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).
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).
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).
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).
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).
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).
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).
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