Polycomb transcripts are most abundant in unfertilized eggs and early cleavage embryos, when PC mRNA is homogeneously distributed. At blastoderm stages PC transcripts are still uniformly distributed but there is less staining at anterior and posterior poles. PC transcripts are found in almost all cells through gastrulation [Images] and organogenesis though at a much lower level than in earlier syncytial stages. In late stages PC transcripts are predominantly expressed in the central nervous system (Paro, 1992).

The subcellular three-dimensional distribution of three polycomb-group (PcG) proteins (Polycomb, Polyhomeotic and Posterior sex combs) in fixed whole-mount Drosophila embryos was analyzed by multicolor confocal fluorescence microscopy. All three proteins are localized in complex patterns of 100 or more loci throughout most of the interphase nuclear volume. The rather narrow distribution of the protein intensities in the vast majority of loci argues against a PcG-mediated sequestration of repressed target genes by aggregation into subnuclear domains. In contrast to the case for PEV repression, there is a lack of correlation between the occurrence of PcG proteins and high concentrations of DNA, demonstrating that the silenced genes are not targeted to heterochromatic regions within the nucleus. There is a clear distinction between sites of transcription in the nucleus and sites of PcG binding, supporting the assumption that most PcG binding loci are sites of repressive complexes. Although the PcG proteins maintain tissue-specific repression for up to 14 cell generations, the proteins studied here visibly dissociate from the chromatin during mitosis, and disperse into the cytoplasm in a differential manner. Quantitation of the fluorescence intensities in the whole mount embryos demonstrate that the dissociated proteins are present in the cytoplasm. Less than 2% of Ph remains attached to late metaphase and anaphase chromosomes. Each of the three proteins that were studied has a different rate and extent of dissociation at prophase and reassociation at telophase. These observations have important implications for models of the mechanism and maintenance of PcG- mediated gene repression. The findings reported in this paper do not exclude the possibillity that the minor fraction of the PcG proteins, which remains bound to mitotic chromosomes, may be associated with specific nucleation sites within the repressed genes. Repression could then be initiated in telophase from these sites via cooperative binding of previously dispersed PcG protein complexes, insuring that promoters are blocked before reassembly of functional transcription complexes. A similar marking mechanism has been proposed for active genes by residual transcription factors on mitotic chromosomes. In the BX-C, possible candidates for repression nucleation sites are regulatory elements, which are preferentially associated with PC (Buchenau, 1998).

Polycomb group proteins (PcG) repress homeotic genes in cells where these genes must remain inactive during Drosophila and vertebrate development. This repression depends on cis-acting silencer sequences, called Polycomb group response elements (PREs). Pleiohomeotic (Pho), the only known sequence-specific DNA-binding PcG protein, binds to PREs, but pho mutants show only mild phenotypes compared with other PcG mutants. pho-like, a gene encoding a protein with high similarity to Pho, has been characterized. Pho-like binds to Pho-binding sites in vitro and pho-like; pho double mutants show more severe misexpression of homeotic genes than do the single mutants. These results suggest that Pho and Pho-like act redundantly to repress homeotic genes. The distribution of five PcG proteins on polytene chromosomes was examined in pho-like, pho double mutants. Pc, Psc, Scm, E(z) and Ph remain bound to polytene chromosomes at most sites in the absence of Pho and Pho-like. At a few chromosomal locations, however, some of the PcG proteins are no longer present in the absence of Pho and Pho-like, suggesting that Pho-like and Pho may anchor PcG protein complexes to only a subset of PREs. Alternatively, Pho-like and Pho may not participate in the anchoring of PcG complexes, but may be necessary for transcriptional repression mediated through PREs. In contrast to Pho and Pho-like, removal of Trithorax-like/GAGA factor or Zeste, two other DNA-binding proteins implicated in PRE function, do not cause misexpression of homeotic genes or reporter genes in imaginal discs (Brown, 2003).

The distribution of the PcG proteins Pc, Psc, Polyhomeotic (Ph), Sex combs on midleg (Scm) and Enhancer of zeste [E(z)] on polytene chromosomes was examined. Pc, Ph and Psc are all core components of the PcG protein complex called PRC1. Scm has also been reported to co-purify with PRC1. Scm and Ph may also be present in protein complexes other than PRC1. E(z) is a component of the Esc-E(z) complex, which is distinct from PRC1. The analysis focused on PcG protein binding sites on the X chromosome and on the right arm of chromosome 3, which includes the bithorax and Antennapedia gene complexes (BXC and ANTC) (Brown, 2003).

Pho, Pc, Psc, Ph and Scm all bind the same three sites in wild-type chromosomes. As expected, in phol; pho double mutants, no Pho protein is detected. Binding of Pc, Psc and Scm is lost at polytene subdivision 2D in phol; pho double mutants; binding of these proteins to all other sites on the X chromosome is unaffected. Binding of Ph is completely unaffected in phol; pho double mutants. In particular, the Ph signal at 2D is present, suggesting that Ph can bind at this site even if other PcG proteins are removed. Pc binding to 2D is not lost in either pho or phol single mutants, suggesting that the presence of either of these two proteins is sufficient for Pc to bind to this site (Brown, 2003).

Taken together, the immunolocalization data suggest that binding of PcG proteins to most sites is unaltered in the absence of Pho and Phol protein, but that two proteins are redundantly required for PcG protein binding at a few specific sites. Intriguingly, it appears that all PcG proteins tested in this study are still associated with the BXC and ANTC loci. Nevertheless, the BXC genes Ubx and Abd-B are derepressed in phol; pho double mutant wing discs. Several different explanations for this paradox are proposed. (1) Derepression of homeotic genes and binding of PcG proteins were not assayed in the same tissues. It was not possible to detect derepression of Ubx in salivary gland cells of phol, pho double mutants. (2) Pho and Phol may only be required for anchoring PcG proteins at some PREs in the BXC. Different DNA-binding proteins may provide this function at other PREs. This is supported by the finding that binding of PcG proteins is lost at some sites in phol; pho double mutants. Moreover, several different PREs have been identified in the Ubx gene. The resolution of antibody signals on polytene chromosomes is not refined enough to resolve distinct PREs in a single gene and, hence, loss of only a fraction of PcG protein complexes may not be detectable. Finally, Phol and Pho may not be necessary for the anchoring of PcG protein complexes to the DNA, but may confer the actual transcriptional repression mediated by PREs in imaginal discs, while the PcG protein complexes might function in the propagation and memory of the repression. Thus, PcG protein complexes might serve to recruit Phol and Pho or their co-repressors to the DNA (Brown, 2003).

These results show a strong requirement for the DNA-binding proteins Pho and Pho-like in homeotic gene silencing in imaginal discs. In fact, the strong misexpression of homeotic genes observed in phol; pho double mutant imaginal cells is comparable with that seen in imaginal disc clones mutant for Pc, Scm, Sce or Pcl. The loss of PcG protein binding at only a small number of sites in phol, pho polytene chromosomes is consistent with the idea that Phol and Pho are required to recruit PcG protein complexes at only a subset of PREs. Alternatively, Phol and Pho may be required for transcriptional repression mediated by PREs, but not for anchoring of PcG protein complexes (Brown, 2003).

Polycomb group protein complexes exchange rapidly in living Drosophila

Fluorescence recovery after photobleaching (FRAP) microscopy was used to determine the kinetic properties of Polycomb group (PcG) proteins Polycomb and Polyhomeotic in whole living Drosophila organisms (embryos) and tissues (wing imaginal discs and salivary glands). Translational diffusion constants of PcG proteins, dissociation constants and residence times for complexes were determined in vivo at different developmental stages. In polytene nuclei, the rate constants suggest heterogeneity of the complexes. Computer simulations with new models for spatially distributed protein complexes were performed in systems showing both diffusion and binding equilibria, and the results compared with the experimental data. Forward and reverse rate constants for complex formation were determined. Complexes exchange within a period of 1-10 minutes, more than an order of magnitude faster than the cell cycle time, ruling out models of repression in which access of transcription activators to the chromatin is limited and demonstrating that long-term repression primarily reflects mass-action chemical equilibria (Ficz, 2005).

Most FRAP studies of nuclear proteins have involved components in transcription complexes or transcriptional activators that exchange in less than 2 minutes. The only repressor protein that has previously been investigated is heterochromatin protein 1 (HP1), a protein targeted to heterochromatin in higher eukaryotes. Although HP1 is loaded directly onto the chromatin during replication, it was found by FRAP to bind only transiently to chromatin with a maximum residence time of 60 seconds. Thus, both HP1 and PcG repression complexes appear to function by dynamic competition with other chromatin-binding proteins rather than by formation of a static, higher-order chromatin structure with immobilized bound repressors. FRAP measurements on polytene chromosomes revealed differences in the dissociation rate constants between individual bands -- this implies that a flexible repression system of complexes with various compositions that influence the binding affinity of other members and whose turnover is in the order of a few minutes. It is concluded that: (1) activation and repression can be dynamically controlled by simple chemical equilibria; (2) reduction in PcG levels will facilitate epigenetic change and may explain why non-cycling cells can be reprogrammed more easily than cycling cells, and (3) PcG complexes are exchangeable protein assemblies that maintain repression over many cell cycles by simple chemical equilibria (Fitz, 2005).

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).

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).


In vivo Polycomb kinetics and mitotic chromatin binding distinguish stem cells from differentiated cells

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

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

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

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

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

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

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

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

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

Genome-wide and cell-specific epigenetic analysis challenges the role of polycomb in Drosophila spermatogenesis

The Drosophila spermatogenesis cell differentiation pathway involves the activation of a large set of genes in primary spermatocytes. Most of these genes are activated by testis-specific TATA-binding protein associated factors (tTAFs). In the current model for the activation mechanism, Polycomb plays a key role silencing these genes in the germline precursors, and tTAF-dependent activation in primary spermatocytes involves the displacement of Polycomb from gene promoters. This study investigated the genome-wide binding of Polycomb in wild type and tTAF mutant testes. According to the model, a clear enhancement in Polycomb binding at tTAF-dependent spermatogenesis genes was expected in tTAF mutant testes. However, little evidence was found for such an enhancement in tTAF mutant testes compared to wild type. To avoid problems arising from cellular heterogeneity in whole testis analysis, the model was further tested by analysing Polycomb binding in purified germline precursors, representing cells before tTAF-dependent gene activation. Although Polycomb was found associated with its canonical targets, little or no evidence of Polycomb was found at spermatogenesis genes. The lack of Polycomb at tTAF-dependent spermatogenesis genes in precursor cells argues against a model where Polycomb displacement is the mechanism of spermatogenesis gene activation (El-Sharnouby, 2013).

Identification of regulators of the three-dimensional polycomb organization by a microscopy-based genome-wide RNAi screen

Polycomb group (PcG) proteins dynamically define cellular identities through epigenetic repression of key developmental genes. PcG target gene repression can be stabilized through the interaction in the nucleus at PcG foci. This study report the results of a high-resolution microscopy genome-wide RNAi screen that identifies 129 genes that regulate the nuclear organization of Pc foci. Candidate genes include PcG components and chromatin factors, as well as many protein-modifying enzymes, including components of the SUMOylation pathway. In the absence of SUMO, Pc foci coagulate into larger aggregates. Conversely, loss of function of the SUMO peptidase Velo disperses Pc foci. Moreover, SUMO and Velo colocalize with PcG proteins at PREs, and Pc SUMOylation affects its chromatin targeting, suggesting that the dynamic regulation of Pc SUMOylation regulates PcG-mediated silencing by modulating the kinetics of Pc binding to chromatin as well as its ability to form Polycomb foci (Gonzalez, 2014).

Effects of Mutation or Deletion

The brahma gene is required for the activation of multiple homeotic genes in Drosophila. Loss-of-function brm mutations suppress mutations in Polycomb, a repressor of homeotic genes, and cause developmental defects similar to those arising from insufficient expression of the homeotic genes of the Antennapedia and Bithorax complexes (Tamkun, 1992).

Polyhomeotic immunoprecipitates in a multimeric complex that includes Polycomb. Duplications of ph suppress homeotic transformations of Pc and Polycomb-like, supporting a mass-action model for Pc-G function. ph alleles have been crossed to all members of the Polycomb group. Synergistic effects are found suggesting that these gene products might interact directly with ph. Embryonic phenotypes of ph mutant embryos that are lethal when heterozygous or homozygous for other mutations suggest that ph may perform different functions in conjunction with differing subsets of Pc group genes (Cheng, 1994).

moira (mor) is a member of the trithorax group of homeotic gene regulators in Drosophila. moira is required for the function of multiple homeotic genes of the Antennapedia and bithorax complexes (HOM genes) in most imaginal tissues. Heterozygous mor mutations suppress the following Polycomb-induced phenotypes:

  1. Derepression of the Antp gene in the eye-antennal disc causes replacement of adult antennal structures with leg structures.
  2. Derepression of the Scr gene in the second and third leg discs causes the appearance of first leg structures in the second and third legs of the adults.
  3. Derepression of the Ubx gene in the wing discs causes the appearance of haltere tissue in the adult wing.
  4. Derepression of the genes in the BXC (abd-A and Abd-B) causes cells of the fourth abdominal segment of the adult to differentiate structures of a more posterior identity.
moira mutations suppress the derepression phenotypes caused by mutations in another Pc group gene, Polycomblike. moira mutant clones in the haltere differentiate large bristles, characteristic of the anterior wing margin, and often lead to absence or duplication of halteres. Homozygous mor mutations in the posterior wing result in a distorted wing shape; the venation is disrupted and large socketed bristles appear along the posterior wing margin. Leg clones result in the femur and tibia being short and twisted and enlargement of the tarsal segment. Clones of the head cause the shape of the head to be abnormal in the dorsal region and sometimes cause the ocellus to be abnormal or absent. Embryos homozygous for moira mutations have defects in head structures, including truncated lateralgraten and defects in the mouth hooks and dorsal bridge. The first and second midgut constrictions are shifted posterior to their wild-type positions (Brizuela, 1997).

The requirement for moira function is at the level of transcription. The ability of moira mutations to supppress Antp homeotic phenotypes is dependent on the promoter. moira is also required for transcription of the engrailed segmentation gene in the imaginal wing disc. Because homozygous mor clones have phenotypes similar to those seen in clones of cells that have lost en function, en transcription was examined in clones of cells in the posterior wing. In the absence of transcriptional activation by mor, the pattern of en is altered. Greatly reduced en expression is found in wing clones. The abnormalities caused by the loss of moira function in germ cells suggest that at least one other target gene requires moira for normal oogenesis (Brizuela, 1997).

Recently, the gene encoding a large protein related to the trithorax group protein brahma has been cloned and maps to the same three polytene chromosome bands as moira (Goldman-Levi, 1996). This new gene 89B helicase, is missing in a cytologically-invisible moira deletion. Since this small deletion also affects at least one other vital gene in this region, attempts are being made to determine whether moira encodes the 89B helicase, and if so, whether the Mor protein is part of a large protein complex similar to the SWI/SNF complex that may interact with specific cis-regulatory regions of its target genes (Brizuela, 1997).

Arresting cell division using the string mutation or blocking DNA replication with aphidicolin failed to prevent ectopic expression of the homeotic gene Ultrabithorax in Polycomb mutants. Thus, even in the absence of DNA replication, Pc is required to maintain spatially restricted patterns of homeotic gene expression (Gould, 1990).

The observation of a shared pathway in the function of a chromatin insulator and trithorax group (trxG) and Polycomb group (PcG) gene activation and silencing is suggestive of a common mechanism at work. If this is the case, mutations in trxG and PcG genes, known to be involved in activation and silencing, might also affect the ability of the insulators to interfere with enhancer-promoter interactions. To test this possibility, the effect of trxG and PcG mutations on the abdominal coloration of flies carrying the yellow2 mutation (affecting coloration) was measured using insertion of an insulator-containing gypsy retrotransposon. Males hemizygous for the y2 allele show brown abdominal pigmentation in the fifth and sixth abdominal segments, instead of the black pigmentation observed in wild-type males, due to the effect of the insulator on the upstream body cuticle enhancer. This insulator effect on the body enhancer is altered by hypomorphic mutations in mod(mdg4), which gives rise to a variegated phenotype resulting from different expression levels of the yellow gene in adjacent groups of cells. In some cuticle cells, the effect of the insulator is reversed, resulting in normal expression of the yellow gene; in other cells, the effect of the insulator on enhancer-promoter communication appears to be enhanced, further repressing yellow gene expression. To examine the effect of trxG mutations on insulator function, the partially nonfunctional insulator, renderend such by hypomorphic alleles of mod(mdg4), was tested. An examination was carried out of the consequence of mutations in trxG genes, such as trithorax, on the frequency and severity of a mod(mdg4) phenotype engendered by Mod(mdg4) action at the gypsy insulator (Gerasimova, 1998).

Both the penetrance and severity of a variegated phenotype due to insulator function are enhanced by mutations in trxG genes. trx mutation results in a decrease in the number of dark spots with respect to that observed in hypomorphilc mod(mdg4) males, with only a few spots visible in a light brown-colored background. A stronger effect can be seen when trx is combined with brahma or ash1. Mutations in polycomb cause the opposite result, reversing the effect of the insulator on enhancer-promoter interactions and resulting in a wild-type expression of the yellow gene in the body cuticle. These results indicate that mutations in trxG genes cause an enhancement of the variegated phenotype induced by mod(mdg4) mutations in the yellow gene, suggesting that decreased levels of these proteins enhance the inhibitory effect of the insulator on enhancer-promoter interactions. In contrast, mutations in Pc impair the ability of the insulator to inhibit enhancer-promoter interactions, restoring normal expression of the gene. The effects of trxG and PcG mutations on insulator function at the yellow gene are not a result of homeotic transformations in abdominal segments that cause changes in the pigmentation of the cuticle, since these effects are not observed in flies carrying a wild-type copy of the yellow gene. In addition, the same effect can be observed with other gypsy-induced mutations such as scute-1 and cut-6. Flies of the genotype ct6; brm+ trx+ mod(mdg4)T16/brm2 trxB11 mod(mdg4)+ display a much stronger cut phenotype than ct6; mod(mdg4)T16/mod(mdg4)+ individuals, suggesting that the effect of TrxG and PcG proteins on gypsy insulator function is general and does not depend on the nature of the affected gene. A similar result was obtained with the sc1 mutation. The effects of trxG and PcG mutations on insulator function suggest that the proteins encoded by these genes might be structural components of the gypsy insulator or they might regulate its function (Gerasimova, 1998).

Drosophila Mi-2 protein binds to a domain in the gap protein Hunchback which is specifically required for the repression of HOX genes. dMi-2 protein was tested to see if it participates in PcG repression. As in the case of dMi-2, maternally deposited PcG product often rescues homozygous mutant PcG embryos to a considerable extent. Extensive derepression of HOX genes can be observed if such homozygous embryos are also mutant for another PcG gene. Thus embryos homozygous for the PcG gene Posterior sex combs (Psc) and dMi-2 were examined and it was found that Ubx and Abd-B are derepressed more extensively in this double mutant than in Psc homozygotes alone. A similar result was found if dMi-2 is combined with other PcG mutations; these double mutants consistently lead to much enhanced homeotic transformations compared with the single PcG mutants. Thus, there is a synergy between dMi-2 and PcG genes. dMi-2 behaves like the PcG mutations Enhancer of Polycomb and Suppressor 2 of zeste, neither of which on their own cause a homeotic phenotype but do so in combination with other PcG mutations. This suggests that dMi-2 functions in PcG repression (Kehle, 1998).

Imaginal discs were examined for derepression of HOX genes as well as the phenotypes of their adult derivatives. Clonal analysis suggests that dMi-2 is required for the survival of somatic cells. Do dMi-2 mutations exhibit gene-dosage interactions with PcG mutations? While larvae heterozygous for Polycomb (Pc) mutations show slight derepression of Ubx, larvae transheterozygous for both Pc and dMi-2 mutations show more extensive derepression. Furthermore, derepression of the HOX gene Sex combs reduced (Scr) in the second and third leg discs of Pc heterozygotes results in the formation of a first leg structure, the sex comb, on the second and third legs. The extent of this homeotic transformation reflects the number of cells that misexpress Scr protein. This homeotic transformation is far stronger in dMi-2/Pc transheterozygotes than in adults heterozygous for Pc alone, which is consistent with more extensive derepression of Scr in the double mutant. These results are further evidence that dMi-2 acts together with PcG proteins to repress HOX genes (Kehle, 1998).

It has been proposed that Hb directly or indirectly recruits PcG proteins to DNA to establish PcG silencing of homeotic genes. The present data suggest that dMi-2 might function as a link between Hb and PcG repressors. Although dMi-2 contains two motifs with similarity to DNA-binding domains (the myb and HMG domains), dMi-2 does not seem to bind to DNA on its own. Therefore, Hb may recruit dMi-2 to DNA. Xenopus Mi-2 was recently purified as a subunit of a histone deacetylase complex with nucleosome remodeling activity. In yeast and in vertebrates, several transcription factors repress transcription by recruiting histone deacetylases. It is possible that in Drosophila, nucleosome remodeling and deacetylase activities of a dMi-2 complex, recruited to homeotic genes by Hb, may result in local chromatin changes that allow binding of PcG proteins to the nucleosomal template. Alternatively, the proposed Hb-dMi-2 complex might directly bind a PcG protein and recruit it to DNA. Finally, the involvement of dMi-2 in PcG silencing suggests that this process may involve deacetylation of histones (Kehle, 1998 and references).

Animals doubly mutant for two different PcG genes often show phenotypes more extreme than either single mutant alone. It has been suggested that this phenotypic enhancement results from PcG protein complexes that are more severely impaired by simultaneous reduction or alteration of multiple components. Having defined the molecular lesions and relative strengths of many Sex combs on midleg (Scm) mutations, it was of interest to test the Scm alleles for interactions with other PcG mutations. In particular, a comparison was made of genetic interactions exhibited by mutations affecting different parts of SCM protein (Bornemann, 1998).

Animals were generated that were doubly heterozygous for each of the Scm alleles and for a lethal allele of Polycomb (Pc3). Transheterozygous Pc3/Scm adults display more severe homeotic phenotypes than Pc3/+ adults, and this enhancement is seen with all Scm alleles tested. These phenotypes include transformations of wing to haltere, antenna to leg, second and third leg to first leg, and fourth abdominal segment to fifth. However, three Scm alleles, Su(z)302, R5-13, and ET50, produce much stronger interactions with Pc3 than do other Scm alleles. These three are the only Scm alleles to cause partial lethality in combination with Pc3. The transheterozygous progeny classes for Su(z)302, R5-13, and ET50 are reduced to about one-third that expected for full viability. In contrast, the Scm null alleles H1 and M56 are fully viable with Pc3. The surviving Su(z)302, R5-13, and ET50 transheterozygous progeny also exhibit more severe homeotic phenotypes than do other Pc3/Scm combinations. A marked male sex bias was observed among these survivors. In the most severe case, only about 5% of the surviving Pc3/ScmSu(z)302 progeny were female. Similarly, partial lethality and a male sex bias were seen with the reciprocal crosses consisting of Su(z)302, R5-13, or ET50 females mated to Pc3 males. These interactions likely result from the Scm lesions rather than mutations at other loci because these three Scm alleles produce similar phenotypic effects and were isolated independently on different genetic backgrounds. Each of the three alleles maps to the first mbt repeat in SCM protein. Thus, Su(z)302, R5-13, and ET50 are hypomorphic mutations based upon their behavior as homozygotes, yet they interact with Pc3 more strongly than do null Scm alleles (Bornemann, 1998).

Posttranscriptional gene silencing and the function of Polycomb chromatin-repressive proteins

Two types of transgene silencing were found for the Alcohol dehydrogenase (Adh) transcription unit. Transcriptional gene silencing (TGS) is Polycomb dependent and occurs when Adh is driven by the white eye color gene promoter. Full-length Adh transgenes are silenced posttranscriptionally at high copy number or by a pulsed increase over a threshold. The posttranscriptional gene silencing (PTGS) exhibits molecular hallmarks typical of RNA interference (RNAi), including the production of 21-25 bp length sense and antisense RNAs homologous to the silenced RNA. Mutations in piwi, which belongs to a gene family with members required for RNAi, block PTGS and one aspect of TGS, indicating a connection between the two types of silencing (Pal-Bhadra, 2002).

Despite the fact that posttranscriptional silencing appears to be a matter of RNA metabolism, some indications of chromatin modification of the homologous endogenous gene under certain circumstances have emerged. (1) Transgene copies of viral genes present in the nucleus only become methylated upon infection of the plant by the homologous virus, which has a double-stranded RNA genome and which does not enter the plant nucleus. (2) In plants and rodent cells, the introduction of DNA constructs into cells can trigger the RNA degradation reaction. (3) Mutations in Arabidopsis selected for reduced DNA methylation, ddm1, a SWI2/SNF chromatin component, and met1, the major DNA methyltransferase, will relieve gene silencing, including a stochastic reversal of posttranscriptional silencing. (4) Transgene arrays in the C. elegans germline are desilenced and appear less condensed in mutant backgrounds for mut-7 and rde-2, which are both required for RNAi. (5) The ectopic transcription of promoter sequences or their introduction to the plant cell in a virus will trigger transcriptional silencing of another reporter construct in the same cell with a homologous promoter, which also becomes hypermethylated. Evidence is presented for a link between posttranscriptional and transcriptional modes of gene silencing in Drosophila (Pal-Bhadra, 2002 and references therein).

The silencing of the promoter-reporter construct white-Alcohol dehydrogenase (w-Adh) and full-length Adh transgenes occurs in Drosophila . The w-Adh effect is modulated by mutations in the Polycomb group (Pc-G) of chromatin-repressive proteins, and the silenced transgenes are associated with the Pc-G complex, whereas single highly expressed copies are not. The endogenous Adh gene is drawn into the silencing pool and is capable of further extending the effect to an Adh-w construct, which has no portion in common with w-Adh but which does share homology with the endogenous Adh 5' sequences. The silenced Adh-w construct also accumulates the Pc-G complex. Deletion of the endogenous Adh gene eliminates the silencing interaction between the two nonhomologous, reciprocally constructed transgenes, w-Adh and Adh-w (Pal-Bhadra, 2002 and references therein).

The full-length Adh transgene silencing operates posttranscriptionally as determined by nuclear run-on transcription assays that directly assess the distribution of transcriptionally-engaged Pol II. In contrast, the w-Adh and Adh-w silencing is transcriptional. The posttranscriptional silencing of Adh correlates with the appearance of 21-25 bp sense and antisense RNAs as occurs with virus and PTGS silencing in plants and RNAi in flies (Pal-Bhadra, 2002).

Mutations have been recovered in C. elegans that are defective for RNAi. Homologs to these genes exist throughout the plant and animal kingdoms. One of these mutations, rde1, has several related gene family members in flies. A homozygous viable member of this group was tested for its impact on transgene silencing. The piwi mutation drastically reduces the magnitude of posttranscriptional silencing. Surprisingly, piwi also had a strong impact on the transcriptional silencing of Adh-w by w-Adh. This result indicates that under certain circumstances the two types of silencing can be mechanistically related (Pal-Bhadra, 2002).

When one to five copies of full-length Adh transgenes are introduced in the genome, the steady-state RNAs accumulate in direct correlation with copy number. However, at higher dosage (six to ten), Adh RNA levels depart dramatically from a linear relationship with transgene copy number. The transgene analyzed contains all the Adh sequences required for normal function. Five single insert strains showing minimal positional effects were selected. By combining these insertions via genetic crosses, a series of Adh stocks was generated that carry one to ten copies. Each stock is also homozygous for the endogenous transformation recipient allele, Adhfn6. This allele is defective at the 3' splicing acceptor of the first intron, resulting in a longer transcript. The endogenous allele produces only 5%-10% of the steady-state level of a normal Adh gene (Pal-Bhadra, 2002).

To determine whether the endogenous gene was silenced in concert with the transgenes, an RNase protection assay was used that distinguishes the two RNAs. The normal full-length Adh RNA protects two fragments, 142 and 160 bp, while the Adhfn6 RNA protects a 355 bp fragment due to defective splicing. To correct for loading differences, a ß-tubulin probe was included, which protects a 70 bp fragment. The level of each protected fragment (142/70 or 160/70 ratio) was reduced at higher dosage. The amount of endogenous Adhfn6 transcripts did not show any significant difference in one to five copies, suggesting an equal expression. However, these transcripts followed a similar trend as those from the transgenes at higher dosage (Pal-Bhadra, 2002).

To test for any positional influence on Adh transgene silencing, separate sets of one to nine copy stocks were examined. A similar level of Adh expression at any one dosage suggests that the silencing is not significantly affected by the insertion sites. RNA in situ analyses in embryos the Adhfn6 strain, as well as with one, five, and seven Adh transgene copies, indicates a linear increase of Adh expression to five copies. Silencing of the seven copy stock was already evident during blastoderm, the stage where Adh RNA is accumulated initially (Pal-Bhadra, 2002).

To determine whether the Adh transgene silencing is transcriptional or posttranscriptional, nuclear run-on assays were performed. ß-tubulin was included as an internal control in order to determine the relative amount of Adh transcription, while lacZ acted as a negative control (Pal-Bhadra, 2002).

Transcription levels were estimated in adult flies carrying zero to ten copies of full-length Adh transgenes. The results reveal that the endogenous Adhfn6copies, present in each stock, are transcribed similarly to two normal copies. In the one to ten copy series, the amount of transcription increases proportionally to the transgene copy number relative to ß-tubulin (Pal-Bhadra, 2002).

A similar experiment was performed using flies that contain selected genotypes from zero to six copies of the w-Adh hybrid constuct. In the absence of transgenes, the Adhfn6 allele is transcribed at the normal level. When one copy of the w-Adh transgene is present, the transcript level is increased as expected. In contrast, two copies of w-Adh exhibit a plateau of transcript accumulation. In the presence of more w-Adh copies (four to six), total Adh transcription is reduced progressively. The transcribed RNA in four to six copy stocks is below the level produced by the Adhfn6 alleles alone. The amount of Adh transcription in males is always greater than in females with equal dosage (Pal-Bhadra, 2002).

Adh transcriptional level (as detected using run-on assays), produced by multiple full-length Adh or w-Adh transgenes, was compared to steady-state mRNA levels produced by the same genotypes, as determined in Northern analyses. In the Adh series, the amount of mRNA is proportional to dosage from one to five, while in six to ten copies, the steady-state levels depart from linearity. This difference from the run-on assays indicates that silencing of full-length Adh transgenes is posttranscriptional. On the contrary, with the w-Adh dosage series, a similar curve was found when the transcription level and steady-state RNA were compared. A parallel pattern of reduction of both nascent and mature RNA suggests that w-Adh silencing, which includes an effect upon the endogenous Adh gene, is transcriptional. The reason for the different modes of silencing of the two types of transgenes is not known (Pal-Bhadra, 2002).

The silencing of w-Adh and the endogenous Adh locus can also be extended to an Adh-white (Adh-w) transgene that is the reciprocal construct of w-Adh. Run-on transcription analysis of the Adh-w transgene in a w deficiency background with varying numbers of w-Adh indicates that this type of silencing also occurs on the transcriptional level. A comparison of the run-on data with a previous Northern analysis of Adh-w with increasing w-Adh copies shows a similar relationship. The Adh-w + 1 w-Adh and Adh-w + 4 w-Adh genotypes have nearly identical levels of reduction in gene expression in the two assays. There is a residual amount of transcription in the Adh-w + 2 w-Adh genotype but no detectable RNA in the Northern analysis, which might suggest that a combination of PTGS and TGS is operating. However, these data points lie near the limit of detection for the two techniques and likely differ by chance due to exposure time because the same genotype assayed subsequently by both methods shows a similar low level of expression. Thus, at the developmental stage examined (adults), the silencing is predominantly, if not exclusively, transcriptional (Pal-Bhadra, 2002).

Biochemical experiments on RNA interference (RNAi) in Drosophila have shown that double-stranded RNAs in the form of 21-25 nt fragments are generated and these fragments are used in the sequence-specific degradation of mRNA. Therefore, the in vivo existence of such RNAs was tested in the case of Adh silencing. The 21-25 nt antisense Adh RNAs were strongly accumulated in the stocks that contain six to ten copies of the Adh transgene. At the lower doses, the same RNA was not detected or was present at very low amounts in the four and five copy stocks. Using an antisense probe for the detection of sense fragments, a similar-sized RNA was found in the silencing doses (Pal-Bhadra, 2002).

Whether transcriptional silencing induced by w-Adh transgenes was associated with small RNAs was tested by analyzing stocks containing zero to six copies of w-Adh. Hybridization with sense or antisense Adh probes revealed that low molecular weight RNA was not found in abundance at any w-Adh dosage (Pal-Bhadra, 2002).

To investigate the nature of PTGS, it was reasoned that an induced hsp70-Adh construct with four Adh transgenes would bring the Adh expression over the silencing threshold. The hsp70-Adh construct contains a heat shock promoter and the Adh reporter gene. Adh RNA from adult flies was measured relative to ß-tubulin controls in three different circumstances: no heat, heat shock (37°C for 45 min), and 20 hr after heat shock. In flies with five full-length Adh copies, the amount of RNA in heat shock and 20 hr after heat shock did not change relative to that of the untreated flies (Pal-Bhadra, 2002).

The heat treatment of adult flies carrying two copies of the hsp70-Adh gene increased their RNAs as expected. In contrast, the heat treatment of a stock that carries four copies of the Adh transgene and in addition an hsp70-Adh gene causes a rapid loss of Adh mRNA. A similar response was found in embryos. The heat shock induced increase in Adh RNA appears to surpass the threshold limit that triggers silencing. A time course sampling of RNA after heat shock showed that Adh mRNA levels were partially recovered 20 hr subsequent to heat treatment. Interestingly, the transient silencing in this case is in contrast to the systemic spread and prolonged silencing that occurs in C. elegans and plants following a localized induction of posttranscriptional silencing (Pal-Bhadra, 2002).

Whether pulsed threshold induced RNA degradation is correlated with the small species of Adh RNA was tested. The small RNAs were found at a high level in flies with the combination genotype after heat shock. Treatment of the control stock composed only of two hsp70-Adh copies does not produce the small RNAs. This result suggests a threshold level must be exceeded to initiate the synthesis of the small RNAs, which in turn participate in the destruction of the homologous mRNA (Pal-Bhadra, 2002).

Because Polycomb and Polycomb-like (Pcl) mutations have a significant effect on w-Adh transcriptional silencing and Pc-G proteins are bound at the sites of the transgenes under silencing conditions, binding of Pc-G proteins at the Adh transgene insertion sites was tested cytologically. Those sites that do not overlap (1CD and 53B) normal positions of Pc labeling were examined for evidence of binding in the single insert stocks and for the same insert in the ten copy larvae. The Pc proteins were not observed at these sites in either case. Therefore, Pc protein recruitment is not a consequence of posttranscriptional silencing of Adh (Pal-Bhadra, 2002).

A group of genes has been characterized that affects PTGS or RNAi in fungi, plants, and animals. Some of the mutations show sequence similarity to members of the piwi/sting/eIF2C/argonaute gene family conserved from plants to vertebrates. Members of this family, including piwi, have been characterized in Drosophila . To determine whether this mutation has any effect on PTGS and TGS of Adh, the role of this mutation on posttranscriptional silencing was tested. Two or four copies of fully functional Adh genes (two normal endogenous copies ± two transgenes) plus a hsp70-Adh transgene in combination with either heterozygotes or homozygotes of piwi were examined. In the stocks analyzed, a normal Adh allele, rather than Adhfn6, resides on both the balancer chromosome present in heterozygotes and on the chromosome carrying the piwi mutations. These endogenous Adh copies contribute greater amounts of mRNA to the total pool than Adhfn6 (Pal-Bhadra, 2002).

The heat shock and nonheat shock RNAs of the respective stocks were compared in quantitative Northern blots. The stocks that are heterozygous for the piwi 1 or piwi 2 alleles with two copies of the normal Adh gene plus hsp70-Adh exhibit an increase of RNA after heat incubation. In contrast, a sharp reduction of the Adh RNA was found in the five copy (four Adh + hsp70-Adh) stock following heat shock. In piwi homozygotes, the presence of two endogenous genes + hsp70-Adh has a similar pattern of expression as in heterozygotes following heat shock. However, the Adh mRNA level in the four Adh + hsp70-Adh stocks that are homozygous for piwi alleles is restored to nearly normal after heat shock. An almost equal level of Adh expression in nonheat shock and heat shock flies suggests that the piwi mutation disrupts the threshold-based posttranscriptional Adh silencing. The two separate piwi alleles examined as well as their heteroallelic combination, which was used to minimize any influence of linked modifiers, produced the same results (Pal-Bhadra, 2002).

Examination of 21-25 nt RNAs in the piwi-segregating classes shows that the small RNAs are present in the heat shocked class with four Adh genes plus a hsp70-Adh construct when the flies are heterozygous for piwi. In contrast, these RNAs are diminished in the piwi homozygotes that carry the same constellation of Adh genes and that were subjected to heat shock. This result suggests that piwi acts before or during the formation of these small RNAs (Pal-Bhadra, 2002).

As a first measure of the effect on transcriptional silencing, the interaction between the reciprocal w-Adh and Adh-w transgenes was examined because the expression of the latter can be observed phenotypically. One copy of w-Adh reduces and two copies nearly eliminate the expression of Adh-w. The w-Adh transgenes among themselves participate in silencing and include endogenous Adh. The w-Adh/Adh-w silencing interaction is eliminated when the endogenous Adh is deleted from the genome, suggesting that it mediates the reaction via mutually homologous sequences. The expression of the Adh-w transgene can be assayed on the RNA level by probing for white RNA because the transgene is present in a stock with the normal white gene deleted. In the same RNA populations, the silencing of w-Adh and its effect on endogenous Adh can be assayed by probing for total Adh messenger RNA (Pal-Bhadra, 2002).

piwi mutations were introduced into a background with an Adh-w and a w-Adh transgene. The eye color of Adh-w flies, which is reduced in the interaction with one w-Adh copy, is restored to a normal level in piwi1 or piwi2 homozygotes. The heteroallelic combination piwi1/piwi2 shows a similar level of restoration. These combinations of piwi alleles did not have any effect on the eye color of Adh-w in the absence of w-Adh, indicating that their effect is due to a relief of silencing (Pal-Bhadra, 2002).

w mRNA was measured in genotypes with Adh-w and zero to two copies of w-Adh segregating for the piwi alleles. The accumulation of white transcripts in the Adh-w/Y flies without a w-Adh transgene was not significantly different in the presence of the mutations. As expected, the w transcripts from Adh-w were reduced from the normal level in the presence of one copy of w-Adh. However, the presence of the homozygous mutations restored the levels significantly but not completely to the normal amount. Moreover, the w transcripts from Adh-w were significantly restored in mutant homozygotes carrying two w-Adh copies (Pal-Bhadra, 2002).

The same RNA blots were hybridized using an Adh probe to estimate the effect on w-Adh/endogenous Adh silencing. The data revealed that the chromosomes carrying either allele of piwi slightly increased the Adh mRNA in the Adh-w flies without w-Adh copies compared to the balancer chromosomes. The effect of the mutant-bearing chromosome may reflect variation at linked modifiers or at Adh itself. In the Adh-w/Y;w-Adh/+ and Adh-w/Y;w-Adh/w-Adh flies, the total Adh expression is significantly reduced as expected when piwi is heterozygous. This Adh expression is minimally increased in piwi homozygotes, although the increase in these classes is likely due to the variation on chromosome 2, rather than a release from silencing. The magnitude of this slight increase is similar with Adh-w alone and in the presence of one or two w-Adh copies, suggesting that the mutations do not interfere with silencing at this step. These data suggest that piwi has no effect on w-Adh/Adh silencing in contrast to the effect on Adh-w (Pal-Bhadra, 2002).

Run-on analysis was conducted on heterozygous and homozygous genotypes to determine the influence of piwi on the transcriptional silencing of Adh-w. The heteroallelic mutant combination has no impact on Adh-w expression in the absence of w-Adh transgenes. In the heterozygous genotypes, introduction of one or two w-Adh copies causes a progressive reduction of Adh-w expression. The magnitude of reduction in expression in the transcriptional assay is quite similar to the Northern analysis, again indicating that the silencing of Adh-w at the adult stage does not appear to have a posttranscriptional component. In the heteroallelic piwi mutant flies, Adh-w transcription is only slightly diminished by the addition of one or two copies of w-Adh. These results indicate that piwi mutations interfere with the transcriptional silencing of Adh-w (Pal-Bhadra, 2002).

Thus, a single transcription unit, namely Alcohol dehydrogenase, can experience two types of transgene silencing: transcriptional and posttranscriptional. The w-Adh/Adh/Adh-w silencing is transcriptional as might have been anticipated from the involvement of the Polycomb chromatin complex. In contrast, the full-length Adh transgene silencing is posttranscriptional. The molecular features of this silencing follow those established from biochemical studies of RNAi. In other words, the specific loss of Adh messenger RNA is accompanied by the appearance of small sense and antisense 21-25 nt length RNAs (referred to as small interfering RNAs, siRNAs). The appearance of these RNAs and the degradation of Adh messenger RNA require a certain threshold over which the silencing is triggered. When these in vivo data are considered together with in vitro analyses of RNAi, it is reasonable to suggest that at a certain concentration of Adh messenger RNA, a double-stranded RNA moiety is transiently formed, presumably by a form of RNA-dependent, RNA polymerase activity. These molecules would then be cleaved to form the siRNAs by an RNase type III nuclease and subsequently incorporated into a larger RNase complex (RISC) to specifically target the homologous mRNA for enzymatic destruction. It is noted, however, that until further study is performed, it remains a formal possibility that the siRNAs form by an alternative pathway that does not involve double-stranded RNA (Pal-Bhadra, 2002).

The piwi gene is a member of a family including the RNAi defective 1 (rde1) gene of C. elegans, which was isolated for its failure to support RNAi. Family members contain a PAZ domain (Cerutti, 2000) that is characteristic of several gene products involved with RNAi. This study demonstrates that piwi mutation blocks the posttranscriptional silencing of Adh, including the production of siRNAs. In contrast, rde1 does not inhibit siRNA formation during RNAi. Another member of this gene family, the aubergine locus, interferes with the germline silencing of the Stellate genes present on the X chromosome (Aravin, 2001). This silencing must occur for male fertility and is accomplished by repeated genes on the Y chromosome that generate siRNAs in conjunction with Stellate. The product of another family member, the argonaute2 gene, is associated with the RISC complex. Thus, several members of this gene family have been implicated in PTGS at various steps (Pal-Bhadra, 2002).

It was of interest to determine whether genes involved with posttranscriptional silencing might also have an impact on transcriptional silencing. Several lines of evidence from plant research have suggested a connection between silencing via RNA in the cytoplasm and changes in the nucleus, although no previous data in animal species have indicated such a relationship. For example, cDNAs of plant viruses (or other sequences incorporated into the virus) transformed into the nucleus are undermethylated until infection by the corresponding virus. The presence of the virus in the cytoplasm undergoing silencing causes a hypermethylation of the sequences in the nucleus. These DNA modifications are coincident in length with the homologous portion carried in the virus. While the mechanism of this RNA-directed DNA methylation is unknown, it is presumed to involve an RNA-DNA interaction. In addition, ectopic transcription of promoter sequences will cause transcriptional silencing of transgenes with a homologous promoter driving another reporter gene (Pal-Bhadra, 2002).

The piwi mutations inhibit the transcriptional silencing of Adh-w. Two aspects of the Adh-w silencing can be assayed by probing for either white or Adh messenger RNA. The silencing of w-Adh and its effect on endogenous Adh can be monitored by examining the amount of Adh RNA. With increasing dosage of w-Adh, the total Adh RNA declines. This trend is not affected by the mutations. The silencing of Adh-w itself can be determined phenotypically and by measuring the amount of w RNA. Typically, the expression of Adh-w declines with increasing dosage of w-Adh, but this response is strongly diminished in homozygotes of piwi (Pal-Bhadra, 2002).

While the evidence presented here suggests a relationship between posttranscriptional and transcriptional silencing, the basis for this connection is unknown. The step in the silencing of Adh-w that is affected by piwi requires the presence in the nucleus of the 5' regulatory sequences of Adh. These sequences are not known to be transcribed under normal circumstances (Pal-Bhadra, 2002).

There are several possibilities to draw a connection between an RNAi-like mechanism and this transcriptional silencing. First, there may be undetected, transient transcription of the Adh regulatory sequences, which in turn form homologous siRNAs. These may act in the nucleus to trigger a chromatin change at the complementary regulatory regions in an analogous fashion to experimental transcription of promoters in tobacco. The piwi mutations might block such a mechanism by inhibiting the formation of the siRNAs homologous to the regulatory sequences. Considering this scenario, in the case of the w-Adh/Adh-w interaction, one must postulate that increased dosage of w-Adh would increase the amount of ectopic transcription of the endogenous Adh regulatory sequences and that the Polycomb complex becomes targeted to Adh-w in conjunction with a homologous interaction between the siRNAs and the regulatory region of Adh-w (Pal-Bhadra, 2002).

If small RNAs trigger transcriptional silencing, it is of interest why w-Adh/Adh silencing is unaffected by piwi. One potential explanation is that the RNA involvement is needed only for the establishment of transcriptional silencing but not its maintenance through to the adult stage. The initiation of w-Adh/Adh silencing in early embryogenesis precedes that of Adh-w. It is possible that maternal contributions of piwi product in early embryos are sufficient for initiation of w-Adh/Adh silencing but are depleted to effective levels by the time of the establishment of Adh-w silencing (Pal-Bhadra, 2002).

Another explanation suggests that the piwi gene product may play dual roles in an RNAi-like mechanism and transcriptional silencing. One possibility is that it could affect some aspect of gene-to-gene association that might trigger transcriptional silencing. Pairing of transgenes or other intranuclear interactions appears to have an impact on transcriptional gene silencing. Indeed, the w-Adh transgene is silenced much more effectively when paired between homologs than when two dispersed copies are present in the nucleus. If the piwi product participates in some aspect of sequence recognition, nucleic acid associations or protein-nucleic acid interactions, its elimination might block certain steps of both posttranscriptional and transcriptional silencing. It remains a possibility that gene-to-gene associations might trigger silencing as well as dsRNA-to-gene interactions with the two acting independently but by a related mechanism. Finally, the connection between the two types of silencing could be indirect, with piwi blocking a function secondarily removed from the transcriptional silencing but that is required nevertheless (Pal-Bhadra, 2002).

Certainly, members of this gene family have diverse roles in the cell. A previously identified function of the nucleoplasmic product of piwi indicates its requirement for germline stem cell renewal. The aubergine product is required for dorsoventral patterning of the early embryo, which is mediated by an enhancement of the translation of the oskar mRNA. The aubergine product is also implicated with the expression of the Stellate (Ste) repeats and Suppressor of Stellate [Su(Ste)] genes on the X and Y chromosomes. The argonaute1 gene is required early in Drosophila embryogenesis for proper development. In mammals, a member of this gene family, eIF2C, functions in translation initiation via tRNA association with messenger RNA, while in Arabidopsis the argonaute mutation affects the functions of the meristem. In C. elegans, Drosophila, and human cells, related genes play a role in the maturation of small regulatory RNAs involved in the temporal control of development. The data presented here suggest that they can also play a direct or indirect role in transcriptional regulation. Clearly, many aspects of development and cellular metabolism use these gene products, raising the possibility that the transposon and virus defense functions may have been co-opted early in evolution from genes involved with other regulatory processes (Pal-Bhadra, 2002).

Mutations in Drosophila heat shock cognate 4 are enhancers of Polycomb

The homeotic genes controlling segment identity in Drosophila are repressed by the Polycomb group of genes (PcG) and are activated by genes of the trithorax group (trxG). An F1 screen for dominant enhancers of Polycomb yielded a point mutation in the heat shock cognate gene, hsc4, along with mutations corresponding to several known PcG loci. The new mutation is a more potent enhancer of Polycomb phenotypes than is an apparent null allele of hsc4, although even the null allele occasionally displays homeotic phenotypes associated with the PcG. Previous biochemical results had suggested that HSC4 might interact with Brahma, a trxG member. Further analyses now show that there is no physical or genetic interaction between HSC4 and the Brahma complex. HSC4 might be needed for the proper folding of a component of the Polycomb repression complex, or it may be a functional member of that complex (Mollaaghababa, 2001).

The hsc4 gene encodes the major heat shock cognate protein, which is expressed at all times of development, but is particularly abundant in ovaries and embryos. A variety of phenotypes have been associated with hsc4 mutations. Removal of both the zygotic and maternal contributions of hsc4 causes embryonic lethality accompanied by variable segmental deletions. Loss of zygotic activity alone leads to larval lethality. These dying larvae develop melanotic tumors and are defective in the development and projection of the larval optic nerve, referred to as Bolwig's nerve. Heterozygotes for all alleles show malformations of the fourth abdominal tergite in a small fraction of adults. A dominant negative allele of hsc4, hsc4195, modulates Notch signaling during development. The same allele enhances the dominant adult phenotypes of mutations in the ecdysone receptor. This study describes the interaction with mutations in the PcG, that constitutes another distinctive class of phenotypes. A seemingly contradictory enhancement of trithorax mutation, itself an antagonist of Polycomb, is reported. One mechanism that might connect all of these phenotypes is the one suggested by the protein sequence of HSC4, namely, the protein folding process. Protein targets of the HSC4 chaperone function might be involved in many diverse developmental pathways (Mollaaghababa, 2001).

The interaction of the hsc454.1 allele with Polycomb is distinctive. The HSC4 protein is not particularly limiting, because heterozygotes for a null allele appear to be nearly wild type. Heterozygotes for hsc454.1 do show a phenotype, suggesting that the hsc454.1 protein product might act as a poison. Perhaps the hsc454.1 protein product sequesters a target protein or misfolds it into a nonfunctional product. If Polycomb were the target, this could inactivate 50% of the Polycomb protein, assuming half of the Polycomb nascent chains interact with the mutant form of HSC4. A 50% reduction in functional Polycomb would explain the weak phenotypes of hsc454.1/+, and the lethality of hsc4 alleles with ph410, because another ph-proximal null, ph409, is lethal in a Pc-/+ background (Mollaaghababa, 2001).

An alternative explanation for the hsc454.1 phenotype is that HSC4 is more directly involved in the process of Polycomb group repression. HSC4 has been identified among the components of the Polycomb complex purified from Drosophila embryos. Because HSC4 is so abundant, it could be merely a contaminant, but the purification includes immunoprecipitation of an epitope-tagged complex, and it was demonstrated that some HSC4 protein coelutes with the complex on a sizing column. It is possible that HSC4 remains bound to one of its targets and becomes incorporated into the complex with that protein. It is also possible that HSC4 has a catalytic role. HSC4 is related to a variety of ATPases, including actin. A bacterial homolog of HSC4, DnaK, is thought to rearrange the protein complex involved in phage lambda DNA replication. Other actin-related proteins have been found in the SWI/SNF and RSC chromatin remodeling complexes, and it has been suggested that they catalyze conformational changes in those complexes or their substrates. HSC4 could have a similar function in PRC1 repression (Mollaaghababa, 2001).

Telomeric associated sequences of Drosophila recruit Polycomb-group proteins in vivo and can induce pairing-sensitive repression

In Drosophila, relocation of a euchromatic gene near centromeric or telomeric heterochromatin often leads to its mosaic silencing. Nevertheless, modifiers of centromeric silencing do not affect telomeric silencing, suggesting that each location requires specific factors. Previous studies suggest that a subset of Polycomb-group (PcG) proteins could be responsible for telomeric silencing. This study presents the effect on telomeric silencing of 50 mutant alleles of the PcG genes and of their counteracting trithorax-group genes. Several combinations of two mutated PcG genes impair telomeric silencing synergistically, revealing that some of these genes are required for telomeric silencing. In situ hybridization and immunostaining experiments on polytene chromosomes reveal a strict correlation between the presence of PcG proteins and that of heterochromatic telomeric associated sequences (TASs), suggesting that TASs and PcG complexes could be associated at telomeres. Furthermore, lines harboring a transgene containing an X-linked TAS subunit and the mini-white reporter gene can exhibit pairing-sensitive repression of the white gene in an orientation-dependent manner. Finally, an additional binding site for PcG proteins was detected at the insertion site of this type of transgene. Taken together, these results demonstrate that PcG proteins bind TASs in vivo and may be major players in Drosophila telomeric position effect (TPE) (Boivin, 2003).

Among the 50 mutant alleles of PcG and trxG genes tested, <10 behave as dominant modifiers of TPE. By contrast, combination analyses reveal that 10 alleles that have no effect alone have synergistic effects on TPE. Interestingly, the subgroup of dominant suppressors that act alone on TPE (Pc, ph, Psc, and Scm) are members of the PRC1 complex that has been purified from embryonic nuclear extracts. Some other PcG mutations, such as Asx, E(z), Pcl, or Sce, act as suppressors in combination, suggesting that the products of these genes participate with a specific telomeric PcG complex. Strikingly, this subgroup of eight PcG genes was already highlighted in a genetic interaction study showing that Pc, Scm, Psc, Pcl, Sce, and Asx are lethal when heterozygous with ph2, a temperature-sensitive mutation, all combinations leading to similar phenotypes in the dying embryos (Boivin, 2003).

It has been shown that telomeric inserts are less accessible than euchromatic inserts to restriction enzymes and to DAM methylase. In addition, the accessibility of telomeric inserts to DAM methylase increases in a ph410 background and this is correlated to derepression of the white gene. This result is similar to that obtained with the ph PRE-mini-white transgenes suggesting that PcG products adopt a similar chromatin-based mechanism to repress their euchromatic and telomeric targets (Boivin, 2003).

PREs were initially identified by their ability to prevent ectopic activation of a Hox reporter gene construct. This capacity depends on the dose of the PcG proteins. Placed in a transgene, PREs can also induce mosaic expression of the flanking reporter gene, a phenotype resembling that of PEV and TPE. Moreover, PRE-mediated repression often exhibits pairing sensitivity, defined as the lower expression of the flanking reporter gene in a homozygous state than in a heterozygous one. This study shows that a 1.2-kb fragment of the 1.8-kb X-chromosome TAS induces variegation or pairing-sensitive repression in an orientation-dependent manner and creates new binding sites for the PcG proteins as detected by immunostaining on polytene chromosomes. These results demonstrate that this TAS fragment mimics some properties of a PRE and thus reinforce the parallels that can be made between telomeric silencing and PcG-mediated euchromatic repression. TASs from the left tip of chromosome 2 (2L-TAS) retain aspects of telomeric silencing in ectopic positions. At this telomere, TASs are composed of repeats of 457 bp that present only limited homology with TASs present at the X, 2R, and 3R telomeres. Analysis of the sequence of one repeat (457 bp) revealed nine GAF-binding sites but no PHO-binding site. Several transgenic lines have been establised carrying different constructs made up of 6 kb of 2L-TAS (~13 repeats) adjacent to the mini-white reporter gene and flanked by Su(Hw) insulator sequences. Depending on the orientation of the TASs inside the transgene, some lines present reduced expression of the mini-white gene when compared to lines carrying a similar transgene without TASs or with TASs in the opposite orientation. Such orientation-dependent silencing has been described for the Fab7 PRE of the Ubx gene, but does not appear to be a general property of PREs since another PRE from Ubx (Mcp) has been shown to function in both orientations . From this study, the more efficient orientation for the 1.2-kb X-TAS-induced repression appears to be the same as that described for the 2L-TASs: repression appears to be stronger from the centromere-proximal side (Boivin, 2003).

Repression induced by the 2L-TAS when inserted within a transgene is weakly sensitive to Su(z)25. Surprisingly, no effect of PcG mutations on the repression induced by the 1.2-kb X-TASs could be detected, except a slight suppressor effect of Su(z)25 on P-CoT-1 in a homozygous state. At the moment, no explanation is available for why the repression induced by the 1.2-kb X-TASs in a euchromatic environment is not sensitive to modification of the dose of PcG proteins that could otherwise affect TPE (Boivin, 2003).

Increasing the distance between the 2L-TAS and the mini-white gene with 2.4 kb of unrelated DNA in another transgene did not change the silencing capacity of 2L-TAS. In this study, the 1.2 kb of X-TAS is located >5 kb away from the mini-white gene, thus showing the silencing capability of TASs over an extended distance. Similar results were obtained with transgenes containing the Fab7 PRE. According to chromatin-immunoprecipitation experiments, PcG products can spread as far as 10-15 kb from PREs and repression could be expected to occur over such a distance (Boivin, 2003).

In fact, what was observed with the 1.2 kb of X-TAS in the pCoT- transgenes resembles what has been observed with PREs from the Bithorax complex. Using Fab7-mini-white transgenes, it has been shown that some insertion sites present pairing sensitivity (as observed with P-CoT-2 and P-CoT-3), while others present variegation with darker spots (as observed with P-CoT-1). The Fab7 PRE has been shown to convey a derepressed state through meiosis after being activated in the embryonic stage by the UAS/GAL4 system. In the case of TPE, the derepressed state observed in a PcG mutant background is not transmitted to the next generation. A fundamental difference between these studies is that the suppressor effect observed in the case of TPE is due to the lack of one PcG partner. It is hyperactivation (forced activation) induced by GAL4 via the UAS sequences that abolishes the repressor capacity of the Fab7 PRE. This activation likely involves fundamental changes in chromatin conformation and/or epigenetic marks (such as hyperacetylation) that may be different from the effect of a decrease in the dosage of a repressor. To compare TPE and the Fab7 PRE it would thus be interesting to test transmission through meiosis of the derepressed state of the UAS-Fab7 transgene induced by a PcG mutation rather than upon activation by GAL4. Different PREs thus share properties but also present particularities that likely depend on their sequence. Indeed, the dissection of Mcp, another PRE from the Bithorax complex, revealed that repression in cis and pairing-sensitive repression could be separated. This shows that PREs may combine several regulatory properties and future dissection of the different TASs will tell which functions telomeric PREs combine (Boivin, 2003).

A mosaic genetic screen reveals distinct roles for trithorax and Polycomb group genes in Drosophila eye development

The wave of differentiation that traverses the Drosophila eye disc requires rapid transitions in gene expression that are controlled by a number of signaling molecules also required in other developmental processes. A mosaic genetic screen has been used to systematically identify autosomal genes required for the normal pattern of photoreceptor differentiation, independent of their requirements for viability. In addition to genes known to be important for eye development and to known and novel components of the Hedgehog, Decapentaplegic, Wingless, Epidermal growth factor receptor, and Notch signaling pathways, several members of the Polycomb and trithorax classes of genes, encoding general transcriptional regulators, were identified. Mutations in these genes disrupt the transitions between zones along the anterior-posterior axis of the eye disc that express different combinations of transcription factors. Different trithorax group genes have very different mutant phenotypes, indicating that target genes differ in their requirements for chromatin remodeling, histone modification, and coactivation factors (Janody, 2004).

Very similar phenotypes were observed in clones mutant for Pc or E(z), which encode components of two distinct complexes implicated in transcriptional repression. Although likely null alleles for both genes were used, the phenotype of E(z) clones appeared slightly stronger, with a greater likelihood of derepressing hth in posterior regions of the eye disc. The E(z) protein has been shown to act as a histone methyltransferase for H3 K27 within a complex that also includes Extra sex combs (Esc), Suppressor of zeste 12 [Su(z)12], and the histone-binding protein NURF-55. esc appears to act only early in embryonic development, while E(z) and Su(z)12 are continuously required to repress inappropriate homeotic gene expression in wing imaginal discs. The core PRC1 complex contains Pc, as well as Ph, Psc, and dRing1, and can prevent SWI/SNF complexes from binding to a chromatin template. Pc, Psc, and ph are all required to prevent homeotic gene misexpression in wing discs; however, Psc and ph act redundantly with closely related adjacent genes. The two complexes are thought to be linked through binding of the Pc chromodomain to K27-methylated H3. The stronger phenotype of E(z) mutations in the eye disc might suggest that methylation of H3 K27 can recruit other proteins in addition to Pc (Janody, 2004).

In the eye disc, loss of E(z) or Pc leads to misexpression of the homeotic gene Ubx, but this does not seem to account for the entire phenotype. Although Ubx is sufficient to turn on tsh ectopically, misexpression of hth and tsh can occur in E(z) or Pc clones in which Ubx is not misexpressed. This suggests that hth and tsh are either direct targets of Pc/E(z)-mediated repression or targets of a downstream gene other than Ubx, possibly one of the homeotic genes not examined. Tsh misexpression would be sufficient to explain the suppression of photoreceptor differentiation in clones close to the morphogenetic furrow, since it is able to maintain expression of Hth and Ey and, in combination with them, to repress eya. Misexpression of Tsh can also account for overgrowth of Pc or E(z) mutant cells at the posterior margin of the eye disc (Janody, 2004).

trithorax group genes were initially identified as suppressors of Polycomb phenotypes and are therefore thought to contribute to the activation of homeotic gene expression. Some members of the group encode components of the Brahma chromatin-remodeling complex, others encode components of the mediator coactivation complex, and still others encode histone methyltransferases. In addition to their distinct biochemical functions, members of the trithorax group act on different sets of target genes during eye development and can also have different effects on the same target genes. Components of the Brahma complex are strongly required for cell growth and/or survival; brm and mor, but not osa, are also absolutely required for photoreceptor differentiation. However, these three genes do not seem to be required for the restricted expression in anterior-posterior domains of the eye disc of the transcription factors examined. In contrast, the mediator complex subunits encoded by skd and kto are not required for cell proliferation, although they are strongly required for photoreceptor differentiation. trx, which encodes a histone methyltransferase, is required primarily for the normal development of marginal regions of the disc. No significant effect on photoreceptor differentiation were seen in clones mutant for kismet1, which encodes chromodomain proteins, or ash21, which encodes a PHD protein. These differences are unlikely to be due to different expression patterns of the trithorax group genes, since Trx, Skd, Kto, and Osa are ubiquitously expressed in the eye disc (Janody, 2004).

The effects of these genes on the rapid transitions between domains of expression of different transcription factors are of particular interest. In the most anterior region of the eye disc, hth expression is enhanced by skd and kto. The domain just posterior to this also expresses tsh and ey, and activation of both of these genes requires trx. However, skd and kto have opposite effects on the two genes, enhancing tsh expression and preventing the maintenance of ey expression in posterior cells. Since Hth and Tsh can positively regulate each other's expression, it is possible that only one of these genes is directly dependent on skd and kto. Next, dac and h are activated transiently and eya is activated and sustained. The establishment of both dac and eya is delayed in trx mutant clones, and h expression is reduced. This delay in establishing the preproneural domain may be due to the failure to activate ey and tsh earlier in development, since Ey and Tsh combine to activate eya. The effect of Pc or E(z) mutations on eya, dac, and h appears very similar to the effect of trx mutations. However, in Pc or E(z) clones, the delay in eya and dac expression is likely to be caused by the failure to repress tsh and hth, since the combination of these two proteins has been shown to repress genes expressed in the preproneural domain. skd and kto clones also show a reduction in h and anterior eya expression, but an inappropriate maintenance of dac and dpp. These mediator complex components may thus contribute both to the activation of genes such as h in the preproneural domain and to the activation of unknown genes that shut off the expression of ey, dac, and dpp. Alternatively, skd and kto could be directly involved in the repression of these genes. Finally, trx is important to prevent misexpression of hth in cells near the posterior and lateral margins. Although Dpp normally represses hth, in trx mutant clones dpp and hth are both inappropriately expressed in marginal cells. This may reflect a role for trx in the process of morphogenetic furrow initiation, perhaps contributing to the ability of dpp to control gene expression (Janody, 2004).

Further study will be needed to determine which genes are direct targets of each trithorax group protein. However, the results point to a strong specificity of these general transcriptional regulators, suggesting that they may be specialized to mediate the effects of particular signaling pathways or to control specific subsets of downstream genes (Janody, 2004).

The Drosophila kismet gene is related to chromatin-remodeling factors and is required for both segmentation and segment identity

The Drosophila trithorax group gene kismet (kis) was identified in a screen for extragenic suppressors of Polycomb (Pc) and subsequently shown to play important roles in both segmentation and the determination of body segment identities. One of the two major proteins encoded by kis (Kis-L) is related to members of the SWI2/SNF2 and CHD families of ATP-dependent chromatin-remodeling factors. To clarify the role of Kis-L in gene expression, its distribution on larval salivary gland polytene chromosomes was examined. Kis-L is associated with virtually all sites of transcriptionally active chromatin in a pattern that largely overlaps that of RNA Polymerase II (Pol II). The levels of elongating Pol II and the elongation factors SPT6 and CHD1 are dramatically reduced on polytene chromosomes from kis mutant larvae. By contrast, the loss of Kis-L function does not affect the binding of PC to chromatin or the recruitment of Pol II to promoters. These data suggest that Kis-L facilitates an early step in transcriptional elongation by Pol II (Srinivasan, 2005).

The Drosophila kismet gene was identified in a screen for dominant suppressors of Polycomb, a repressor of homeotic genes. kismet mutations suppress the Polycomb mutant phenotype by blocking the ectopic transcription of homeotic genes. Loss of zygotic kismet function causes homeotic transformations similar to those associated with loss-of-function mutations in the homeotic genes Sex combs reduced and Abdominal-B. kismet is also required for proper larval body segmentation. Loss of maternal kismet function causes segmentation defects similar to those caused by mutations in the pair-rule gene even-skipped. The kismet gene encodes several large nuclear proteins that are ubiquitously expressed along the anteriorposterior axis. The Kismet proteins contain a domain conserved in the trithorax group protein Brahma and related chromatin-remodeling factors, providing further evidence that alterations in chromatin structure are required to maintain the spatially restricted patterns of homeotic gene transcription (Daubresse, 1999).

The genetic interactions between kis and Pc provided the first clue that kis plays an important role in the determination of body segment identity. kis mutations suppress the adult Pc phenotype by preventing the ectopic transcription of homeotic genes. Thus, kis is a member of the trithorax group of homeotic gene activators. Mosaic analyses reveal that loss of kis function causes homeotic transformations, including the transformation of first leg to second leg and the fifth abdominal segment to a more anterior identity. These phenotypes are identical to those associated with loss-of-function Scr and Abd-B mutations, respectively. Taken together, these findings suggest that kis acts antagonistically to Pc to activate the transcription of both Scr and Abd-B. It is intriguing that kis mutations alter the fate of only the fifth abdominal segment, since the identities of the fifth through ninth abdominal segments are determined by a single homeotic gene, Abd-B (Daubresse, 1999).

Variations in the levels of Abd-B protein result in the differences between these abdominal segments, with Abd-B expression being lowest in the fifth abdominal segment. Parasegment-specific cis-regulatory regions, termed infra-abdominal (iab) regions control Abd-B expression. Each iab region is named for the segment that it affects (iab-5 through iab-9). Mutations in both iab-5 and kis affect the identity of only the fifth abdominal segment, suggesting that the Kis protein may interact specifically with the iab-5 cis-regulatory element of Abd-B (Daubresse, 1999).

kis probably interacts not only with Scr and Abd-B, but with other homeotic genes as well. For example, the isolation of kis mutations as enhancers of loss-of-function Deformed (Dfd) mutations suggests that kis is probably also required to activate transcription of this ANTC homeotic gene. Furthermore, kis duplications strongly enhance the transformation of wing to haltere in Pc heterozygotes, a phenotype caused by the ectopic transcription of Ubx in the wing imaginal disc. However, kis mutations do not cause haltere-to-wing transformations due to decreased Ubx transcription. A possible explanation for the lack of homeotic transformations in kis clones in segments other than the prothoracic and fifth abdominal segment is that the mutations used in these studies are not null alleles. kis1 is a strong loss-of-function mutation. It has not been characterized at the molecular level, however, and may not completely eliminate kis function. It is also possible that sufficient levels of Kis protein persist in homozygous mutant tissue following mitotic recombination to support normal development. Further genetic studies, including the analysis of conditional kis alleles, will be necessary to distinguish between these possibilities (Daubresse, 1999).

Germline clonal analysis has revealed an unanticipated role for kis in segmentation. Embryos from mosaic kisS females exhibit a deletion or alteration of every other segment, while mutant embryos from mothers bearing germline clones of the stronger kis1 allele usually develop only half of the normal number of segments. This variation in phenotypic severity is closely correlated with the extent to which en expression is disrupted. The phenotypes associated with loss of maternal kis function resemble those caused by mutations in pair-rule segmentation genes that cause the deletion of the odd-numbered parasegments. kis thus appears to be necessary for the expression (or function) of one or more pair-rule genes. Recent genetic studies have suggested that kis may also be involved in the Notch signaling pathway. Thus it appears that kis plays roles in addition to the regulation of homeotic genes (Daubresse, 1999).

What pair-rule genes might require kis for their activity? Based on the kis mutant phenotype, perhaps the best candidates are eve and hairy (h), both of which are required for the formation of odd-numbered parasegments. Unlike eve, h and most other segmentation genes, kis is uniformly expressed in the early embryo. This raises the possibility that Kis functions as an essential cofactor or modifier of Eve or other pair-rule proteins. It is also possible that loss of kis function might result in pair-rule genes being transcribed outside of their normal expression domains. Additional work will be necessary to determine the molecular basis of the segmentation defects resulting from loss of maternal kis function (Daubresse, 1999).

The MYST domain acetyltransferase Chameau functions in epigenetic mechanisms of transcriptional repression

Reversible acetylation of histone tails plays an important role in chromatin remodelling and regulation of gene activity. While modification by histone acetyltransferase (HAT) is usually linked to transcriptional activation, evidence is provided for HAT function in several types of epigenetic repression. Chameau (Chm), a new Drosophila member of the MYST HAT family, dominantly suppresses position effect variegation (PEV), is required for the maintenance of Hox gene silencing by Polycomb group (PcG) proteins, and can partially substitute for the MYST Sas2 HAT in yeast telomeric position effect (TPE). Finally, in vivo evidence is provided that the acetyltransferase activity of Chm is required in these processes, since a variant protein mutated in the catalytic domain no longer rescues either PEV modification, telomeric silencing of SAS2-deficient yeast cells, or lethality of chm mutant flies. These findings emphasize the role of an acetyltransferase in gene silencing, which supports, according to the histone code hypothesis, the observation that transcription at a particular locus is determined by a precise combination of histone tail modifications rather than by overall acetylation levels (Grienenberger, 2002).

To examine whether Chm and PcG proteins act together to maintain Hox gene repression, the effect of a reduction of chm dosage was tested on homeotic transformations that result from mutations affecting either PcG transregulators or a PRE cis-regulatory element. The first PcG dominant phenotype examined was a T2 into T1 transformation. In the second leg disc of Pc male heterozygotes, derepression of Sex comb reduced leads to the formation on the second leg of a sex comb, a structure normally found on the first leg only. The mutation of one copy of chm significantly enhances this phenotype. chm and PcG gene interactions in the specification of adult abdomen identities were tested. In parasegment 9 (PS9) of males heterozygous for PcXT109 or for the ph410 allele of polyhomeotic (ph), inappropriate expression of Abdominal-B (Abd-B) produces a mild transformation of the fourth abdominal segment into the fifth (A4 into A5), as evidenced by patches of pigmentation in the anterior part of A4. This phenotype, which is never observed in a wild-type context, occurs at low frequency in males heterozygous for chm (less than 1%). Double heterozygotes for chm and for ph or Pc exhibit increased A4 into A5 transformation and/or increased number of transformed individuals, compared to single PcG mutants. Finally, the homeotic transformation induced by a PRE mutation was examined. McpB116 affects Abd-B silencing in PS9, giving rise to incomplete A4 into A5 transformations. This homeotic phenotype is stronger in double heterozygotes for chm14 and McpB116 and becomes further enhanced by the mutation of one copy of Pc. In the various genetic contexts reported here, chm was therefore found to genetically interact with PcG genes and the Mcp element in a positive manner. These synergistic effects strongly suggest that Chm collaborates with PcG proteins for PRE-mediated repression at Hox gene loci (Grienenberger, 2002).

Direct evidence for a role of Chm in Hox gene silencing was obtained from the examination of Ubx expression in imaginal discs. Whereas Ubx is not detected in the columnar epithelium of a wild-type wing disc, derepression is observed in few cells from discs heterozygous for Pc, and a more extended activation occurs in discs heterozygous for both chm and Pc. These results confirm that Chm and PcG proteins act together to repress Hox genes. No misexpression of Ubx could be detected, however, in discs from chm homozygous larvae. Thus, chm can be classified as an enhancer of PcG mutations instead of a novel PcG gene (Grienenberger, 2002).

Characterization of the grappa gene, a Drosophila histone H3 lysine 79 methyltransferase that interacts genetically with polycomb

grappa (gpp) is the Drosophila ortholog of the Saccharomyces cerevisiae gene Dot1, a histone methyltransferase that modifies the lysine (K)79 residue of histone H3. gpp is an essential gene identified in a genetic screen for dominant suppressors of pairing-dependent silencing, a Polycomb-group (Pc-G)-mediated silencing mechanism necessary for the maintenance phase of Bithorax complex (BX-C) expression. Surprisingly, gpp mutants not only exhibit Pc-G phenotypes, but also display phenotypes characteristic of trithorax-group mutants. gpp dominantly enhances the phenotypic effects of mutations in Sex combs extra, Polycomblike, Sex combs on the midleg, and Polycomb. Mutations in gpp also disrupt telomeric silencing but do not affect centric heterochromatin. These apparent contradictory phenotypes may result from loss of gpp activity in mutants at sites of both active and inactive chromatin domains. Unlike the early histone H3 K4 and K9 methylation patterns, the appearance of methylated K79 during embryogenesis coincides with the maintenance phase of BX-C expression, suggesting that there is a unique role for this chromatin modification in development (Shanower, 2005).

Regulation of cellular plasticity in Drosophila imaginal disc cells by the Polycomb group, trithorax group and lama genes

Drosophila imaginal disc cells can switch fates by transdetermining from one determined state to another. The expression profiles of cells induced by ectopic Wingless expression to transdetermine from leg to wing were examined by dissecting transdetermined cells and hybridizing probes generated by linear RNA amplification to DNA microarrays. Changes in expression levels implicated a number of genes: lamina ancestor, CG12534 (a gene orthologous to mouse augmenter of liver regeneration), Notch pathway members, and the Polycomb and trithorax groups of chromatin regulators. Functional tests revealed that transdetermination was significantly affected in mutants for lama and seven different PcG and trxG genes. These results validate the described methods for expression profiling as a way to analyze developmental programs, and they show that modifications to chromatin structure are key to changes in cell fate. These findings are likely to be relevant to the mechanisms that lead to disease when homologs of Wingless are expressed at abnormal levels and to the manifestation of pluripotency of stem cells (Klebes, 2005).

When prothoracic (1st) leg discs are fragmented and cultivated in vivo, cells in a proximodorsal region known as the 'weak point' can switch fate and transdetermine. These 'weak point' cells give rise to cuticular wing structures. The leg-to-wing switch is regulated, in part, by the expression of the vestigial (vg) gene, which encodes a transcriptional activator that is a key regulator of wing development. vg is not expressed during normal leg development, but it is expressed during normal wing development and in 'weak point' cells that transdetermine from leg to wing. Activation of vg gene expression marks leg-to-wing transdetermination (Klebes, 2005).

Sustained proliferation appears to be a prerequisite for fate change, and conditions that stimulate growth increase the frequency and enlarge the area of transdetermined tissue. Transdetermination was discovered when fragments of discs were allowed to grow for an extensive period of in vivo culture. More recently, ways to express Wg ectopically have been used to stimulate cell division and cell cycle changes in 'weak point' cells (Sustar, 2005), and have been shown to induce transdetermination very efficiently. Experiments were performed to characterize the genes involved in or responsible for transdetermination that is induced by ectopic Wg. Focus was placed on leg-to-wing transdetermination because it is well characterized, it can be efficiently induced and it can be monitored by the expression of a real-time GFP reporter. These attributes make it possible to isolate transdetermining cells as a group distinct from dorsal leg cells, which regenerate, and ventral leg cells in the same disc, which do not regenerate; and, in this work, to directly define their expression profiles. This analysis identified unique expression properties for each of these cell populations. It also identified a number of genes whose change in expression levels may be significant to understanding transdetermination and the factors that influence developmental plasticity. One is lamina ancestor (lama), whose expression correlates with undifferentiated cells and is shown to control the area of transdetermination. Another has sequence similarity to the mammalian augmenter of liver regeneration (Alr; Gfer -- Mouse Genome Informatics), which controls regenerative capacity in the liver and is upregulated in mammalian stem cells. Fifteen regulators of chromatin structure [e.g. members of the Polycomb group (PcG) and trithorax group (trxG)] are differentially regulated in transdetermining cells, and mutants in seven of these genes have significant effects on transdetermination. These studies identify two types of functions that transdetermination requires -- functions that promote an undifferentiated cell state and functions that re-set chromatin structure (Klebes, 2005).

The importance of chromatin structure to the transcriptional state of determined cells makes it reasonable to assume that re-programming cells to different fates entails reorganization of the Polycomb group (PcG) and trithorax group (trxG) protein complexes that bind to regulatory elements. Although altering the distribution of proteins that mediate chromatin states for transcriptional repression and activation need not involve changes in the levels of expression of the PcG and trxG proteins, the array hybridization data was examined to determine if they do. The PcG Suppressor of zeste 2 [Su(z)2] gene had a median fold repression of 2.1 in eight TD to DWg/VWg comparisons, but the cut-off settings did not detect significant enrichment or repression of most of the other PcG or trxG protein genes with either clustering analysis or the method of ranking median ratios. Since criteria for assigning biological significance to levels of change are purely subjective, the transdetermination expression data was re-analyzed to identify genes whose median ratio changes within a 95% confidence level. Fourteen percent of the genes satisfied these conditions. Among these genes, 15/32 PcG and trxG genes (47%) had such statistically significant changes. Identification of these 15 genes with differential expression suggests that transdetermination may be correlated with large-scale remodeling of chromatin structure (Klebes, 2005).

To test if the small but statistically significant changes in the expression of PcG and trxG genes are indicative of a functional role in determination, discs from wild-type, Polycomb (Pc), Enhancer of Polycomb [E(Pc)], Sex comb on midleg (Scm), Enhancer of zeste [E(z)], Su(z)2, brahma (brm) and osa (osa) larvae were examined. The level of Wg induction was adjested to reduce the frequency of transdetermination and both frequency of transdetermination and area of transdetermined cells was determined. The frequency of leg discs expressing vg increased significantly in E(z), Pc, E(Pc), brm and osa mutants, and the frequency of leg to wing transdetermination in adult cuticle increased in Scm, E(z), Pc, E(Pc) and osa mutants. Remarkably, Su(z)2 heterozygous discs had no vg expression, suggesting that the loss of Su(z)2 function limits vg expression (Klebes, 2005).

Members of the PcG and trxG are known to act as heteromeric complexes by binding to cellular memory modules (CMMs). The functional tests demonstrate that mutant alleles for members of both groups have the same functional consequence (they increase transdetermination frequency). The findings are consistent with recent observations that the traditional view of PcG members as repressors and trxG factors as activators might be an oversimplification, and that a more complex interplay of a varying composition of PcG and trxG proteins takes place at individual CMMs. Furthermore the opposing effects of Pc and Su(z)2 functions are consistent with the proposal that Su(z)2 is one of a subset of PcG genes that is required to activate as well as to suppress gene expression. In addition to measuring the frequency of transdetermination, the relative area of vg expression was examined in the various PcG and trxG heterozyogous mutant discs. The relative area decreased in E(Pc), brm and osa mutant discs, despite the increased frequency of transdetermination in these mutants. There is no evidence to explain these contrasting effects, but the roles in transdetermination of seven PcG and trxG genes that were identified by these results support the proposition that the transcriptional state of determined cells is implemented through the controls imposed by the regulators of chromatin structure (Klebes, 2005).

The determined states that direct cells to particular fates or lineages can be remarkably stable and can persist after many cell divisions in alien environments, but they are not immune to change. In Drosophila, three experimental systems have provided opportunities to investigate the mechanisms that lead to switches of determined states. These are: (1) the classic homeotic mutants; (2) the PcG and trxG mutants that affect the capacity of cells to maintain homeotic gene expression, and (3) transdetermination. During normal development, the homeotic genes are expressed in spatially restricted regions, and cells that lose (or gain) homeotic gene function presumably change the transcriptional profiles characteristic of the particular body part. In the work reported here, techniques of micro-dissection, RNA amplification and array hybridization were used to monitor the transcription profiles of cells in normal leg and wing imaginal discs, in leg disc cells that regenerate and in cells that transdetermine from leg to wing. The results validate the idea that changing determined states involves global changes in gene expression. They also identify genes whose function may be unrelated to the specific fates of the cells characterized, but instead may correlate with developmental plasticity (Klebes, 2005).

Overlap between the transcriptional profiles in the wing and transdetermination lists (15 genes) and with genes in subcluster IV (high expression in wing discs) is extensive. The overlap is sufficient to indicate that the TD leg disc cells have changed to a wing-like program of development, but interestingly, not all wing-specific genes are activated in the TD cells. The reasons could be related to the incomplete inventory of wing structures produced (only ventral wing) or to the altered state of the TD cells. During normal development, vg expression is activated in the embryo and continues through the 3rd instar. Although the regulatory sequences responsible for activation in the embryo have not been identified, in 2nd instar wing discs, vg expression is dependent upon the vgBE enhancer, and in 3rd instar wing discs expression is dependent upon the vgQE enhancer. Expression of vg in TD cells depends on activation by the vgBE enhancer, indicating that cells that respond to Wg-induction do not revert to an embryonic state. Recent studies of the cell cycle characteristics of TD cells support this conclusion (Sustar, 2005), but the role of the vgBE enhancer in TD cells and the incomplete inventory of 'wing-specific genes' in their expression profile probably indicates as well the stage at which the TD cells were analyzed: they were not equivalent to the cells of late 3rd instar wing discs (Klebes, 2005).

Investigations into the molecular basis of transdetermination have led to a model in which inputs from the Wg, Dpp and Hh signaling pathways alter the chromatin state of key selector genes to activate the transdetermination pathway. The analyses were limited to a period 2-3 days after the cells switched fate, because several cell doublings were necessary to produce sufficient numbers of marked TD cells. As a consequence, these studies did not analyze the initial stages. Despite this technical limitation, this study identified several genes that are interesting novel markers of transdetermination (e.g., ap, CG12534, CG14059 and CG4914), as well as several genes that function in the transdetermination process (e.g., lama and the PcG genes). The results from transcriptional profiling add significant detail to a general model proposed for transdetermination (Klebes, 2005).

(1) It is reported that ectopic wg expression results in statistically significant changes in the expression of 15 PcG and trxG genes. Moreover, although the magnitudes of these changes were very small for most of these genes, functional assays with seven of these genes revealed remarkably large effects on the metrics used to monitor transdetermination -- the fraction of discs with TD cells, the proportion of disc epithelium that TD cells represent, and the fraction of adult legs with wing cuticle. These effects strongly implicate PcG and trxG genes in the process of transdetermination and suggest that the changes in determined states manifested by transdetermination are either driven by or are enabled by changes in chromatin structure. This conclusion is consistent with the demonstrated roles of PcG and trxG genes in the self-renewing capacity of mouse hematopoietic stem cells, in Wg signaling and in the maintenance of determined states. The results now show that the PcG and trxG functions are also crucial to pluripotency in imaginal disc cells, namely that pluripotency by 'weak point' cells is dependent upon precisely regulated levels of PcG and trxG proteins, and is exquisitely sensitive to reductions in gene dose (Klebes, 2005).

The data do not suggest how the PcG and trxG genes affect transdetermination, but several possible mechanisms deserve consideration. A recent study (Sustar, 2005) reported that transdetermination correlates with an extension of the S phase of the cell cycle. Several proteins involved in cell cycle regulation physically associate with PcG and trxG proteins, and Brahma, one of the proteins that affects the metrics of transdetermination, has been shown to dissociate from chromatin in late S-phase and to reassociate in G1. It is possible that changes in the S-phase of TD cells are a consequence of changes in PcG/trxG protein composition (Klebes, 2005).

Another generic explanation is that transdetermination is dependent or sensitive to expression of specific targets of PcG and trxG genes. Among the 167 Pc/Trx response elements (PRE/TREs) predicted to exist in the Drosophila genome, one is in direct proximity to the vg gene. It is possible that upregulation of vg in TD cells is mediated through this element. Another factor may be the contribution of targets of Wg signaling, since targets of Wg signaling have been shown to be upregulated in osa and brm mutants. These are among a number of likely possible targets, and identifying the sites at which the PcG and trxG proteins function will be necessary if an understand is to be gained of how transdetermination is regulated. Importantly, understanding the roles of such targets and establishing whether these roles are direct will be essential to rationalize how expression levels of individual PcG and trxG genes correlate with the effects of PcG and trxG mutants on transdetermination (Klebes, 2005).

(2) The requirement for lama suggests that proliferation of TD cells involves functions that suppress differentiation. lama expression has been correlated with neural and glial progenitors prior to, but not after, differentiation, and it is observed that lama is expressed in imaginal progenitor cells and in early but not late 3rd instar discs. lama expression is re-activated in leg cells that transdetermine. The upregulation of unpaired in TD cells may be relevant in this context, since the JAK/STAT pathway functions to suppress differentiation and to promote self-renewal of stem cells in the Drosophila testis. It is suggested that it has a similar role in TD cells (Klebes, 2005).

(3) A role for Notch is implied by the expression profiles of several Notch pathway genes. Notch may contribute directly to transdetermination through the activation of the vgBE enhancer [which has a binding site for Su(H)] and of similarly configured sequences that were found to be present in the regulatory regions of 45 other TD genes. It will be important to test whether Notch signaling is required to activate these co-expressed genes, and if it is, to learn what cell-cell interactions and 'community effects' regulate activation of the Notch pathway in TD cells (Klebes, 2005).

(4) The upregulation in TD cells of many genes involved in growth and division, and the identification of DNA replication element (DRE) sites in the regulatory region of many of these genes supports the observation that TD cells become re-programmed after passing through a novel proliferative state (Sustar, 2005), and suggests that this change is in part implemented through DRE-dependent regulation (Klebes, 2005).

There was an interesting correlation between transdetermination induced by Wg mis-expression and the role of Wg/Wnt signaling for stem cells. Wg/Wnt signaling functions as a mitogen and maintains both somatic and germline stem cells in the Drosophila ovary, and mammalian hematopoetic stem cells. Although the 'weak point' cells in the Drosophila leg disc might lack the self-renewing capacity that characterizes stem cells, they respond to Wg mis-expression by manifesting a latent potential for growth and transdetermination. It seems likely that many of the genes are conserved that are involved in regulating stem cells and that lead to disease states when relevant regulatory networks lose their effectiveness (Klebes, 2005).

The prevalence of transcription factors among the genes whose relative expression levels differed most in the tissue comparisons was intriguing. It is commonly assumed that transcription factors function catalytically and that they greatly amplify the production of their targets, so the expectation was that the targets of tissue-specific transcription factors would have the highest degree of tissue-specific expression. In these studies, tissue-specific expression of 15 transcription factors among the 40 top-ranking genes in the wing and leg data sets (38%) is consistent with the large number of differentially expressed genes in these tissues, but these rankings suggest that the targets of these transcription factors are expressed at lower relative levels than the transcription factors that regulate their expression. One possible explanation is that the targets are expressed in both wing and leg disc cells, but the transcription factors that regulate them are not. This would imply that the importance of position-specific regulation lies with the regulator, not the level of expression of the target. Another possibility is that these transcription factors do not act catalytically to amplify the levels of their targets, or do so very inefficiently and require a high concentration of transcription factor to regulate the production of a small number of transcripts. Further analysis will be required to distinguish between these or other explanations, but it is noted that the prevalence of transcription factors in such data sets is neither unique to wing-leg comparisons nor universal (Klebes, 2005).

Polycomb group mutants exhibit mitotic defects in syncytial cell cycles of Drosophila embryos

The Polycomb Group (PcG) of epigenetic regulators maintains the repressed state of Hox genes during development of Drosophila, thereby maintaining the correct patterning of the anteroposterior axis. PcG-mediated inheritance of gene expression patterns must be stable to mitosis to ensure faithful transmission of repressed Hox states during cell division. Previously, two PcG mutants, polyhomeotic and Enhancer of zeste, were shown to exhibit mitotic segregation defects in embryos, and condensation defects in imaginal discs, respectively. polyhomeoticproximal but not polyhomeoticdistal is necessary for mitosis. To test if other PcG genes have roles in mitosis, embryos derived from heterozygous PcG mutant females were examined for mitotic defects. Severe defects in sister chromatid segregation and nuclear fallout, but not condensation are exhibited by Polycomb, Posterior sex combs and Additional sex combs. By contrast, mutations in Enhancer of zeste (which encodes the histone methyltransferase subunit of the Polycomb Repressive Complex 2) exhibit condensation but not segregation defects. It is proposed that these mitotic defects in PcG mutants delay cell cycle progression. Possible mitotic roles for PcG proteins are discussed, and suggest that delays in cell cycle progression might lead to failure of maintenance (O'Dor, 2006).

The data for ph mutations confirm the original observation that ph503 mutations exhibit mitotic defects. These original observations have been confirmed in several ways. First, the observation that strains out-crossed to wild-type flies show similar frequencies of defects compared to heterozygous mutants show that the phenotypes arise from ph mutations rather than background effects. Second, embryos derived from homozygous ph409 mothers show similar frequencies of mitotic defects to those derived from heterozygous mothers. These results suggest that for ph, the severity of the phenotype reaches a plateau when the amount of Ph is reduced below a threshold which must be greater than 50% of the wild-type amount. Third, only php (ph409) is necessary for normal mitosis, because mutations in phd (ph401) have no effect on mitosis. This observation is consistent with data that has shown that only one isoform of Ph-P coimmunoprecipitates with Barren or Topoisomerase II. This observation supports the conclusion that Ph-P and Ph-D have different functions. Fourth, because homozygous ph409 flies are viable, the ph phenotypes reported here represent those of maternal germline nulls (O'Dor, 2006).

The results show that early embryos of PcG and Asx mutants exhibit highly penetrant and expressive mitotic phenotypes in syncytial embryos, consistent with problems in cell cycle progression. Two classes of phenotypes are observed: segregation defects and condensation defects, but no mutant exhibits both phenotypes. In these experiments, with the exception of ph, embryos were scored derived from heterozygous mothers, in which 50% of the wild-type product remain. Therefore, the possibility cannot be ruled out that more severe mitotic phenotypes would be observed in embryos derived from homozygous mothers, resulting in less distinct differences between E(z) and other mutants. Consistent with this caveat, when homozygous E(z)5 (l(3)1902) mutant imaginal disks were examined, both condensation defects and chromosome breakage consistent with problems in segregation were observed, so E(z) may function in both condensation and segregation. The data show that embryos derived from homozygous ph-proximal mutants do not have condensation defects, so at a minimum, E(z) has at least one role in mitosis different from that of ph. To accurately compare the roles of different PcG and ETP genes in mitosis, it will be necessary to examine mutations derived from homozygous mutant mothers, or from germline clones (O'Dor, 2006).

Mutations in the PcG genes Sex combs extra and Sex combs on midleg reduce proliferation of ovarian follicle cells in Drosophila, suggesting that other PcG members are also required for cell cycle progression. It is predicted that other PcG and ETP group mutants not tested here will also exhibit significant mitotic defects (O'Dor, 2006).

Given the high penetrance and expressivity of the chromatin bridge phenotype in PcG mutant embryos, what becomes of the embryos with severe chromatin bridges, and more specifically, what happens to chromatin bridges themselves? Four observations suggest that most chromatin bridges are resolved in PcG mutants. First, nuclear fallout should remove unresolved nuclei, but relatively few fallout nuclei were observed in any embryo. Second, anaphase and telophase embryos together made up 7–9% of the total developed embryos, a proportion that is consistent with the short duration of those mitotic phases. This low proportion of embryos in anaphase or telophase argues that the embryos that exhibited severe chromatin bridges were not developmentally arrested or dead. Third, only a few embryos out of all mutants tested appeared to have bridged prometaphase nuclei. If chromatin bridges did not resolve, one would expect a higher proportion of these prometaphase bridges. Fourth, unresolved chromatin bridges should break. However, fragmented chromosomes, evidence of chromosome breakage and all low-penetrant mitotic defects accounted for only 4–10% of the total mitotic defects observed in mutant embryos (O'Dor, 2006).

C(2)EN embryos carrying an abnormally long second chromosome exhibited chromatin bridges between some nuclei since the extra-long chromosomes were not able to fully segregate during anaphase. These bridged nuclei lagged behind neighboring nuclei and were subsequently removed from the cortex by the fallout mechanism once they reached telophase. Therefore, the fallout nuclei observed in PcG mutants are likely previously-bridged nuclei not able to resolve in time to maintain overall mitotic synchrony. Interestingly, the fallout nuclei were never joined by chromatin bridges. This may be because the delay is only detected once the bridges are resolved, or the bridged nuclei “snap-back” and fuse with each other, as has been observed for bridged nuclei in embryos mutant for grapes, a checkpoint gene required at several cell cycle stages (O'Dor, 2006).

Occasionally, the fallout mechanism may be unable to detect or remove delayed nuclei. If resolved, these nuclei may appear as asynchronous to neighboring nuclei, or, if bridged, they appear as prometaphase bridges, polyploid, giant nuclei or chromosome breaks. The embryos of polyhomeotic mutants develop at a slower rate than those of wild-type flies as judged by timed embryo collections. The slower developmental rate may reflect delays in the mitotic cycles due to segregation defects. In other cases, the most extreme segregation defects overwhelm the fallout mechanisms and continue with the mitotic program until the segregation failures reach a critical point and the embryo dies. In some embryos, the cortex is completely disorganized with very large amorphous nuclei and extensive chromosome breakage. These embryos are probably dead and appear to be the result of cumulative effects of several rounds of segregation defects (O'Dor, 2006).

It remains to be determined whether the length of cell cycle stages in PcG mutants is altered by a checkpoint pathway. In syncytial embryos, the metaphase to anaphase transition is delayed in response to damaged DNA, improper spindle assembly, or faulty centrosome activation. Activation of the spindle checkpoint also delays mitotic progression. It is possible that the mitotic defects of PcG embryos also delay the mitotic cycle by activating a pre-mitotic checkpoint (O'Dor, 2006).

PcG proteins could have a direct structural or enzymatic role in mitosis, separate from their role in silencing. PcG proteins associate with chromatin in a cell cycle-dependent manner. In Drosophila embryos, Polyhomeotic (PH), Polycomb (PC), and Posterior sex combs (PSC) proteins associate with chromatin at S phase, almost completely dissociate by metaphase and reassociate at telophase. BMI1, the human homologue of PSC, shows a similar pattern of association and dissociation in primary and tumor cell lines. Therefore, PcG proteins are present during the key events of mitosis that occur prior to metaphase. An interesting recent report shows that Set1, the yeast homolog of the MP Trithorax, methylates a component of the kinetochore, consistent with the possibility that the methyltransferase activity of E(z) could directly modify proteins needed for mitosis (O'Dor, 2006).

The presence of anaphase bridges in ph, Pc, Psc, and Asx mutants does not necessarily imply that PcG proteins act at anaphase. Mitotic defects may arise at other cell cycle stages but carry forward to manifest as a segregation phenotype. Many different Drosophila cell cycle genes regulating every stage of the cell cycle also have chromatin bridge phenotypes. Some examples include kinesin-like enzymes, a variety of regulatory kinases such as polo kinase and aurora-like kinases, replication checkpoint regulators such as grapes, chk2, and mei-41, and genes involved in sister chromatid segregation such as pimples and three rows (O'Dor, 2006).

PcG proteins could be required at DNA synthesis. Cramped colocalizes with PCNA, which is required for DNA synthesis. Therefore, Cramped, and by extension, other PcG proteins could have a role in elongation of replication forks. RAE28, the homolog of PH, interacts and colocalizes with GEMININ, a replication licensing factor. There have been suggestions in the literature that silenced genes are late-replicating, and this observation has been supported in Drosophila. Therefore, PcG mutations, by interfering with silencing or chromatin structure, could affect replication timing in mitosis. Consistent with this idea, PC tethered near an origin interferes with origin activity. However, it has been reported that an E(z) mutation does not affect replication timing in polytene chromosomes. This may be a reflection of the differences in possible mitotic roles between E(z) and other PcG members. Interestingly, in mammals, heritable gene silencing delays chromatid resolution without affecting timing of DNA replication (O'Dor, 2006).

PcG proteins could also be required for association of sister chromatids. Interaction between PcG Response Elements (PREs), presumably mediated by PcG proteins, is important for repression of PcG targets. Interaction between PcG proteins has been proposed to account for the high likelihood of insertion of PRE-containing transgenes in genomic regions that already contain a PRE. By analogy, PcG proteins could have roles in sister chromatid adhesion or resolution (O'Dor, 2006).

Finally, PcG proteins could be required for chromatin condensation prior to metaphase. This hypothesis is consistent with the E(z) phenotype, in which mitotic chromosomes fail to condense. E(z) is a histone methyltransferase. Histone modifications, notably hypoacetylation and methylation of histone H3 lysine 9 and 27, have been associated with heterochromatin and silencing, consistent with the idea that E(z) might have a role in chromosome condensation. In support of this idea, PH-P coimmunoprecipitates with Barren and Topoisomerase II. Though Barren is a member of the condensin complex, it is not essential for condensation, but is required for sister chromatid resolution. It is speculated that E(z) has a specific role in condensation separate from the role of other PcG proteins, perhaps because its role as a methyltransferase might be required for targets other than histones. It will be interesting to determine if all PRC2 members exhibit condensation defects (O'Dor, 2006).

PcG genes could have indirect effects on mitosis if they are required for regulation of genes that are themselves important for mitosis, or to prevent expression of genes that disrupt mitosis. There are two clear precedents for this possibility. Bmi1, the mammalian Psc homolog originally identified as an oncogene, is also required for regulating lymphoid cell proliferation via repression of the ink4a tumor suppressor locus. Mel18, another mammalian Psc homologue, was originally identified as a tumor suppressor and inhibits cell cycle progression likely via repression of c-myc, leading to downregulation of cyclins and CDKs. If PcG-mediated regulation of proteins important for the cell cycle accounts for the mitotic phenotypes observed in embryos, then this challenges the assumption that maintenance proteins are required only to propagate expression states of genes between cell cycles (O'Dor, 2006).

Steroid hormone-dependent transformation of polyhomeotic mutant neurons in the Drosophila brain

Polyhomeotic (Ph), which forms complexes with other Polycomb-group (PcG) proteins, is widely required for maintenance of cell identity by ensuring differential gene expression patterns in distinct types of cells. Genetic mosaic screens in adult fly brains allow for recovery of a mutation that simultaneously disrupts the tandemly duplicated Drosophila ph transcriptional units. Distinct clones of neurons normally acquire different characteristic projection patterns and can be differentially labeled using various subtype-specific drivers in mosaic brains. Such neuronal diversity is lost without Ph. In response to ecdysone, ph mutant neurons are transformed into cells with unidentifiable projection patterns and indistinguishable gene expression profiles during early metamorphosis. Some subtype-specific neuronal drivers become constitutively activated, while others are constantly suppressed. By contrast, loss of other PcG proteins, including Pc and E(z), causes different neuronal developmental defects; and, consistent with these phenomena, distinct Hox genes are differentially misexpressed in different PcG mutant clones. Taken together, Drosophila Ph is essential for governing neuronal diversity, especially during steroid hormone signaling (Wang, 2006).

Ph is well implicated in maintaining cell fates via controlling transcription of genes in distinct cell type-characteristic manners. Deregulation of multiple genes aberrantly occurs in ph mutant tissues. A similar mechanism probably underlies most of the abnormalities in ph mutant neurons. In particular, there are multiple lines of evidence suggesting mal-expression of various subtype-specific GAL4 drivers in ph mutant clones. First, with respect to GAL4-OK107, GAL4-NP225 and elav-GAL4, the use of various GAL4 drivers results in labeling of similar numbers of clones. Second, clones were induced in the central brain versus the optic lobe, depending on when mitotic recombination was induced; the result is the same as in wild-type mosaic brains. Third, ato-GAL4 and GAL4-EB1 fail to label any clone, arguing against constitutive expression of UAS-transgenes in mutant clones. Finally, examining clones through development reveals no evidence for derivation of some clones from other clones; and, instead, sudden labeling of full-sized clones was constantly observed shortly after a big ecdysone pulse. Apparently, loss of Ph function alone is short of causing the full spectrum of abnormalities. Mass ecdysone is required for the pathological transformation of ph mutant neurons in the Drosophila brain, raising several interesting possibilities about mutual involvement between the epigenetic function of PcG and the global nuclear signaling of steroid hormones (Wang, 2006).

Distinct wild-type cells respond differentially to ecdysone, but ph mutant neurons of distinct origins become no longer distinguishable after ecdysone signaling. Ecdysone mediates diverse biological activities partially via binding to different heterodimeric receptors. Its conventional receptors consist of the nuclear receptor superfamily members ecdysone receptor (EcR) and Ultraspiracle (USP; the Drosophila RXR). There are three documented EcR isoforms; and cells that express different EcR isoforms have been shown to undergo different changes in response to the prepupal ecdysone peak. For example, abundant EcR-B1 exists selectively in the neurons that remodel projections during early metamorphosis. Since no change was observed in EcR expression patterns in ph mutant neurons, it is unlikely that the aberrant responses of ph mutant neurons to the prepupal ecdysone peak occur as a result of derepression of specific EcR isoforms. In addition, derepression of multiple Hox genes appears not to be involved either. Nevertheless, given the involvement of Ph in silencing transcription, it remains possible that derepression of other unidentified genes directly re-programs ecdysone-induced transcriptional hierarchies, leading to transformation of ph mutant neurons. Alternatively, it is possible that loss of the epigenetic function of Ph may permit diffuse activation of prohibited genes by normal transcriptional hierarchies. Moreover, massive steroid hormone signaling might directly modify genomic imprinting when PcG functions are compromised (Wang, 2006).

Ecdysone-dependent transformation of ph mutant neurons provides a possible model system for characterizing the epigenetic functions of steroid hormones. In addition, the demonstration of the unusual potent epigenetic effects of ecdysone in ph mutant neurons suggests complex mechanisms may underlie pathogenesis of other documented PcG loss-of-function phenotypes (Wang, 2006).

Both derepression and inactivation of genes occur in transformed ph mutant neurons, characterization of which offers some molecular insights into this status of transformation. First, the fine-tuning of gene expression in transformed cells was no longer detected; and all the examined drivers appeared either fully on or completely off. Second, on or off could not be simply attributed to the genomic locations of drivers, as evidenced by constitutive silencing of the multiple independently inserted atonal-GAL4 transgenes. Third, transformed cells retained neuron-type morphologies and remained positive for the neuron-specific gene elav; and ph mutant neurons had been earlier reported to acquire normal-looking neurites in culture. Taken together, the transformation leads to loss of subtype identity without affecting basic neuronal fates, abolishes the genomic imprints governing fine controls over gene expression, and locks gene expression in 'on' or 'off' possibly in a promoter-autonomous manner (largely independent of its chromatin environment) (Wang, 2006).

Finally, loss of Ph, Pc, versus E(z) results in distinct phenotypes in the developing fly brain. Differences in their underlying pathological mechanisms are well exemplified by differential derepression of distinct Hox genes in different PcG clones. In addition, for a given PcG mutation, patterns of Hox gene derepression vary from neural clones to wing disc clones and visceral mesoderm. It remains to be elucidated how distinct PcG functions are governed in diverse cell type-characteristic manners (Wang, 2006).

Polycomb group genes are required for neural stem cell survival in postembryonic neurogenesis of Drosophila

Genes of the Polycomb group (PcG) are part of a cellular memory system that maintains appropriate inactive states of Hox gene expression in Drosophila. This study investigates the role of PcG genes in postembryonic development of the Drosophila CNS. Mosaic-based MARCM techniques were used to analyze the role of these genes in the persistent larval neuroblasts and progeny of the central brain and thoracic ganglia. Proliferation in postembryonic neuroblast clones is dramatically reduced in the absence of Polycomb, Sex combs extra, Sex combs on midleg, Enhancer of zeste or Suppressor of zeste 12. The proliferation defects in these PcG mutants are due to the loss of neuroblasts by apoptosis in the mutant clones. Mutation of PcG genes in postembryonic lineages results in the ectopic expression of posterior Hox genes, and experimentally induced misexpression of posterior Hox genes, which in the wild type causes neuroblast death, mimics the PcG loss-of-function phenotype. Significantly, full restoration of wild-type-like properties in the PcG mutant lineages is achieved by blocking apoptosis in the neuroblast clones. These findings indicate that loss of PcG genes leads to aberrant derepression of posterior Hox gene expression in postembryonic neuroblasts, which causes neuroblast death and termination of proliferation in the mutant clones. These findings demonstrate that PcG genes are essential for normal neuroblast survival in the postembryonic CNS of Drosophila. Moreover, together with data on mammalian PcG genes, they imply that repression of aberrant reactivation of Hox genes may be a general and evolutionarily conserved role for PcG genes in CNS development (Bello, 2007).

Genetic analysis indicates that the PcG genes Sce, Scm, Pc, E(z) and Su(z)12 are required for postembryonic neurogenesis in the central brain and thoracic ganglia of Drosophila. In the absence of any one of these genes, several mutant phenotypes are observed in the third-instar CNS: (1) neural proliferation is dramatically reduced and only small numbers of cells are found in neuroblast clones; (2) proliferating postembryonic neuroblasts are absent in most of the mutant clones due to apoptosis; (3) posterior Hox genes are ectopically expressed in the postembryonic neuroblast lineages. It is hypothesized that these phenotypes are causally related, in that loss of PcG genes leads to ectopic Hox gene expression in postembryonic neuroblasts resulting in their premature cell death and, thereby, in drastically reduced neuroblast lineage size. Strong support for this hypothesis is provided by the fact that the mutant lineages proliferate normally if apoptosis is blocked. Corollary support for this notion is provided by the fact that Psc-Su(z)2 mutant clones, which do not show ectopic Hox gene expression, are consistently wild-type-like in size and presence of neuroblast (Bello, 2007).

Numerous genes are required for the continued mitotic activity of neuroblasts during postembryonic life. These findings provide the first demonstration that PcG genes are essential for neuroblast survival and proliferation in the postembryonic CNS. Previous work on PcG gene action during embryonic neurogenesis has demonstrated that the derepression of posterior Hox genes in PcG mutants leads to a change in the segmental determination of neuroblasts and their lineage, but not to their mitotic arrest and death. Thus, the effects of PcG gene loss on neurogenesis are context-dependent and differ during embryonic development as compared with postembryonic development. This is underscored in recent work which indicates that the PcG gene ph is essential for maintaining neuronal identity and diversity during metamorphosis (Bello, 2007).

In postembryonic development of the Drosophila CNS, a remarkable link exists between neuroblast survival and Hox gene expression. In the ventral ganglia, a neuroblast-specific pulse of abd-A during the third instar provides the cue for cell death, which limits the number of progeny produced per neuroblast. These data indicate that this mechanism, which in the wild type relates Hox gene expression to the clone size of neural stem cells, also operates in PcG mutants and is responsible for the PcG mutant phenotypes. Indeed, a general function of PcG genes in postembryonic neurogenesis may be to prevent the premature and widespread operation of this mechanism for temporal regulation of neurogenesis through termination of neuroblast life. It is noteworthy that the Hox gene-dependent activation of apoptosis within the CNS is selective for the neuroblast and does not occur when Hox genes are derepressed in neurons, either during normal development or in misexpression experiments. This explains why the neurons in PcG mutant clones, which were generated before the induction of neuroblast cell death, continue to survive despite the presence of ectopic Hox gene derepression (Bello, 2007).

These data indicate that loss of specific PcG genes in larval neuroblasts leads to ectopic Hox gene expression that is sufficient to cause neuroblast cell death. However, the PcG proteins may also contribute to neuroblast survival by repressing other unidentified target genes which, when derepressed, might result in premature death of postembryonic neuroblasts. Indeed, although deregulation of Hox gene expression is one of the hallmarks of PcG phenotypes in Drosophila, a diverse set of other target genes, including genes involved in cell cycle regulation, are controlled by PcG genes (Bello, 2007).

Interesting parallels to these findings on the role of PcG genes in neural proliferation come from studies of mammalian PcG genes, specifically of the Bmi1 gene. Bmi1 mutant mice develop ataxia, seizures and tremors in early postnatal life, and display a significant reduction in overall brain size, which is particularly severe in the granular and molecular layers of the cerebellum. Strikingly, Bmi1-deficent mice become depleted of cerebellar neural stem cells postnataly, indicating an in vivo requirement for Bmi1 in neural stem cell renewal. Bmi1 deficiency leads to increased expression of the cell cycle regulators p16Ink-4a and p19Arf (both now known as Cdkn2a - Mouse Genome Informatics), and the neurogenesis defect in the mutant mice can be partially rescued by further deleting p16Ink4a. This suggests that one way in which Bmi1 promotes the maintenance of adult stem cells is by repressing the p16Ink4a pathway. However, it is also likely that Hox gene repression through Bmi1 is involved in this process, given that loss of Bmi1 has been shown to cause a deregulation of posterior Hox gene expression in neural stem cells in vitro. Moreover, a direct molecular link between Bmi1 and Hox gene regulation has recently been discovered in mammalian development, in that the promyelocytic leukemia zinc finger (Plzf; Zbtb16 -- Mouse Genome Informatics) protein directly binds Bmi1 and recruits PcG proteins in the HoxD cluster (Bello, 2007 and references therein).

In Drosophila, the homologs of the mammalian Bmi1 gene are the PcG genes Psc and Su(z)2. Psc and Su(z)2 encode very similar proteins and are partially redundant in function, but both genes are eliminated in a deletion in the Psc-Su(z)2 line. Rather surprisingly, mutational loss of Psc-Su(z)2 does not lead to ectopic Hox gene derepression and, in consequence, does not appear to affect neuronal proliferation in the postembryonic CNS of Drosophila. This is in stark contrast to the other five PcG genes investigated, which do play important roles in proliferation control by preventing ectopic Hox gene expression and cell death in postembryonic neuroblasts. The discrepancy between murine Bmi1 and Drosophila Psc-Su(z)2 function in neuronal proliferation suggests that although a general role of PcG genes in neuronal proliferation control may be conserved between mammals and flies, conservation of gene action may not always be retained at the level of individual PcG homologs (Bello, 2007).

In terms of overall development, it is clear that one and the same PcG gene can have very different functions depending on the developmental context in which it acts. For example, as mentioned above, during embryonic neurogenesis the Drosophila Pc gene acts in tagmata-specific differentiation of neuroblasts, in contrast to its role in postembryonic neurogenesis. Moreover, in postembryonic development of imaginal discs, deletions in the Drosophila Psc-Su(z)2 genes have been shown to result in cellular hyperproliferation, which contrasts with the lack-of-proliferation phenotype of Psc-Su(z)2 mutants in postembryonic development of the CNS. Similarly, in the mouse, the Bmi1 gene has been implicated in tumor progression in mantle cell lymphoma, colorectal cancer, liver carcinomas and non-small cell lung cancer, in addition to its role in nervous system development. Nevertheless, in all of the Drosophila and mammalian phenotypes mentioned, deregulation of Hox gene expression appears to be one of the conserved and thus unifying features of PcG gene functional loss (Bello, 2007).

Drosophila Reptin and other TIP60 complex components promote generation of silent chromatin: Interaction between reptin and polycomb

Histone acetyltransferase (HAT) complexes have been linked to activation of transcription. Reptin is a subunit of different chromatin-remodeling complexes, including the TIP60 HAT complex (see Tip60). In Drosophila, Reptin also copurifies with the Polycomb group (PcG) complex PRC1, which maintains genes in a transcriptionally silent state. Genetic interactions have been demonstrated between reptin mutant flies and PcG mutants, resulting in misexpression of the homeotic gene Scr. Genetic interactions are not restricted to PRC1 components, but are also observed with another PcG gene. In reptin homozygous mutant cells, a Polycomb response-element-linked reporter gene is derepressed, whereas endogenous homeotic gene expression is not. Furthermore, reptin mutants suppress position-effect variegation (PEV), a phenomenon resulting from spreading of heterochromatin. These features are shared with three other components of TIP60 complexes, namely Enhancer of Polycomb, Domino, and dMRG15. It is concluded that Drosophila Reptin participates in epigenetic processes leading to a repressive chromatin state as part of the fly TIP60 HAT complex rather than through the PRC1 complex. This shows that the TIP60 complex can promote the generation of silent chromatin (Qi, 2006).

The Drosophila reptin allele l(3)06945 contains a lethal P-element transposon insertion in the 5' untranslated region of the gene. This insertion causes a dramatic reduction in reptin mRNA levels, as determined by in situ hybridization to l(3)06945 homozygous mutant embryos. Whether reptin mutants genetically interact with mutants of PcG genes was tested by creating flies trans-heterozygous for l(3)06945 and PcG genes and male progeny were examined for the presence of ectopic sex combs on the second and third pair of legs. The trans-heterozygous males were compared with their brothers that do not contain mutations in reptin and to PcG mutants crossed to wild-type males. Under the original culture conditions, heterozygotes of the Pc11 allele do not contain extra sex combs, nor do reptin heterozygous flies. However, 47% of males trans-heterozygous for Pc and reptin contain sex combs on the second pair of legs (T2), and 5% additionally contain sex combs on the third pair of legs (T3). Another Pc allele, Df(3L)Pc, causes sex comb development on T2 in 37% of the flies and on T3 in 6% of the flies. The phenotype can be enhanced by reptin to 41% T2 and 25% T3. This genetic interaction indicates that Reptin and Pc participate in the same pathway in vivo (Qi, 2006).

To investigate whether the genetic interaction with PcG genes is restricted to members of the PRC1 complex, trans-heterozygous combinations were examined of reptin with two additional PRC1 complex components, Psc and ph, and with two components of the E(z)–Esc complex, namely esc and Polycomb like (Pcl). 40% of Psc1/+; reptin/+ trans-heterozygotes contain ectopic sex combs on T2 and 2% on T3, whereas 18% of the Psc1/+; TM3/+ brothers have sex combs on T2 and none on T3. Psc1/+ heterozygotes do not contain any extra sex combs. A deficiency that removes Psc, Df(2R)vg-D, only weakly interacts with reptin, and a P-element-induced Psc allele, Psck07834, does not interact with reptin at all. In the ph allele ph-p410, 90% of the males contain sex combs on T2, but none on T3. When crossed to the reptin mutant stock, sex combs were found on both T2 and T3 in all of the Ph410; reptin/+ mutant males, whereas their Ph410 mutant brothers receiving the TM3 balancer chromosome contain extra sex combs on T2 in 100% and on T3 in 22% of the cases, suggesting a specific phreptin interaction. In summary, some alleles of three different PcG genes in the PRC1 complex genetically interact with reptin (Qi, 2006).

Two members of the E(z)–Esc complex, esc and Pcl, were also tested. The esc alleles esc1 and esc21 showed either no or a very weak interaction with reptin mutants. However, the number of flies with extra sex combs in one of the two Pcl alleles, Pcl11, was increased by the reptin mutation. This shows that the ability of reptin to genetically interact with PcG genes is not restricted to components of the PRC1 complex (Qi, 2006).

To confirm that the interactions observed are caused by the P-element insertion in reptin, and not due to unidentified second-site mutations on the l(3)06945 chromosome, precise excisions of the P element were generated. Such excision lines are viable and do not interact with PcG genes. Furthermore, expression of the reptin cDNA using an actin-Gal4 driver transgene could rescue the reptinPc interaction. Under these culture conditions, Pc11 flies contained extra sex combs even in the absence of the reptin mutation. However, the number of sex combs per fly was enhanced by the reptin mutation. Introduction of actin-Gal4 and UAS-reptin transgenes into the Pc11/reptinl(3)06945 trans-heterozygous flies reduced the number of sex combs to below the number observed with Pc11 over the balancer chromosome. From these data, it is concluded that genetic interactions with PcG genes are specifically due to reduced reptin expression in l(3)06945 mutant flies (Qi, 2006).

Sex comb development is under the control of the Hox gene Sex combs reduced (Scr). To determine whether Reptin is involved in regulation of Scr expression, leg imaginal discs of third instar larvae were stained with anti-Scr antibody 6H4.1. Scr protein is found in the first thoracic (T1), but not in the T2 and T3 leg discs in wild-type larvae. reptin/Pc11 and Psc1/+; reptin/+ larvae express Scr protein ectopically in T2 and T3 discs, but reptin, Psc, or Pc heterozygous larvae do not. In conclusion, genetic data show that Reptin interacts with PcG gene products to control Scr expression (Qi, 2006).

Polycomb genes interact with the tumor suppressor genes hippo and warts in the maintenance of Drosophila sensory neuron dendrites

Dendritic fields are important determinants of neuronal function. However, how neurons establish and then maintain their dendritic fields is not well understood. Polycomb group (PcG) genes are required for maintenance of complete and nonoverlapping dendritic coverage of the larval body wall by Drosophila class IV dendrite arborization (da) neurons. In esc, Su(z)12, or Pc mutants, dendritic fields are established normally, but class IV neurons display a gradual loss of dendritic coverage, while axons remain normal in appearance, demonstrating that PcG genes are specifically required for dendrite maintenance. Both multiprotein Polycomb repressor complexes (PRCs) involved in transcriptional silencing are implicated in regulation of dendrite arborization in class IV da neurons, likely through regulation of homeobox (Hox) transcription factors. Genetic interactions and association between PcG proteins and the tumor suppressor kinase Warts (Wts) is demonstrated, providing evidence for their cooperation in multiple developmental processes including dendrite maintenance (Parrish, 2007).

Dendrite arborization patterns are a hallmark of neuronal type; yet how dendritic arbors are maintained after they initially cover their receptive field is an important question that has received relatively little attention. The Drosophila PNS contains different classes of sensory neurons, each of which has a characteristic dendrite arborization pattern, providing a system for analysis of signals required to achieve specific dendrite arborization patterns. Class IV neurons are notable among sensory neurons because they are the only neurons whose dendrites provide a complete, nonredundant coverage of the body wall. This study found tha the function of Polycomb group genes is required specifically in class IV da neurons to regulate dendrite development. In the absence of PcG gene function, class IV dendrites initially cover the proper receptive field but subsequently fail to maintain their coverage of the field. Time-lapse analysis of dendrite development in esc or Pc mutants suggests that a combination of reduced terminal dendrite growth and increased dendrite retraction likely accounts for the gradual loss of dendritic coverage in these mutants. Maintenance of axonal terminals in class IV da neurons is apparently unaffected by loss of PcG gene function, suggesting that PcG genes function as part of a program that specifically regulates dendrite stability (Parrish, 2007).

Establishment of dendritic territories in class IV neurons is regulated by homotypic repulsion, and this process proceeds normally in the absence of PcG function. In PcG mutants, class IV neurons tile the body wall by 48 h AEL, similar to wild-type controls. However, beginning at 48 h AEL, likely as a result of reduced dendritic growth and increased terminal dendrite retraction, class IV neurons of PcG mutants gradually lose their dendritic coverage. In contrast, the axon projections and terminal axonal arbors of PcG mutants show no obvious defects. Although an early role for PcG genes in regulating axon development cannot be ruled out, MARCM studies showed that PcG genes are required for the maintenance of dendrites but not axons in late larval development. Thus, different genetic programs appear to be responsible for the establishment and maintenance of dendritic fields, and for the maintenance of axons and dendrites (Parrish, 2007).

It is well established that PcG genes participate in regulating several important developmental processes including expression of Hox genes for the specification of segmental identity. In comparison, much less is known about the function of PcG genes in neuronal development. Studies of the expression patterns of PcG genes and the consequences of overexpression of PcG genes suggest that PcG genes may affect the patterning of the vertebrate CNS along the anterior-posterior (AP) axis, analogous to their functions in specifying the body plan. A recent study demonstrates that the PcG gene Polyhomeotic regulates aspects of neuronal diversity in the Drosophila CNS. The current study now links the function of PcG genes to maintenance of dendritic coverage of class IV sensory neurons. Thus it will be interesting to determine whether PcG genes play a conserved role in the regulation of dendrite maintenance (Parrish, 2007).

Since Hox genes function in late aspects of neuronal specification and axon morphogenesis, it seems possible that regulation of Hox genes by PcG genes may be important for aspects of post-mitotic neuronal morphogenesis, including dendrite development. The PcG genes esc and E(z) are required for proper down-regulation of BX-C Hox gene expression in class IV neurons. The timing of this change in BX-C expression corresponds to the time frame during which PcG genes are required for dendritic maintenance. Furthermore, post-mitotic overexpression of BX-C genes in class IV da neurons, but not other classes of da neurons, is sufficient to cause defects in dendrite arborization, thus phenocopying the mutant effects of PcG genes. Finally, it was found that Hox genes are required cell-autonomously for dendrite development in class IV neurons, and loss of Hox gene function causes defects in terminal dendrite dynamics that are opposite to the defects caused by loss of PcG genes. Therefore, it seems likely that PcG genes regulate dendrite maintenance in part by temporally regulating BX-C Hox gene expression (Parrish, 2007).

Several recent studies have focused on the identification of direct targets of PcG-mediated silencing, demonstrating that PcG genes regulate expression of distinct classes of genes in different cellular contexts. During Drosophila development, PRC proteins likely associate with >100 distinct loci, and the chromosome-associate profile of PRC proteins appears dynamic. Therefore, identifying the targets of PcG-mediated silencing in a given developmental process has proven difficult. Thus far, alleles of >20 predicted targets of PcG-mediated silencing have been analyzed for roles in establishment or maintenance of dendritic tiling and a potential role has been found for only Hox genes. Future studies will be required to identify additional targets of PcG-mediated silencing in regulation of dendrite maintenance (Parrish, 2007).

PcG genes are broadly expressed, so it seems likely that interactions with other factors or post-translational mechanisms may be responsible for the cell type-specific activity of PcG genes. Indeed, PcG genes genetically interact with components of the Wts signaling pathway to regulate dendrite development specifically in class IV neurons. Based on the observations that wts mutants also show derepression of Ubx in class IV neurons and that Wts can physically associate with PcG components, it seems likely that Wts may directly or indirectly influence the activity of PcG components. In proliferating cells, Wts phosphorylates the transcriptional coactivator Yorkie to regulate cell cycle progression and apoptosis, demonstrating that Wts can directly influence the activity of transcription factors. In support of a possible role for Wts directly modulating PcG function, several recent reports have documented roles for phosphorylation in regulating PcG function both in Drosophila and in vertebrates. Thus, it is possible that some of the components involved in PcG-mediated silencing are regulated by Wts phosphorylation. Alternatively, association of Wts with PcG proteins may facilitate Wts-mediated phosphorylation of chromatin substrates (Parrish, 2007).

The tumor suppressor kinase Hpo regulates both establishment and maintenance of dendritic tiling in class IV neurons through its interactions with Trc and Wts, respectively, but how Hpo coordinately regulates these downstream signaling pathways is currently unknown. Similar to mutations in wts, mutations in PcG genes interact with mutations in hpo to regulate dendrite maintenance but show no obvious interaction with trc, consistent with the observation that PcG gene function is dispensable for establishment of dendritic tiling. Although it is possible that different upstream signals control Hpo-mediated regulation of establishment and maintenance of dendritic tiling, the nature of such signals remain to be determined. Another possibility is that the activity of the Wts/PcG pathway could be antagonized by additional unknown factors that promote establishment of dendritic tiling (Parrish, 2007).

In addition to their interaction in regulating dendrite maintenance, PcG genes and wts interact to regulate expression of the Hox gene Scr during leg development. This finding suggests that the Hpo/Wts pathway may play a general role in contributing to PcG-mediated regulation of Hox gene expression. The presence of ectopic sex combs provides a very simple and sensitive readout of wts/PcG gene interactions and should form the basis for conducting large-scale genetic screens to identify other genes that interact with wts or PcG genes and participate in this genetic pathway (Parrish, 2007).

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

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

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

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

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

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

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

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

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

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

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

Reactive oxygen species prime Drosophila haematopoietic progenitors for differentiation

Reactive oxygen species (ROS), produced during various electron transfer reactions in vivo, are generally considered to be deleterious to cells. In the mammalian haematopoietic system, haematopoietic stem cells contain low levels of ROS. However, unexpectedly, the common myeloid progenitors (CMPs) produce significantly increased levels of ROS. The functional significance of this difference in ROS level in the two progenitor types remains unresolved. This study shows that Drosophila multipotent haematopoietic progenitors, which are largely akin to the mammalian myeloid progenitors, display increased levels of ROS under in vivo physiological conditions, which are downregulated on differentiation. Scavenging the ROS from these haematopoietic progenitors by using in vivo genetic tools retards their differentiation into mature blood cells. Conversely, increasing the haematopoietic progenitor ROS beyond their basal level triggers precocious differentiation into all three mature blood cell types found in Drosophila, through a signalling pathway that involves JNK and FoxO activation as well as Polycomb downregulation. It is concluded that the developmentally regulated, moderately high ROS level in the progenitor population sensitizes them to differentiation, and establishes a signalling role for ROS in the regulation of haematopoietic cell fate. These results lead to a model that could be extended to reveal a probable signalling role for ROS in the differentiation of CMPs in mammalian haematopoietic development and oxidative stress response (Owusu-Ansah, 2009).

The Drosophila lymph gland is a specialized haematopoietic organ which produces three blood cell types -- plasmatocytes, crystal cells and lamellocytes -- with functions reminiscent of the vertebrate myeloid lineage. During the first and early second larval instars, the lymph gland comprises only the progenitor population. However, by late third instar, multipotent stem-like progenitor cells become restricted to the medial region of the primary lymph gland lobe, in an area referred to as the medullary zone; whereas a peripheral zone, referred to as the cortical zone, contains differentiated blood cells. By late third instar, the progenitors within the medullary zone are essentially quiescent, whereas the mature, differentiated population in the cortical zone proliferates extensively. The posterior signalling centre is a group of about 30 cells that secretes several signalling molecules and serves as a stem-cell niche regulating the balance between cells that maintain 'stemness' and those that differentiate (Owusu-Ansah, 2009).

Although several studies have identified factors that regulate the differentiation and maintenance of Drosophila blood cells and the stem-like progenitor population that generates them, intrinsic factors within the stem-like progenitors are less explored. Interrogation of these intrinsic factors is the central theme of this investigation. It was observed that by the third instar, the progenitor population in the normal wild-type lymph gland medullary zone contains significantly increased ROS levels compared with their neighbouring differentiated progeny that express mature blood cell markers in the cortical zone. ROS are not increased during the earlier larval instars but increase as the progenitor cells become quiescent and subside as they differentiate. This first suggested that the rise in ROS primes the relatively quiescent stem-like progenitor cells for differentiation. ROS was reduced by expressing antioxidant scavenger proteins GTPx-1 or catalase, specifically in the progenitor cell compartment using the GAL4/UAS system, and it was found that suppressing increased ROS levels in haematopoietic progenitors significantly retards their differentiation into plasmatocytes. As a corollary, mutating the gene encoding the antioxidant scavenger protein superoxide dismutase (Sod2) led to a significant increase in differentiated cells and decrease in progenitors (Owusu-Ansah, 2009).

ROS levels in cells can be increased by the genetic disruption of complex I proteins of the mitochondrial electron transport chain, such as ND75 and ND42. Unlike in wild type, where early second-instar lymph glands exclusively comprise undifferentiated cells, mitochondrial complex I depletion triggers premature differentiation of the progenitor population. This defect is even more evident in the third instar, where a complete depletion of the progenitors is seen as primary lobes are populated with differentiated plasmatocytes and crystal cells. The third differentiated cell type, the lamellocyte, defined by the expression of the antigen L1, is rarely observed in the wild-type lymph gland but is abundantly seen in the mutant. Finally, the secondary and tertiary lobes, largely undifferentiated in wild type, also embark on a robust program of differentiation upon complex I depletion. Importantly, the phenotype resulting from ND75 disruption can be suppressed by the co-expression of the ROS scavenger protein GTPx-1, which provides a causal link between increased ROS and the premature differentiation phenotype. It is concluded that the normally increased ROS levels in the stem-like progenitors serve as an intrinsic factor that sensitizes the progenitors to differentiation into all three mature cell types. Any further increase or decrease in the level of ROS away from the wild-type level enhances or suppresses differentiation respectively (Owusu-Ansah, 2009).

In unrelated systems, increased ROS levels have been demonstrated to activate the JNK signal transduction pathway. Consequently, it was tested whether the mechanism by which the progenitors in the medullary zone differentiate when ROS levels increase could involve this pathway. The gene puckered (puc) is a downstream target of JNK signalling and its expression has been used extensively to monitor JNK activity. Although puc transcripts are detectable by reverse transcriptase PCR (RT- PCR), the puc-lacZ reporter is very weakly expressed in wild type. After disruption of ND75, however, a robust transcriptional upregulation of puc-lacZ expression can be seen, indicating that JNK signalling is induced in these cells in response to high ROS levels. The precocious progenitor cell differentiation caused by mitochondrial disruption is suppressed upon expressing a dominant negative version of basket (bsk), the sole Drosophila homologue of JNK. This suppression is associated with a decrease in the level of expression of the stress response gene encoding phosphoenol pyruvate carboxykinase; quantitatively a 68% suppression of the ND75 crystal cell phenotype was observed when JNK function was removed as well. Although disrupting JNK signalling suppressed differentiation, ROS levels remain increased in the mutant cells, as would be expected from JNK functioning downstream of ROS (Owusu-Ansah, 2009).

In several systems and organisms, JNK function can be mediated by activation of FoxO as well as through repression of Polycomb activity. FoxO activation can be monitored by the expression of its downstream target Thor, using Thor-lacZ as a transcriptional read-out. This reporter is undetectable in wild-type lymph glands although Thor transcripts are detectable by RT-PCR; however, the reporter is robustly induced when complex I is disrupted, suggesting that the increase in ROS that is mediated by loss of complex I activates FoxO. To monitor Polycomb de-repression, a Polycomb reporter was used that expresses lacZ when Polycomb proteins are downregulated. Although undetectable in wild-type lymph glands, disrupting ND75 leads to lacZ expression suggesting that Polycomb activity is downregulated by the altered ROS and resulting JNK activation. Direct FoxO overexpression causes a remarkable advancement in differentiation to a time as early as the second instar, never seen in wild type. By early third instar, the entire primary and secondary lobes stained for plasmatocyte and crystal cell markers when FoxO is expressed in the progenitor population. Unlike with ROS increase, no a significant increase in lamellocytes was found upon FoxO overexpression. However, downregulating the expression of two polycomb proteins, Polyhomeotic Proximal (Php-x) and Enhancer of Polycomb [E(Pc)], that function downstream of JNK, markedly increased lamellocyte number without affecting plasmatocytes and crystal cells. When FoxO and a transgenic RNA interference (RNAi) construct against E(Pc) are expressed together in the progenitor cell population, differentiation to all three cell types is evident. It is concluded that FoxO activation and Polycomb downregulation act combinatorially downstream of JNK to trigger the full differentiation phenotype: an increase in plasmatocytes and crystal cells due to FoxO activation, and an increase in lamellocytes primarily due to Polycomb downregulation (Owusu-Ansah, 2009).

This analysis of ROS in the wild-type lymph gland highlights a previously unappreciated role for ROS as an intrinsic factor that regulates the differentiation of multipotent haematopoietic progenitors in Drosophila. Any further increase in ROS beyond the developmentally regulated levels, owing to oxidative stress, will cause the progenitors to differentiate into one of three myeloid cell types. It has been reported that the ROS levels in mammalian haematopoietic stem cells is low but that in the CMPs is relatively high. The Drosophila haematopoietic progenitors give rise entirely to a myeloid lineage and therefore are functionally more similar to CMPs than they are to haematopoietic stem cells. It is therefore a remarkable example of conservation to find that they too have high ROS levels. The genetic analysis makes it clear that the high ROS in Drosophila haematopoietic progenitors primes them towards differentiation. It will be interesting to determine whether such a mechanism operates in mammalian CMPs. In mice, as in flies, a function of FoxO is to activate antioxidant scavenger proteins. Consequently, deletion of FoxO increases ROS levels in the mouse haematopoietic stem cell and drives myeloid differentiation. However, even in the mouse haematopoietic system, FoxO function is dose and context dependent, as ROS levels in CMPs are independent of FoxO. Thus, although the basic logic of increased ROS in myeloid progenitors is conserved between flies and mice, the exact function of FoxO in this context may have diverged (Owusu-Ansah, 2009).

Past work has hinted that ROS can function as signalling molecules at physiologically moderate levels. This work supports and further extends this notion. Although excessive ROS is damaging to cells, developmentally regulated ROS production can be beneficial. The finding that ROS levels are moderately high in normal Drosophila haematopoietic progenitors and mammalian CMPs raises the possibility that wanton overdose of antioxidant products may in fact inhibit the formation of cells participating in the innate immune response (Owusu-Ansah, 2009).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Histone H3 lysine(27)-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 shows 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).

Polycomb group genes are required to maintain a binary fate choice in the Drosophila eye

Identifying the mechanisms by which cells remain irreversibly committed to their fates is a critical step toward understanding and being able to manipulate development and homeostasis. Polycomb group (PcG) proteins are chromatin modifiers that maintain transcriptional silencing, and loss of PcG genes causes widespread derepression of many developmentally important genes. However, because of their broad effects, the degree to which PcG proteins are used at specific fate choice points has not been tested. To understand how fate choices are maintained, R7 photoreceptor neuron development has been examined in the fly eye. R1, R6, and R7 neurons are recruited from a pool of equivalent precursors. In order to adopt the R7 fate, these precursors make three binary choices. They: (1) adopt a neuronal fate, as a consequence of high receptor tyrosine kinase (RTK) activity (they would otherwise become non-neuronal support cells); (2) fail to express Seven-up (Svp), as a consequence of Notch (N) activation (they would otherwise express Svp and become R1/R6 neurons); and (3) fail to express Senseless (Sens), as a parallel consequence of N activation (they would otherwise express Sens and become R8 neurons in the absence of Svp). PcG genes were removed specifically from post-mitotic R1/R6/R7 precursors, allowing these genes' roles to be probed in the three binary fate choices that R1/R6/R7 precursors face when differentiating as R7s. This study shows that loss of the PcG genes Sce, Scm, or Pc specifically affects one of the three binary fate choices that R7 precursors must make: mutant R7s derepress Sens and adopt R8 fate characteristics. This fate transformation occurs independently of the PcG genes' canonical role in repressing Hox genes. While N initially establishes Sens repression in R7s, this study shows that N is not required to keep Sens off, nor do these PcG genes act downstream of N. Instead, the PcG genes act independently of N to maintain Sens repression in R1/R6/R7 precursors that adopt the R7 fate. It is concluded that cells can use PcG genes specifically to maintain a subset of their binary fate choices (Finley, 2015).

Identifying the mechanisms by which cells remain irreversibly committed to their fates is a critical step toward understanding and being able to manipulate development and homeostasis. Polycomb group (PcG) proteins are chromatin modifiers that maintain transcriptional silencing, and loss of PcG genes causes widespread derepression of many developmentally important genes. However, because of their broad effects, the degree to which PcG proteins are used at specific fate choice points has not been tested. To understand how fate choices are maintained, R7 photoreceptor neuron development has been examined in the fly eye . R1, R6, and R7 neurons are recruited from a pool of equivalent precursors. In order to adopt the R7 fate, these precursors make three binary choices. They: (1) adopt a neuronal fate, as a consequence of high receptor tyrosine kinase (RTK) activity (they would otherwise become non-neuronal support cells); (2) fail to express Seven-up (Svp) , as a consequence of Notch (N) activation (they would otherwise express Svp and become R1/R6 neurons); and (3) fail to express Senseless (Sens) , as a parallel consequence of N activation (they would otherwise express Sens and become R8 neurons in the absence of Svp). PcG genes were removed specifically from post-mitotic R1/R6/R7 precursors, allowing these genes' roles to be probed in the three binary fate choices that R1/R6/R7 precursors face when differentiating as R7s. This study shows that loss of the PcG genes Sce , Scm , or Pc specifically affects one of the three binary fate choices that R7 precursors must make: mutant R7s derepress Sens and adopt R8 fate characteristics. This fate transformation occurs independently of the PcG genes' canonical role in repressing Hox genes. While N initially establishes Sens repression in R7s, this study shows that N is not required to keep Sens off, nor do these PcG genes act downstream of N. Instead, the PcG genes act independently of N to maintain Sens repression in R1/R6/R7 precursors that adopt the R7 fate. It is concluded that cells can use PcG genes specifically to maintain a subset of their binary fate choices (Finley, 2015).

The GMR-FLP/MARCM system allowed allowed the removal of Sce and Scm function specifically from post-mitotic R1/R6/R7 precursors, allowing these genes' roles to be probed in the limited number of binary fate choices that R1/R6/R7 precursors face. In order to adopt the R7 fate, these precursors must choose to: (1) become neurons in response to high RTK activity-they would otherwise become non-neuronal cells; (2) fail to express Svp in response to N activity-they would otherwise become R1/R6s; and (3) fail to express Sens in response to N activity-they would otherwise become R8s. Loss of Sce or Scm from R7s specifically was found to compromises maintenance of the last of these choices. By contrast, no evidence was found that PcG genes maintain either of the other two choices. Sce mutant R7s were examined throughout larval and pupal development and none were found none misexpressed Svp, nor were Sce or Scm mutant R7s that displayed other R1/R6 characteristics found, such as large rhabdomeres positioned at the periphery of the ommatidium or expression of the R1-R6-specific rhodopsin Rh1. While loss of the Abelson kinase was recently shown to cause R neurons to lose expression of the neuronal marker Elav and switch to a non-neuronal pigment cell fate, this study found that Sce and Scm mutant R1/R6s and R7s maintain expression of Elav and the photoreceptor-specific protein Chaoptin, indicating that their commitment to a neuronal fate is also independent of PcG gene function. It is concluded that R7s use Sce and Scm to maintain repression of one but not all alternative binary fate choices (Finley, 2015).

The Sens-encoding region is bound by Pc in Drosophila embryos and by Sce in Drosophila larvae , suggesting that Sens is directly regulated by these proteins in at least some cell types. However, because of the technical difficulty in isolating sufficient quantities of chromatin specifically from R7 cells, it was not possible to determine whether PcG proteins bind the Sens locus in R7s. It remains possible, therefore, that Sce, Scm, and Pc maintain Sens repression indirectly in R7s-however, the evidence suggests that they do so independently of their canonical role in repressing Hox genes (Finley, 2015).

Considerable differences were observed in the strengths of the R7 defects caused by loss of Sce, Scm, Pc, or Psc. One possibility is that these proteins do not contribute equally to PRC1's gene-silencing ability. Indeed, the fly genome contains a second Psc-related gene that plays a redundant role with Psc in some cells, possibly accounting for the lack of defect in Psc mutant R7s. Alternatively, the different wild-type PcG proteins may perdure to different degrees within the mutant R7 clones (the cells that divide to generate the mutant R1/R6/R7 precursors contain a wild-type copy of the mutant gene). Attempts were made, but it was not possible to measure the time course of Sce and Scm protein levels in Sce and Scm mutant R7s, respectively, to test their perdurance directly. However, this thought that perdurance is likely, as this study found that Gal80 perdures until early pupal development within GMR-FLP/MARCM-induced R7 clones (Finley, 2015).

Sce and Scm were found to be required to maintain Sens repression in R7s generated either in the presence or absence of N activity. What might be regulating the deployment of Sce and Scm in these cells? One possibility is that Sce and Scm repress Sens in R1/R6/R7 precursors by default, since these cells never normally express Sens. However, it was found that neither Sce nor Scm is required to maintain the repression of Sens that is established by Svp. Alternatively, Sce and Scm may be deployed to repress Sens as part of a cell's initial commitment to the R7 fate. As mentioned above, wild-type Sce or Scm protein is likely to perdure in newly created homozygous Sce or Scm mutant R7s, respectively, leaving open the possibility that these genes are required not only for the maintenance but also for the establishment of the R7 fate. Previous work showed that the NF-YC subunit of the heterotrimeric transcription factor nuclear factor Y (NF-Y) is also required to maintain Sens repression in R7s. Like the PcG proteins, NF-YC is broadly expressed in all photoreceptor neurons and is not sufficient to cause R7s to adopt R8 fates, indicating that NF-YC is not responsible for the specific role of PcG proteins in R7s. However, the resemblance between the R7 defects caused by loss of Sce, Scm, and NF-YC suggests that NF-Y may participate in PRC1 function. In support of this possibility, loss of the NF-YA subunit from Caenorhabditis elegans also causes defects similar to those caused by loss of the PcG gene sop-2, including derepression of the Hox gene egl-5 (Finley, 2015).

PcG proteins have been shown to silence many regulators of development in addition to their canonical Hox targets, suggesting that PcG proteins are likely to play broad roles in maintaining cell fate commitments. However, whether PcG proteins are used to maintain specific binary fate choices as cells differentiate is unclear. In fact, the opposite is true during stem cell differentiation, when the repression of terminal differentiation genes by PcG proteins must instead be relieved. This paper has have identified a role for PRC1-associated PcG proteins in maintaining a specific binary fate choice made during adoption of the R7 fate-a choice that does not involve Hox gene regulation or misregulation. The same PRC1-associated proteins are not required to maintain two other binary fate choices that R7s must make. It is concluded that PcG genes are indeed used to maintain some though not all binary fate choices (Finley, 2015).

Polycomb: Biological Overview | Evolutionary Homologs | Regulation | Protein interactions | Developmental Biology | References

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