through a poised state, marked by presence of both active Pol II and repressive Polycomb, before the genes are actively transcribed. Stalled Pol II and abortive transcript initiation are emerging as a common feature in stem/progenitor cells. This mechanism may prime genes to rapidly respond to developmental cues or environmental stimuli. Stalled Pol II could represent transcription events that have initiated elongation but then pause and await further signals, as in the regulation of gene expression by the androgen receptor or by heat shock. Alternatively, Pol II might be trapped at a nascent preinitiation complex, without melting open the DNA, as found in some instances of transcriptional repression by Polycomb. Although ChIP analyses did not have the resolution to distinguish whether Pol II was stalled at the promoter or had already initiated a short transcript, the results with antibodies specific for unphosphorylated Pol II suggest that Pol II is trapped in a nascent preinitiation complex. The PRC1 component dRing has been shown to monoubiquitylate histone H2A on Lys119 near or just downstream of the transcription start site. It is proposed that in early spermatocytes, before expression of the tTAFs and tMAC, the local action of PRC1 in causing H2AK119ub at the terminal differentiation gene promoters might block efficient clearing of Pol II from the preinitiation complex and prevent transcription elongation (Chen, 2011).

Removal of PRC1 from the promoter and full expression of the terminal differentiation genes in spermatocytes require the expression and action of tMAC and tTAFs. Cell type-specific homologs of TFIID subunits have been shown to act gene-selectively to control developmentally programmed gene expression. For example, incorporation of one subunit of the mammalian TAF4b variant into TFIID strongly influences transcriptional activation at selected promoters, directing a generally expressed transcriptional activator to turn on tissue-specific gene expression (Chen, 2011).

The local action of the tTAFs to relieve repression by Polycomb at target gene promoters provides a mechanism that is both cell type specific and gene selective, allowing expression of some Polycomb-repressed genes while keeping others silent. Similar developmentally programmed mechanisms may also reverse PcG-mediated epigenetic silencing in other stem cell systems. Indeed, striking parallels between the current findings and recent results from mammalian epidermis suggest that molecular strategies are conserved from flies to mammals. In mouse epidermis, the mammalian E(z) homolog Ezh2 is expressed in stem/precursor cells at the basal layer of the skin. Strikingly, as was observed for E(z) and Su(z)12 in the Drosophila male GSC lineage, the Ezh2 level declines sharply as cells cease DNA replication and the epidermal differentiation program is turned on. Overexpression of Ezh2 in epidermal precursor cells delays the onset of terminal differentiation gene expression, and removal of the Ezh2-generated H3K27me3 mark by the Jmjd3 (Kdm6b) demethylase is required for epidermal differentiation (Chen, 2011 and references therein).

In particular, the results suggest a possible explanation for the conundrum that, although PcG components are bound at many transcriptionally silent differentiation genes in mammalian ESCs, loss of function of PcG components does not cause loss of pluripotency but instead causes defects during early embryonic differentiation. In Drosophila male germ cells, events during the switch from precursor cell proliferation to differentiation are required to recruit Pol II to the promoters of differentiation genes. Without this differentiation-dependent recruitment of Pol II, loss of Polycomb is not sufficient to precociously turn on terminal differentiation genes in precursor cells. Rather, Polycomb that is pre-bound at the differentiation gene promoters might serve to delay the onset of their transcription after the mitosis-to-differentiation switch. Robust transcription must await the expression of cell type- and stage-specific components of the transcription machinery. These might in turn guide gene-selective reversal of Polycomb repression to facilitate appropriate differentiation gene expression in specific cell types (Chen, 2011).

Replacement of histones by protamines and Mst77F during chromatin condensation in late spermatids and role of Sesame in the removal of these proteins from the male pronucleus

Chromatin condensation is a typical feature of sperm cells. During mammalian spermiogenesis, histones are first replaced by transition proteins and then by protamines, while little of this process is known for Drosophila. This study characterizes three genes in the fly genome, Mst35Ba, Mst35Bb, and Mst77F. The results indicate that Mst35Ba and Mst35Bb encode dProtA and dProtB, respectively. These are considerably larger than mammalian protamines, but, as in mammals, both protamines contain typical cysteine/arginine clusters. Mst77F encodes a linker histone-like protein showing significant similarity to mammalian HILS1 protein. ProtamineA-enhanced green fluorescent protein (eGFP), ProtamineB-eGFP, and Mst77F-eGFP carrying Drosophila lines show that these proteins become the important chromosomal protein components of elongating spermatids, and His2AvDGFP vanishes. Mst77F mutants [ms(3)nc3] are characterized by small round nuclei and are sterile as males. These data suggest the major features of chromatin condensation in Drosophila spermatogenesis correspond to those in mammals. During early fertilization steps, the paternal pronucleus still contains protamines and Mst77F but regains a nucleosomal conformation before zygote formation. In eggs laid by sesame-deficient females, the paternal pronucleus remains in a protamine-based chromatin status but Mst77F-eGFP is removed, suggesting that the sesame gene product is essential for removal of protamines while Mst77F removal is independent of Sesame (Raja, 2005).

For mammals, the somatic set of histones are modified, as these are in part replaced by specific variants during meiotic prophase. After meiosis, histones are replaced by major transition proteins TP1 and TP2 and subsequently by highly basic protamines to ensure the remodeling of chromatin to a typically highly condensed and transcriptionally silent state of mature sperm. These replacements leads to a shift from histone-based nucleosomal conformation to a radically different conformation, resembling stacked doughnut structures containing protamines as major chromatin condensing proteins and DNA. Some mammals have only one protamine gene, while mice and humans have two genes encoding two different protamines, both of which are essential for fertility and are haploinsufficient. HILS1 (spermatid-specific linker histone H1-like protein) has been proposed to participate in chromatin remodeling in mouse and human spermiogenesis. The transition between histone removal and its replacement by protamines in mice and humans is characterized by small 6- to 10-kDa transition proteins acting as a short-term chromosomal proteins. In mice, the transition proteins TP1 and TP2 are redundant in function. In fishes and birds, transition proteins are missing and protamines directly reorganize the chromatin. In annelids and echinoderms, the nucleosomal configuration is maintained in sperms, while protamine-like proteins have been described for mussels. These protamine-like proteins lack the typical high cysteine content necessary for disulfide bridges. Therefore, a doughnut-type chromatin structure as in mammals is unlikely to occur in mussels. It has been proposed that the protamine-like proteins in mussels belong to the histone H1 family. The sperm chromatin of mussels contain core histones and thus a nucleosomal configuration, but histone H1 is replaced by protamine-like molecules which organize the higher order structure of the chromatin (Raja, 2005).

For Drosophila melanogaster, chromatin reorganization after meiosis has not been studied at the molecular level. At the light microscopic level, the Drosophila spermatid nucleus is initially round after meiosis and then is shaped to a thin needle-like structure with highly condensed chromatin, so that the volume of the nucleus is condensed over 200-fold. In mammals, the volume of the nucleus is reduced over 20-fold. In the mature sperms of Drosophila, core histones are not detectable by immunohistology. There is histochemical evidence for the presence of very basic proteins in sperm, but it still remains an open question whether histones are replaced by protamine-like basic proteins in Drosophila. The analysis of the Drosophila genome sequence revealed that the proteins encoded by two genes show similarity to mammalian protamines for which the male-specific transcripts Mst35Ba and Mst35Bb have been found and have been proposed to encode protamine-like proteins. Another male specifically transcribed gene, Mst77F, is a distant relative of the histone H1/H5 (linker histone) family and has been proposed to play a role either as a transition protein or as a replacement protein for compaction of the Drosophila sperm chromatin. With enhanced green fluorescent protein (eGFP) fusion for these abovementioned proteins, this study shows that Mst35Ba and Mst35Bb indeed encode protamines and Mst77F encodes a linker histone-like protein. The expression pattern of Mst77F overlaps the pattern of protamines as a chromatin component. Furthermore, during fertilization, the removal of protamines from the male pronucleus requires the function of the maternal component, Sesame, but not for the removal of Mst77F. It has been shown that sesame mutants cause impairment of the entry of histones into the male pronucleus (Raja, 2005).

Mst35Ba and Mst35Bb are present at cytological position 35B6 and 35B6-7, respectively, on the chromosome arm 2L. These two genes are arranged in tandem, and both consist of three exons. The 5'UTR, coding region, and the 3'UTR of these genes are highly identical; they probably arose from a recent gene duplication. The encoded protamines show over 94% identity to each other (Raja, 2005).

A remarkable feature of protamines is their ability to form intermolecular disulfide bridges, which is reflected by the conserved cysteine residues within mammalian protamines. The dProtA and dProtB are of 146 amino acids (aa) and 144 aa, respectively, and thus longer than even the human and mouse Protamine-2, which are 102 aa and 107 aa, respectively. Both Drosophila protamines contain 10 cysteines each and show significant similarity, particularly with respect to a high cysteine, lysine, and arginine content to mammalian protamines. Human and mouse Protamine-1 aligns to the N-terminal half of the Drosophila protamines (from aa positions 27 to 82), and four cysteine residues are conserved and regularly spaced. In contrast, Protamine-2 of human and mouse shows relatively high similarity to the C-terminal half of the Drosophila protamines, with four cysteines in this region that are conserved and regularly spaced, whereas one cysteine is shared with the mouse and human Protamine-1 (Raja, 2005).

Mst77F is present at the cytological position 77F on the chromosome arm 3L and lies within the large intron of PKA-R1. Mst77F is also male specifically transcribed, and the encoded protein has been proposed to be a linker histone H1/H5 type, which could also play the role of a transition protein or a protamine. The Mst77F protein shares a significant similarity to the HILS1 protein of mouse and human HILS1, where the percentages of cysteine, lysine, and arginine are similar to that of mHILS1 and hHILS1. HILS1 protein has been recently described as a component of the mammalian sperm nucleus. Drosophila Mst77F encodes a protein of 215 aa with a molecular mass of 24.5 kDa and with a pI of 9.86. mHILS1 is of 170 aa and shows 39% similarity to Mst77F. Mst77F contains 10 cystine residues as in Drosophila protamines, and mHILS1 contains eight cystine residues, of which four residues are conserved (Raja, 2005).

As there are considerable differences between the mammalian protamines as well as between the mammalian HILS1 proteins and the presumptive Drosophila homologue Mst77F, additional experiments are essential to clarify if these proteins are indeed involved in the condensation of sperm chromatin (Raja, 2005).

Drosophila protamine mRNAs are transcribed at the primary spermatocyte stage, whereas in mammals protamine mRNAs are synthesized at the round spermatid stage and translationally repressed until the elongated stage, which is mediated by 3'UTR. The Drosophila ProtamineA-eGFP and ProtamineB-eGFP constructs do not contain the 3'UTR of the respective protamine genes. Nevertheless, the transgenic flies carrying these constructs still show repression of translation. So, in Drosophila, the region responsible for the translational repression is most likely in the 5'UTR. Deletion constructs of Mst35Bb and Mst77F 5'UTRs fused to the reporter lacZ show that the translation repression element is indeed present in the 5'UTR. This holds true also for the mRNA of the Mst77F-eGFP fusion gene, as is the case for all mRNAs investigated concerning translational repression so far in male germ lines of Drosophila. In contrast to mammalian spermatogenesis, in Drosophila transcription ceases already with the entry into meiotic divisions. Since the protamines are made in the elongated spermatids, the transcriptional silencing in Drosophila spermatogenesis seems to be independent of protamines (Raja, 2005).

When primary amino acid sequences of Drosophila protamines are compared to mammalian protamines, it is quite evident that Drosophila protamines are relatively large. dProtA and dProtB are over 94% identical to each other. This could explain that both the protamines may be functionally redundant. Human and mouse Protamine-1 aligns with the N terminus of both Drosophila protamines, and Protamine-2 aligns more to the C terminus. It is possible that the Drosophila protamines undergo posttranslational cleavage at the N terminus, as is known for mammals. The cytoplasmic eGFP fused at the C terminus shows clear nuclear localization, indicating that the tagged protamine is functionally intact. Drosophila protamines each contain 10 cysteine residues at identical positions, while over 4 of 10 cysteines at the N terminus and the C terminus are conserved with human and mouse Protamine-1 and Protamine-2, respectively. With nine cysteines, the content is highest in Protamine-1 of mice. Inter- or intra-disulfide bridges can be formed between the cysteine-rich protamines to condense the DNA. For mice it is shown that mutation in protamine-1 or protamine-2 is haploinsufficient and causes male sterility. A haploid situation was analyzed for the Mst35Ba and Mst35Bb genes with the deficiency Df(2L)Exel8033/+; these flies are fertile males and show normal spermatogenesis. The large amount of identity that both dProtA and dProtB exhibit can contribute to the functional redundancy (Raja, 2005).

Chromatin reorganization is an essential feature during spermiogenesis. The functional significance of chromatin compaction during spermiogenesis is still unknown. The main explanation seems to be that compaction of the sperm nucleus is an essential factor for its mobility as well as for the penetration of sperm into the egg and genomic stability. In mammals, somatic histones are in part replaced by spermatid-specific variants during meiotic prophase, later by major transition proteins TP1 and TP2, and subsequently by highly basic protamines to ensure the remodeling of chromatin to a typically highly condensed and transcriptionally silent state of mature sperm. These replacements lead to a shift from histone-based nucleosomal conformation to a radically different conformation, resembling stacked doughnut structures containing major chromatin condensing proteins and DNA in the nucleus (Raja, 2005).

In Drosophila, so far no proteins have been identified that are involved in the packaging of the genome in the mature sperm nucleus. One observation, that Histone3.3 variant and the somatic H3 isoform in Drosophila are vanishing at the time of chromatin condensation, supports the view of histone displacement, but it was still a question of whether it is the real absence of histones at this stage in Drosophila or whether the antibodies are not accessible to the mature sperm due to the tight packaging of the chromatin. To circumvent this problem, the GFP fusion approach was chosen, use was made of the existing His2AvDGFP, and Protamine-eGFP and Mst77F-eGFP fusion transgenic flies were generated in order to analyze the situation in Drosophila. The results clearly show that histone His2AvD is lost from the spermatid nuclei at the time of appearance of protamines and Mst77F during later stages of spermatid differentiation. The exact molecular mechanisms underlying the histone displacement, degradation, and incorporation of protamines onto the chromatin are poorly understood. For mammals, evidence has been obtained that histone H2A is ubiquitinated in mouse spermatids around the developmental time period when histones are removed from the chromatin. The mammalian HR6B ubiquitin-conjugating enzyme is the homologue of yeast RAD6, and both can ubiquitinate histones in vitro. Thus far, the mechanism of histone displacement and protamine incorporation is unknown during spermiogenesis in Drosophila. In flies as well as in mammals, many questions remain unanswered that need to be addressed about these underlying mechanisms of chromatin remodeling during spermiogenesis (Raja, 2005).

In mammals, transition proteins act as intermediates in the histone-to-protamine transition. In mice, the onset of HILS1 and transition proteins TP1 and TP2 (major forms) overlaps with the pattern of Protamine-1 and later with Protamine-2 but HILS1 and the transition proteins are no longer present in the mature sperm. Mice lacking both TP1 and TP2 show normal transcriptional repression, histone displacement, nuclear shaping, and protamine deposition but show the loss of genomic integrity with large numbers of DNA breaks leading to male sterility. In Drosophila, histones are displaced with synchronous accumulation of protamines and Mst77F. Mst77F, a distant relative of the histone H1/H5 (linker histone) family, has been proposed to play a role either as a transition protein or as a protamine for compaction of the Drosophila sperm chromatin. Mst77F shows highest similarity to HILS1 with respect to the cysteines and basic amino acid content but not to mouse TP1, TP2, or H1t. Moreover, the results show that the pattern of expression of Mst77F in the nucleus is similar to that of mHILS1 in the nucleus, with the exception that Mst77F is also transiently detected in the flagella and persists in mature sperm nuclei, unlike mHILS1. In mammalian mature sperm nuclei, it is only the protamines that are the chromatin condensing proteins which persist. This again raises the question of whether Mst77F could also play the role of protamines. However, one additional copy of dProtB (dProtA and dProtB showing 94% identity may be functionally redundant) does not rescue the ms(3)nc3 phenotype, indicating that the role of Mst77F may be completely or partially different from that of protamines in the nucleus. However, a null mutation for Mst77F is required to answer this question with respect to chromatin condensation. In ms(3)nc3 mutants, the chromatin condensation with the native protamines continues to take place. When a closer look was taken at the deposition of ProtamineB-eGFP in ms(3)nc3/Df(3L)ri-79c trans-heterozygotes, it revealed that the condensed chromatin in the tid-shaped nuclei is concentrated at the two opposite ends, with a lightly stained chromatin spaced in the center. So the chromatin condensation takes place but may not be complete with the incorporation of the mutant Mst77F protein. The large amount of chromatin compaction or condensation seen in Drosophila mature sperm when compared to that of mouse and human sperm possibly could be the result of persistence of Mst77F in the mature sperm nuclei. It remains to be clarified whether the sperm nucleus contains further protamines that have not yet been properly annotated (Raja, 2005).

ms(3)nc3 is a second-site noncomplementation (nc) mutation that was isolated in an ethylmethanesulfonate screen to identify interacting proteins involved in microtubule function in Drosophila. This study shows that ms(3)nc3 is a single missense mutation from a T>A transition, causing the substitution of threonine instead of serine at aa position 149. Mst77F shows a pattern of expression similar to protamines in the nucleus and was also seen in the flagella until the individualization stage. Since ms(3)nc3 fails to complement class I alleles at the ß2 tubulin locus, it is possible that Mst77F has a dual role to play as a chromatin condensing protein in the nucleus and for the normal nuclear shaping. Nuclear shaping is a microtubule-based event. ms(3)nc3 leads to a tid-shaped nuclear phenotype, where the nucleus fails to shape into a needle-like nuclei. Similar defective nuclear shaping is seen with the few homozygous and heteroallelic combinations of class I alleles of ß2 tubulin. The incorporation of the defective subunit encoded by ms(3)nc3 may interfere with the function of the resulting complex. These data suggest the involvement of an Mst77F (a linker histone variant) in the microtubule dynamics during the nuclear shaping. This again complements the role of sea urchin histone H1 in the stabilization of flagellar microtubules (Raja, 2005).

After the first steps in the fertilization process, the male gamete is still in the highly compact protamine-based chromatin structure. In a wild-type egg, the paternal pronucleus changes the shape from the needle-like to a spherical structure. Furthermore, the male pronucleus acquires a nucleosome-based structure before zygote formation and thus is transformed into a replication-competent male pronucleus. sesame is a maternal effect mutation in HIRA and had been mapped to 7C1. HIRA family of genes (named after yeast HIR genes; HIR is an acronym for 'histone regulator') includes the yeast HIR1 and HIR2 repressors of histone gene transcription in S. cerevisiae, human TUPLE-1/HIRA, chicken HIRA, and mouse HIRA. In Drosophila, HIRA is expressed in the female germ line and a high level of HIRA mRNA is deposited in the egg. Human HIRA is shown to bind to histone H2B and H4. The WD repeats present at the N-terminal part of HIRA could probably function as a part of a multiprotein complex. Xenopus HIRA proteins are also known in promoting chromatin assembly that is independent of DNA synthesis in vitro. The corresponding maternal effect mutant sesame, in which the sperm fertilizes the egg but no zygote is formed, has been analyzed. Although the shape change of the nucleus to the spherical structure occurs in these mutants, maternal histones are not incorporated into the male pronucleus, which strengthens the function of HIRA in binding to the core histones. This study shows that neither Drosophila protamine is removed from the male pronucleus in sesame mutants. This leads to the proposal that the transport and incorporation of histones onto the chromatin in some manner is coupled to the removal of protamines in which HIRA could play an important role in the multiprotein complex required in this chromatin reconstitution process. Mst77F removal from the male pronucleus in contrast to protamines is independent of HIRA (Raja, 2005). During spermiogenesis, chromatin reorganization of the complete genome is an essential feature for male fertility. This process leads to an extremely condensed state of the haploid genome in the sperm and requires a reorganization of the paternal genome in the male pronucleus during fertilization and before zygote formation. With the characterization of the chromatin condensing proteins in Drosophila, it would be possible to gain more insight into the mechanisms of sperm chromatin reorganization during spermiogenesis and fertilization (Raja, 2005).

Nucleosomal chromatin in the mature sperm of Drosophila melanogaster

During spermiogenesis in mammals and many other vertebrate classes, histone-containing nucleosomes are replaced by protamine toroids, which can repackage chromatin at a 10 to 20-fold higher density than in a typical somatic nucleus. However, recent evidence suggests that sperm of many species, including human and mouse retain a small compartment of nucleosomal chromatin, particularly near genes important for embryogenesis. As in mammals, spermiogenesis in the fruit fly, Drosophila melanogaster has also been shown to undergo a programmed substitution of nucleosomes with protamine-like proteins. Using chromatin immunoprecipitation (ChIP) and whole-genome tiling array hybridization (ChIP-chip), supported by immunocytochemical evidence, this stuy shows that in a manner analogous to nucleosomal chromatin retention in mammalian spermatozoa, distinct domains packaged by the canonical histones H2A, H2B, H3 and H4 are present in the fly sperm nucleus. Evidence was also found for the retention of nucleosomes with specific histone H3 trimethylation marks characteristic of chromatin repression (H3K9me3, H3K27me3) and active transcription (Elnfati, 2016).

Tissue-specific TAFs counteract Polycomb to turn on terminal differentiation

Polycomb transcriptional silencing machinery is implicated in the maintenance of precursor fates, but how this repression is reversed to allow cell differentiation is unknown. Testis-specific TAF (TBP-associated factor) homologs required for terminal differentiation of male germ cells may activate target gene expression in part by counteracting repression by Polycomb. Chromatin immunoprecipitation revealed that testis TAFs bind to target promoters, reduce Polycomb binding, and promote local accumulation of H3K4me3, a mark of Trithorax action. Testis TAFs also promoted relocalization of Polycomb Repression Complex 1 components to the nucleolus in spermatocytes, implicating subnuclear architecture in the regulation of terminal differentiation (Chen, 2005).

Male germ cells differentiate from adult stem cell precursors, first proliferating as spermatogonia, then converting to spermatocytes, which initiate a dramatic, cell type–specific transcription program. In Drosophila, five testis-specific TAF homologs (tTAFs) encoded by the can, sa, mia, nht, and rye genes are required for meiotic cell cycle progression and normal levels of expression in spermatocytes of target genes involved in postmeiotic spermatid differentiation. Requirement for the tTAFs is gene selective: Many genes are transcribed normally in tTAF mutant spermatocytes. Tissue-specific TAFs have also been implicated in gametogenesis and differentiation of specific cell types in mammals. In addition to action with TBP (TATA box–binding protein) in TFIID, certain TAFs associate with HAT (histone acetyltransferase) or Polycomb group (PcG) transcriptional regulatory complexes. To elucidate how tissue-specific TAFs can regulate gene-selective transcription programs during development, the mechanism of action of the Drosophila tTAFs was investigated in vivo (Chen, 2005).

The tTAF proteins were concentrated in a particular subcompartment of the nucleolus in primary spermatocytes. Expression of a functional green fluorescence protein (GFP)–tagged genomic sa rescuing transgene revealed that expression of Sa-GFP turns on specifically in male germ cells soon after initiation of spermatocyte differentiation and persists throughout the remainder of the primary spermatocyte stage, disappearing as cells entered the first meiotic division. Some Sa-GFP was detected associated with condensing chromatin. However, most Sa-GFP localized to the nucleolus, in a pattern complementary with Fibrillarin, which marks a fibrillar nucleolar subcompartment. Staining with antibodies against endogenous Sa, Can, Nht, or Mia proteins showed similar temporal expression and nucleolar localization in primary spermatocytes, consistent with collaborative function of the tTAFs. In contrast, the generally expressed sa homolog TAF8 and its binding partner TAF10b are excluded from the nucleolus (Chen, 2005).

Several components of the Polycomb Repression Complex 1 (PRC1) transcriptional regulator appear in the nucleolus in spermatocytes, coincident with tTAF expression and dependent on tTAF function. Polycomb (Pc) protein expresses from a Pc-GFP genomic transgene localized on chromatin, but in addition becomes concentrated in the nucleolus in primary spermatocytes. Both Pc-GFP and staining of endogenous protein with antibody against Pc (anti-Pc) revealed localization to the same nucleolar subcompartment as the one containing tTAFs. Recruitment of Pc to the nucleolus exactly coincides with onset of expression of the tTAFs after early G2 phase in spermatocytes. Relocalization of Pc depends on wild-type tTAF activity: Pc localizes to chromatin but is not concentrated in the nucleolus in tTAF mutant spermatocytes. Two other components of the PRC1 core complex, Polyhomeotic (Ph) and Drosophila Ring protein (dRing), also become concentrated in the nucleolus in primary spermatocytes dependent on tTAF function. Failure of PRC1 components to localize to the nucleolus in tTAF mutants is not caused by nucleolar loss because Fibrillarin staining appears normal in the mutants. H3K27me3 laid down by action of the PRC2 complex acts as a docking site for the Pc chromodomain to recruit PRC1 and block transcription initiation. H3K27me3 localizes on chromatin in spermatocytes, along with Pc. However, no H3K27me3 was detected in the nucleolus in spermatocytes, suggesting that PRC1 components may be recruited to the nucleolus by a different mechanism independent of chromatin (Chen, 2005).

The tTAFs are required for activation of robust transcription of several spermatid differentiation genes, whereas the PcG proteins are known to mediate transcriptional repression. Chromatin immunoprecipitation (ChIP) suggested that the tTAFs might allow robust transcription of spermatid differentiation genes in part by counteracting repression by Pc, perhaps causing dissociation of PRC1 from cis-acting control sequences at target genes (Chen, 2005).

ChIP from wild-type testes using anti-Sa revealed enrichment of tTAF binding at three different known target genes (fzo, Mst87F, and dj), compared with binding at intergenic regions 10 to 20 kb away or at a tTAF-independent gene expressed in the same cell type (cyclin A or sa itself), suggesting that the tTAFs are in occupancy at target genes. Real-time polymerase chain reaction (PCR) analysis revealed ~10-fold enrichment of Sa at a target (mst87F) compared with a non-target gene (sa) (Chen, 2005).

ChIP analysis also revealed that Pc protein binds to tTAF-dependent target genes in tTAF mutant testes, and that wild-type function of the tTAFs reduce Pc binding. ChIP with anti-Pc from can mutant testes preferentially precipitates the three tTAF target promoters, compared with intergenic regions or promoters from two different nontarget controls. Quantification by real-time PCR showed more than 50-fold enrichment of Pc at the target gene mst87F compared with the tTAF-independent control sa. In contrast, relative occupancy of Pc at the tTAF targets was not significantly different from that at the non-targets in wild-type testes (Chen, 2005).

The tTAFs may act near the promoter of target genes (fzo) to allow expression by directly or indirectly reducing nearby binding of PRC1. ChIP using primer pairs across the promoter region of fzo revealed that the tTAF enrich most strongly for sequences just upstream of the transcription start site. In contrast, Pc-containing protein complexes (in tTAF mutant testes) enrich for a broader distribution, including sequences near and downstream of the transcription start site, consistent with localization of Pc at Ultrabithorax (Ubx) locus in wing discs and on the hsp26 promoter in vivo (Chen, 2005).

Binding of the tTAFs at target promoters may allow expression through recruitment or activation of the Trithorax group (TrxG) transcriptional activation complex, which often acts in opposition to repression by PcG proteins. Trx, like its mammalian homolog MLL, creates an H3K4me3 epigenetic mark. ChIP from wild-type testes revealed H3K4me3 at or near the promoter regions of the three tTAF targets tested, as well as at nontargets. Analysis using primer pairs across the tTAF target fzo region revealed that H3K4me3 associated most strongly with sequences spanning the promoter. In contrast, ChIP with anti-H3K4me3 from can mutant testes did not enrich for the tTAF target promoters. Quantitative PCR revealed 36-fold enrichment of the promoter region of the tTAF-dependent mst87F gene by ChIP for H3K4me3 in wild-type compared with can mutant testes (Chen, 2005).

Consistent with the presence of H3K4me3 at target promoters in wild-type testes, trx function appears to be required for continued expression of two different kinds of tTAF-dependent targets. Boule triggers the G2/M transition in meiosis I by allowing translation of twine and requires tTAFs for protein accumulation, setting up a cross-regulatory mechanism so that meiotic cell cycle progression awaits expression of terminal differentiation genes. When temperature-sensitive trx1 flies grown at permissive temperature were shifted to nonpermissive temperature as adults, the Boule protein level in mutant testes substantially decreased over time at nonpermissive temperature compared with the level in wild-type flies shifted in parallel or trx1 flies held at permissive temperature. Likewise, analysis of mRNA levels by semiquantitative PCR revealed a ~40% decrease in transcript level for the tTAF target gene fzo, but not for the tTAF-independent gene cyclin A, in testes from trx1 mutant flies shifted to non-permissive temperature compared with the level in testes from similarly treated wild-type flies (Chen, 2005).

In summary, occupancy of tTAFs and Pc at target promoters appears to be mutually exclusive in wild-type and tTAF mutant spermatocytes, suggesting that the tTAFs may turn on target gene expression by counteracting repression by Polycomb, either directly or indirectly reducing Pc binding and allowing local action of Trx. Loss of function of Pc in marked clones of homozygous mutant cells does not restore terminal differentiation in a tTAF mutant background, suggesting that in addition to counteracting repression by Pc, tTAFs may also be required at the promoter region independent of Pc, possibly to recruit Trx or other cofactors for transcription activation. Transcriptional derepression by sequestration of PcG proteins has been observed during HIV-1 infection, when the viral Nef protein recruits the PRC2 component Eed to the plasma membrane. Likewise, the tTAFs may sequester Pc to the nucleolus. The tTAFs Nht, Can, and Mia are homologs of the generally expressed TAF4, TAF5, and TAF6, which are found as stoichiometric components of the PRC1 complex purified from fly embryos, raising the possibility that the tTAFs might associate with a population of Pc-, Ph-, and dRing-containing complexes in the nucleolus. If so, interactions in the nucleolus are likely to differ from interactions at the promoters of target genes, because the ChIP results indicate immunoprecipitation of tTAFs without Pc (Chen, 2005).

The PcG and TrxG proteins act to maintain cell fates set during embryogenesis throughout development. Emerging evidence indicates that PcG and TrxG complexes also play critical roles in decisions between proliferating precursor cell fates and terminal differentiation, for example, in the blood cell lineages. In particular, the mammalian PcG protein Bmi-1 promotes proliferation and blocks differentiation of normal and leukemic stem cells, and is required for establishment or maintenance of adult hematopoietic stem cells in mouse. Transcriptional silencing by PcG action may allow self-renewal and continued proliferation of precursor cells by blocking expression of terminal differentiation genes. This repression must be reversed to allow production of terminally differentiated cells, whereas failure may allow overproliferation of precursors and eventually cancer. Although central for both normal development and understanding the genesis of cancer, little is known about the mechanisms that reverse such epigenetic silencing to allow expression of the terminal differentiation program. These findings in the male germ line provide an example of how cell type– and stage–specific transcriptional regulatory machinery, turned on as part of the developmental program, might allow onset of terminal differentiation by counteracting repression by the PcG and highlight the importance of subnuclear localization in regulation of transcriptional regulation (Chen, 2005).

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

Stem cells can self-renew and generate differentiating daughter cells. It is not known whether these cells maintain their epigenetic information during asymmetric division. Using a dual-color method to differentially label 'old' versus 'new' histones in Drosophila male germline stem cells (GSCs), it was shown that preexisting canonical H3, but not variant H3.3, histones are selectively segregated to the GSC, whereas newly synthesized histones incorporated during DNA replication are enriched in the differentiating daughter cell (see Experimental design and potential results). The asymmetric histone distribution occurs in GSCs but not in symmetrically dividing progenitor cells. Furthermore, if GSCs are genetically manipulated to divide symmetrically, this asymmetric mode is lost. This work suggests that stem cells retain preexisting canonical histones during asymmetric cell divisions, probably as a mechanism to maintain their unique molecular properties (Tran, 2013).

Although all cells in an organism contain the same genetic material, different genes are expressed in specific cell types, allowing them to differentiate along distinct pathways. Epigenetic mechanisms regulate gene expression and maintain a specific cell fate through many cell divisions. Stem cells have the remarkable ability to both self-renew and generate daughter cells that enter differentiation. Epigenetic mechanisms have been reported to regulate stem cell activity in multiple lineages. However, there has been little direct in vivo evidence demonstrating whether stem cells retain their epigenetic information (Tran, 2013).

The Drosophila male GSCs are well characterized in terms of their physiological location, microenvironment (i.e., niche), and cellular structures). Male GSCs can be identified precisely by their distinct anatomical positions and morphological features. A GSC usually divides asymmetrically to produce a self-renewed GSC and a daughter cell gonialblast (GB) that undergoes differentiation. Therefore, GSCs can be examined at single-cell resolution for a direct comparison (Tran, 2013).

In eukaryotes, the basic unit of chromatin called nucleosome contains histone octamer [2×(H3, H4, H2A, H2B)] and DNA wrapping around them. Indeed, histones are one of the major carriers of epigenetic information. To address how histones are distributed during the GSC asymmetric division, a switchable dual-color method was developed to differentially label 'old' versus 'new' histones that uses both spatial (by Gal4; UAS system) and temporal (by heat shock induction) controls to switch labeled histones from green [green fluorescent protein (GFP)] to red [monomeric Kusabira-Orange (mKO)]. Heat shock treatment induces an irreversible DNA recombination to shut down expression of GFP-labeled old histones and initiate expression of mKO-labeled new histones. If the old histones are partitioned nonselectively, the GFP will initially exhibit equal distribution in the GSC and GB, and will be gradually replaced by the mKO. However, if the old histones are preferentially retained in the GSCs to constitute potentially GSC-specific chromatin structure, the GFP will be detected specifically in the GSCs. During DNA replication-dependent canonical histone deposition, histones H3 and H4 are incorporated as a tetramer, and histones H2A and H2B are incorporated as dimers. Therefore, independent transgenic strains were generated for H3 and H2B, respectively. On the other hand, histone variants are incorporated into chromatin in a transcription-coupled but DNA replication-independent manner. Therefore, the histone variant H3.3 was used as a control for canonical histones (Tran, 2013).

To avoid potential complications caused by heat shock-induced DNA recombination on either one or both chromosomes in GSCs, each of the three transgenes (H3, H2B, and H3.3) was integrated as a single copy and analyzed in heterozygous flies. Examination of testes with the transgenes revealed nuclear GFP but little mKO signal before heat shock. After heat shock, mKO signals were detectable. Different GSCs undergo mitosis asynchronously, and an average cell cycle length of GSCs is approximately 12 to 16 hours. Among all GSCs, 75% to 77% are in G2 phase, 21% are in S phase, fewer than 2% are in mitosis, and G1-phase GSCs are almost negligible. Moreover, the GSC and GB arising from an asymmetric division remain connected after mitosis by a cellular structure known as the spectrosome, when they undergo the next G1 and S phases synchronously (Tran, 2013).

To examine the distribution of old versus new histones in GSC and GB after a round of DNA replication-dependent histone deposition, testes were studied 16 to 20 hours after heat shock. In particular, GSC-GB pairs connected by spectrosomes were examined. On the basis of cell cycle length of GSCs, these GSC-GB pairs were from GSCs that switched from histone-GFP to histone-mKO genetic code during their G2 phase and then underwent the first mitosis followed by G1, S, and G2 phase and the second mitosis. Within this time frame, both old histones and new histones were detectable in GSCs at the second G2 phase because new histones had been synthesized and incorporated during the first S phase. For histone H3, the GFP signal was detected primarily in the GSC but not in the GB. By contrast, the mKO signals were present in both the GSC and the GB, with a relatively higher level in the GB. The asymmetric distribution of histone H3 was specific for GSC divisions, because both the GFP and mKO signals were equally distributed in spermatogonial cells derived from a symmetric division of the GB in the same testis samples. Quantification of fluorescence intensity revealed that the old H3 (GFP-labeled) signal was more enriched in the GSC than in the GB by a factor of ~5.7, whereas new H3 (mKO-labeled) signal was more enriched in the GB than in the GSC by a factor of ~1.6. By contrast, this differential distribution of old versus new histone was not detected for symmetrically dividing spermatogonial cells (Tran, 2013).

In contrast to the asymmetric distribution pattern for the canonical histone H3, the histone variant H3.3 did not show this asymmetry during GSC divisions, by fluorescence images and by quantification. The symmetry of the histone variant H3.3 suggests that the asymmetric mode is specific for canonical histone H3 (Tran, 2013).

Fewer than 2% of all GSCs are undergoing mitosis; thus, all analyses above were based on postmitotic GSC-GB pairs. To further examine the histone segregation pattern during mitosis, a screen was carried out for mitotic GSCs. Indeed, old histones were mainly associated with the chromatids segregated to the GSC side at metaphase, anaphase, and telophase. By contrast, new histones were more enriched at the chromatids segregated to GB side). These results suggest that the sister chromatids preloaded with old histones are preferentially retained in GSCs and that the ones enriched with new histones are partitioned to GBs during GSC mitosis (Tran, 2013).

Next, the histone distribution pattern was examined during the first GSC division by recovering GSCs for 4 to 6 hours after heat shock. An asymmetric distribution pattern was also found in the GSC-GB pairs with the H3 transgene. By contrast, a symmetric distribution pattern was observed for both dividing spermatogonial cells with the H3 transgene and H3.3 during GSC division. Quantification of fluorescence intensity revealed that the old H3-GFP signal was enriched in the GSC by a factor of ~13 relative to the GB, whereas the new H3-mKO signal was enriched in the GB by a factor of ~2.4 relative to the GSC. By contrast, there was no differential distribution of the old versus new histone for the symmetrically dividing spermatogonial cells, or H3.3 during GSC division. Although an asymmetric histone distribution pattern was detected in postmitotic GSC-GB pairs, examination of the mitotic GSC at this stage did not show any asymmetry. These data suggest that the asymmetric segregation mode relies on replication-dependent histone incorporation prior to mitosis. However, the factor of >10 difference of GFP signal between GSC and GB could be contributed by faster turnover of old histones in GBs, probably as a mechanism to reset the chromatin for differentiation. By contrast, the difference of mKO in GSC and GB was less substantial, probably as a result of new histone synthesis in both cells. Furthermore, the H2B transgene showed a similar pattern to H3 after the first GSC division (Tran, 2013).

The consistent asymmetric cell divisions of GSCs could be lost under certain conditions, such as ectopic activation of the key JAK-STAT signaling pathway in the niche. It has been shown that overexpression of the JAK-STAT ligand unpaired (OE-upd) induces overpopulation of GSCs. Consistent with the loss of asymmetry in expanded GSCs, the asymmetric distribution pattern of the histone H3 was not observed in OE-upd testes 16 to 20 hours after heat shock. These results demonstrate that the asymmetric histone distribution pattern is dependent on GSC asymmetric divisions. A two-step process is proposed as the favored explanation: Old and newly synthesized histones are incorporated to different sister chromatids during S phase; then, during mitosis, the sister chromatid preloaded with old histones is preferentially segregated to GSC (Tran, 2013).

These data reveal that stem cells preserve preexisting histones through asymmetric cell divisions. The JAK-STAT signaling pathway required for the asymmetric GSC divisions contributes to the asymmetric histone distribution pattern. This work provides a critical first step toward identifying the detailed molecular mechanisms underlying old histone retention during GSC asymmetric division. These findings in the well-characterized GSC model system will facilitate understanding of how epigenetic information could be maintained by stem cells or reset in their sibling cells that undergo cellular differentiation (Tran, 2013).

Chromatin remodeling in the aging genome of Drosophila

Chromatin structure affects the accessibility of DNA to transcription, repair, and replication. Changes in chromatin structure occur during development, but less is known about changes during aging. This study examined the state of chromatin structure and its effect on gene expression during aging in Drosophila at the whole genome and cellular level using whole-genome tiling microarrays of activation and repressive chromatin marks, whole-genome transcriptional microarrays and single-cell immunohistochemistry. Dramatic reorganization of chromosomal regions was found with age. Mapping of H3K9me3 and HP1 signals to fly chromosomes reveals in young flies the expected high enrichment in the pericentric regions, the 4th chromosome, and islands of facultative heterochromatin dispersed throughout the genome. With age, there is a striking reduction in this enrichment resulting in a nearly equivalent level of H3K9me3 and HP1 in the pericentric regions, the 4th chromosome, facultative heterochromatin, and euchromatin. These extensive changes in repressive chromatin marks are associated with alterations in age-related gene expression. Large-scale changes in repressive marks with age are further substantiated by single-cell immunohistochemistry that shows changes in nuclear distribution of H3K9me3 and HP1 marks with age. Such epigenetic changes are expected to directly or indirectly impinge upon important cellular functions such as gene expression, DNA repair, and DNA replication. The combination of genome-wide approaches such as whole-genome chromatin immunoprecipitation and transcriptional studies in conjunction with single-cell immunohistochemistry as shown in this study provide a first step toward defining how changes in chromatin may contribute to the process of aging in metazoans (Wood, 2010).

Akirin specifies NF-kappaB selectivity of Drosophila innate immune response via chromatin remodeling

The network of NF-kappaB-dependent transcription that activates both pro- and anti-inflammatory genes in mammals is still unclear. As NF-kappaB factors are evolutionarily conserved, Drosophila was used to understand this network. The NF-kappaB transcription factor Relish activates effector gene expression following Gram-negative bacterial immune challenge. This study shows, using a genome-wide approach, that the conserved nuclear protein Akirin is a NF-kappaB co-factor required for the activation of a subset of Relish-dependent genes correlating with the presence of H3K4ac epigenetic marks. A large-scale unbiased proteomic analysis revealed that Akirin orchestrates NF-kappaB transcriptional selectivity through the recruitment of the Osa-containing-SWI/SNF-like Brahma complex (BAP). Immune challenge in Drosophila shows that Akirin is required for the transcription of a subset of effector genes, but dispensable for the transcription of genes that are negative regulators of the innate immune response. Therefore, Akirins act as molecular selectors specifying the choice between subsets of NF-kappaB target genes. The discovery of this mechanism, conserved in mammals, paves the way for the establishment of more specific and less toxic anti-inflammatory drugs targeting pro-inflammatory genes (Bonnay, 2014).

Genomewide identification of target genes of histone methyltransferase dG9a during Drosophila embryogenesis

Post-translational modification of the histone plays important roles in epigenetic regulation of various biological processes. Among the identified histone methyltransferases (HMTases), G9a is a histone H3 Lys 9 (H3K9)-specific example active in euchromatic regions. Drosophila G9a (dG9a) has been reported to feature H3K9 dimethylation activity in vivo. This study shows that the time required for hatching of a homozygous dG9a null mutant and heteroallelic combination of dG9a null mutants is delayed, suggesting that dG9a is at least partially responsible for progression of embryogenesis. Immunocytochemical analyses of the wild-type and the dG9a null mutant flies indicated that dG9a localizes in cytoplasm up to nuclear division cycle 7 where it is likely responsible for di-methylation of nucleosome-free H3K9. From cycles 8-11, dG9a moves into the nucleus and is responsible for di-methylating H3K9 in nucleosomes. RNA-sequence analysis utilizing early wild-type and dG9a mutant embryos showed that dG9a down-regulates expression of genes responsible for embryogenesis. RNA fluorescent in situ hybridization analysis further showed temporal and spatial expression patterns of these mRNAs did not significantly change in the dG9a mutant. These results indicate that dG9a controls transcription levels of some zygotic genes without changing temporal and spatial expression patterns of the transcripts of these genes (Shimaji, 2015).

This study shows that the time required for hatching is extended by the lack of dG9a. Immunocytochemical studies suggested that cytoplasmic dG9a is responsible for di-methylation of nucleosome-free H3K9 in cytoplasm up to nuclear division cycle 7, whereas from cycle 8 to cycle 11, it moves into nucleus and di-methylates H3K9 in the nucleosome. Across these cycles, dG9a could be responsible for regulating the expression level of many genes required for embryogenesis and transcription (Shimaji, 2015).

Human G9a localizes in nuclei of HEK293 cells and is also reported to be present in nuclei in salivary glands and in cultured Drosophila Kc cells. However, this study found that dG9a almost exclusively localizes in cytoplasm of early embryos up to nuclear division cycle 7 and is likely for a determinant of H3K9me2 levels in cytoplasm. To support rapid progression of nuclear division cycles in Drosophila, large amounts of DNA replication enzymes are maternally stored, along with histone mRNAs and histone proteins. Therefore, the available results indicate that dG9a methylates nucleosome-free histone H3K9 stored in cytoplasm during early stages of nuclear division cycles. It should be noted that dG9a can di-methylate histone H3K9 that is free from nucleosomes in vitro (Stabell, 2006). Some of the di-methylated H3K9 may be selectively transported into nuclei from cycle 8, although further analysis is necessary to clarify this point (Shimaji, 2015).

In later stages, dG9a signals rather accumulate in nuclei from nuclear division cycles 8 to 12 and appear to be responsible for di-methylation of nuclear H3K9. Nuclear localization signals that are distinct from those of mammalian G9a in their positions and amino acid sequences may be responsible for the nuclear localization of dG9a during cycle 8 (Kato, 2011). It is well known that a first wave of ZGA occurs around cycle 8 and activates genes required for cellularization. Nuclear localized dG9a may confer specificity to the zygotic gene transcription by di-methylating H3K9 at some specific genomic regions during these stages (Shimaji, 2015).

In embryos at nuclear division cycle 13 and at cellular blastoderm stage, dG9a mainly localizes in cytoplasm although its accumulation in some nuclei was observed. Furthermore, dG9a appears to be not responsible for H3K9me2 in either cytoplasm or nuclei from cycle 12. Drosophila has three HMTases specific to H3K9: SU(VAR)3-9, DmSETDB1 and dG9a. SU(VAR)3-9 is reported to catalyze H3K9me2 at the chromocenter and tri-methylation of H3K9 (H3K9me3) at the core of the chromocenter, but is not functional for mono-methylation, either at the chromocenter or along chromosome arms, nor for di-methylation at chromosome arms and telomeres of the salivary gland polytene chromosomes. DmSETDB1 catalyzes H3K9me2 and H3K9me3 on chromosome 4, and dG9a acts for H3K9me1 and H3K9me2 in euchromatic regions of the polytene chromosomes (Kato, 2008; Lee, 2010). However, despite dG9a is localized in nuclei, it appears to be not responsible for methylation of H3K9 in elongation stages of Drosophila spermatogenesis. In contrast, DmSETDB1 is responsible for H3K9me1 and H3K9me3 and SU(VAR)3-9 for H3K9me1 in these stages. RNA-sequence transcriptome profiles collected at 2-h intervals for embryogenesis of Drosophila showed that expression of dG9a reaches a peak in 0–2 h embryos, although those of DmSETDB1 and Su(var)3-9 are at 2–4 and 4–6 h, respectively. Therefore, in cycle 13 and cellular blastoderm stages, SU(VAR)3-9 and DmSETDB1 instead of dG9a may be responsible for H3K9 methylation. It has in fact been reported that SU(VAR)3-9 functions prominently during cellular blastoderm stages. However, to address contribution of each of these three H3K9-specific HMTases during early embryogenesis, further analyses with double or triple mutants would be necessary (Shimaji, 2015).

This study determined expression of genes affected by the lack of dG9a in embryogenesis. Previous research focused on differentially expressed genes during the larval stage in dG9aDD1, most likely a dG9a null mutant, by microarray analysis (Kramer, 2011). Among the genes differentially expressed 2.5-fold or more in dG9aDD1 larvae, no example was detected that was differentially expressed in dG9aRG5 embryos.The expression of dG9a in whole body shows its peak during 0–2 h embryo and reduces to approximately 10% by the larval stages (Graveley, 2011). In contrast, expression of other H3K9-specific methyltransferases such as DmSETDB1 and Su(var)3-9 increases by the larval stages. These results suggest that the target genes of dG9a vary greatly between embryonic and larval stages, although the possibility cannot be excluded that the difference may be caused by allelic difference (Shimaji, 2015).

Gene ontology analyses in the present study indicate that up-regulated genes in dG9aRG5 are highly involved in embryogenesis and transcription, in line with the conclusion that dG9a regulates expression of zygotically activated genes from cycle 8 which are important for processes like cellularization. However, RNA FISH analysis showed that spatial and temporal expression patterns of mRNAs of representative up-regulated genes in dG9a-depleted embryos were not significantly changed. Maternal depletion of Zelda, a key activator of the early ZGA, changes expression patterns of genes required for cellular blastoderm formation and causes lethal phenotype during embryogenesis. In contrast, dG9a regulates the amount of expression of specific genes without affecting spatial and temporal expression patterns of their mRNAs, which may be the reason why depletion of dG9a does not affect viability during embryogenesis. Among up-regulated genes in dG9a-depleted embryos, there are four genes, ci, glass bottom boat, patched and wntD, involved in Hedgehog signaling pathway. Among these genes, patched is a negative regulator of Wnt signaling pathway and wntD is a target and also an inhibitor of the Dorsal/Twist/Snail network that functions for ventral cell invagination. In the dG9a null mutant, over-expression of such negative regulators may be responsible for delay of embryogenesis, although further analyses are necessary to more precisely address molecular mechanisms (Shimaji, 2015).

Effects of Mutation

Histone H3 lysine-to-methionine mutants as a paradigm to study chromatin signaling

Histone H3 lysine27-to-methionine (H3K27M) gain-of-function mutations occur in highly aggressive pediatric gliomas. This study established a Drosophila animal model for the pathogenic histone H3K27M mutation and showed that its overexpression resembles polycomb repressive complex 2 (PRC2) loss-of-function phenotypes, causing derepression of PRC2 target genes and developmental perturbations. Similarly, an H3K9M mutant depletes H3K9 methylation levels and suppresses position-effect variegation in various Drosophila tissues. The histone H3K9 demethylase KDM3B/JHDM2 associates with H3K9M-containing nucleosomes, and its misregulation in Drosophila results in changes of H3K9 methylation levels and heterochromatic silencing defects. This study has established histone lysine-to-methionine mutants as robust in vivo tools for inhibiting methylation pathways that also function as biochemical reagents for capturing site-specific histone-modifying enzymes, thus providing molecular insight into chromatin signaling pathways (Herz, 2014).

Histone proteins constitute the core of eukaryotic chromatin. SET domain-containing histone methyltransferase complexes such as complex of proteins associated with Set1 (COMPASS) and polycomb repressive complex 2 (PRC2) methylate lysine residues within the histone H3 amino-terminal tail and are essential for normal development. Establishing direct functions for modified lysine residues in histones is difficult because there are multiple histone gene copies in metazoans. Moreover, histone methyltransferase enzymes occur in multimember families with potential redundant activities and histone methylation-independent functions. In Drosophila, replacing all copies of histone H3 with H3 Lys27-to-Arg27 (H3K27R) in a clonal substitution recapitulates the phenotype of mutating E(z), the PRC2 H3 Lys27 methyltransferase gene, suggesting this mark is indeed required for PRC2-mediated repression. Single-allele mutations of histone H3.3 Lys27-to-Met27 (H3.3K27M) occur in a subtype of aggressive pediatric brain cancers and act in a dominant manner to deplete H3K27 methylation by inhibiting PRC2 methyltransferase activity. Other histone H3 lysine-to-methionine mutants also possess dominant gain-of-function activities, making them attractive tools for in vivo functional studies of histone lysine modifications. Trimethylation of histone H3 Lys27 (H3K27me3) and Lys9 (H3K9me3) are associated with distinct forms of transcriptionally silenced chromatin. Histone H3K27me3 catalyzed by PRC2 is enriched at so-called facultative heterochromatin and is implicated in the silencing of key developmental genes, in particular the homoeotic gene clusters. By contrast, H3K9me3 is associated with 'constitutive' heterochromatin at telomeres and centromeres (Herz, 2014).

This study established wild-type histone H3.3, H3.3K27M, and H3.3K9M constructs with a C-terminal FLAG-hemagglutinin (HA) tag for tissue-specific overexpression in Drosophila. Overexpression of H3.3K27M in the posterior compartment of wing imaginal discs driven by engrailed-GAL4 caused a strong reduction in all three H3K27 methylation states and derepression of the PRC2 target gene Ultrabithorax (Ubx), thus phenocopying knockdown of the catalytic PRC2 subunit E(z). Also, increased H3K27 acetylation was observed for H3.3K27M overexpression in Drosophila and mammalian cells and E(z)-RNAi in Drosophila. Genome-wide RNA sequencing (RNA-seq) analysis of H3.3K27M-overexpressing wing imaginal discs revealed up-regulated RNA transcripts for known polycomb target genes, including Ubx, wingless (wg), and the PRC1 subunits Posterior sex combs (Psc) and Suppressor of zeste 2 ([Su(z)2]). Other Homeobox (Hox)-containing genes such as engrailed (en) and invected (in), and signaling pathway components such as cubitus interruptus (ci), were down-regulated upon H3.3K27M overexpression. Moreover, flies expressing H3.3K27M under a tissue-specific Distal-less-GAL4 driver exhibit gross morphological defects-such as severe leg malformations and fusion phenotypes and malformed, reduced, or missing proboscis-and die around eclosion, phenocopying E(z)-RNAi under the same conditions (Herz, 2014).

Trimethylation of histone H3K9me3 by suppressor of variegation 3-9 [Su(var)3-9] proteins is a hallmark of constitutive heterochromatin. Histone H3K9me3 serves as a binding substrate for heterochromatin protein 1 α (HP1α, also known as CBX5) and establishes a transcriptionally repressed state. Euchromatic genes that become abnormally juxtaposed to heterochromatic regions are subject to transcriptional silencing through position-effect variegation (PEV). Less is known about the direct role of H3K9 methylation in the regulation of gene expression. Indeed, studies in fission yeast point to H3K9 methylation-independent functions for the Su(var)3-9 homolog Clr4 in chromatin silencing. To test a direct role for H3K9 methylation in the regulation of gene expression in metazoans, H3.3K9M was overexpressed in Drosophila wing imaginal discs and mammalian cells, and a global depletion of H3K9 methylation levels was observed but no effect was seen on H3K27 methylation. In contrast, H3K9 mono- and dimethylation were slightly reduced when H3.3K27M was overexpressed. Mononucleosomes were purified from wild-type H3.3-, H3.3K9M-, and H3.3K27M-overexpressing human embryonic kidney (HEK) 293 cells, and these samples were subjected to multidimensional protein identification technology (MudPIT) mass spectrometry). The bindings of HP1α (CBX5), HP1β (CBX1), and HP1γ (CBX3) were substantially reduced for H3.3K9M-containing mononucleosomes, as were the interactions of the HP1-associated proteins chromatin assembly factor 1a (CHAF1A/p150) and CHAF1B/p60. Substantially increased association of the H3K9 demethylase KDM3B and the H3K9/K56 deacetylase SIRT6 were found with H3K9M-containing mononucleosomes (Herz, 2014).

Reduced dosage of Drosophila HP1 α [also known as Su(var)205] and Su(var)3-9 results in suppression of PEV. By using a heat shock-inducible lacZ construct inserted within Y-chromosomal heterochromatin, this study found that overexpression of H3.3K9M results in suppression of PEV in both Drosophila salivary glands and eye-antenna imaginal discs. Bulk histone H3K9 methylation levels were decreased in H3.3K9M-overexpressing salivary glands. The effects of H3.3K9M overexpression on heterochromatic silencing were assessed in Drosophila ovaries. The gypsy-lacZ construct is normally silenced in almost all follicle cells but is up-regulated upon loss of heterochromatin function. Overexpression of H3.3K9M results in derepression of lacZ. Thus, the H3.3K9M mutation disrupts heterochromatic silencing of retroelements (Herz, 2014).

KDM3B is a JumonjiC domain-containing histone demethylase that shows specificity toward H3K9 and is involved in gene activation in leukemia cells. Because KDM3B specifically interacts with H3.3K9M-containing nucleosomes, it was of interest to test whether changes in KDM3B levels would alter H3K9 methylation by knocking down or overexpressing its Drosophila homolog, JHDM2, in wing imaginal discs. Depletion of JHDM2 results in increased H3K9 mono- and dimethylation. Conversely, the overexpression of JHDM2 in wing imaginal discs results in depletion in H3K9 dimethylation levels and, to a lesser extent, H3K9 trimethylation and suppresses PEV in both Drosophila salivary glands and eye-antenna imaginal discs. JHDM2 and SIRT6 also globally affect H3K9 acetylation to a similar degree as H3.3K9M overexpression. Sirt6 is not a major regulator of PEV in eye-antenna imaginal discs and salivary glands, but Sirt6-RNAi results in a somewhat modest derepression of the gypsy-lacZ reporter (Herz, 2014).

Histone lysine-to-methionine mutants were used to globally modulate histone methylation in vivo. A Drosophila animal model of the H3K27M mutation was established, that may help elucidate the molecular pathogenesis of pediatric gliomas. To gain mechanistic insight into the molecular function of these mutants, an unbiased proteomic strategy was used to identify histone lysine-to-methionine-interacting partners. Biochemical studies do not identify PRC2 components, such as EZH2, SUZ12, and EED, as significantly enriched on H3.3K27M-containing nucleosomes as previously suggested. However, an increase in H3K27 acetylation levels was detected, along with association of bromodomain-containing protein 1 (BRD1) and BRD4 to H3.3K27M-containing nucleosomes. These findings suggest that inhibitors of H3K27 acetylation or BRD4 inhibitors, such as JQ1 and iBET, could be useful for the treatment of the H3.3K27M-mutated subtype of aggressive pediatric glioblastomas (Herz, 2014).

It was also demonstrated that H3K9M globally depletes H3K9 methylation levels in vivo, disrupts interaction of HP1 proteins, and thus suppresses PEV. Via an unbiased proteomic strategy, KDM3B/JHDM2 and Sirt6 were identified as regulators of H3K9 methylation-dependent heterochromatic silencing. Indeed, JHDM2 acts as a suppressor of variegation in multiple tissues in these assays, whereas Sirt6 function seems to be restricted to retroelement silencing. Mutations of histone H3.3K36M were recently discovered in a subtype of bone cancer. Thus, histone lysine-to-methionine mutations are associated with highly tissue-specific cancer types. Given the importance of heterochromatin in maintaining genomic stability, it is plausible that as-yet-uncharacterized H3K9M mutations might occur in some cancers. The system that was established will provide a powerful tool to inhibit histone lysine modifications at specific residues in vivo and allow to biochemically capture the molecular players involved in chromatin signaling pathways (Herz, 2014).


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

date revised: 1 June 2024

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