Polycomb group (PcG) and trithorax Group (trxG) proteins maintain the 'OFF' and 'ON' transcriptional states of HOX genes and other targets by modulation of chromatin structure. In Drosophila, PcG proteins are bound to DNA fragments called Polycomb group response elements (PREs). The prevalent model holds that PcG proteins bind PREs only in cells where the target gene is 'OFF'. Another model posits that transcription through PREs disrupts associated PcG complexes, contributing to the establishment of the 'ON' transcriptional state. These two models were tested at the PcG target gene engrailed. engrailed exists in a gene complex with invected, which together have four well-characterized PREs. The data show that these PREs are not transcribed in embryos or larvae. Tests were performed to see Whether PcG proteins are bound to an engrailed PRE in cells where engrailed is transcribed. By FLAG-tagging PcG proteins and expressing them specifically where engrailed is 'ON' or 'OFF', it was determined that components of three major PcG protein complexes are present at an engrailed PRE in both the 'ON' and 'OFF' transcriptional states in larval tissues. These results show that PcG binding per se does not determine the transcriptional state of engrailed (Langlais, 2012).
In this study sought to learn more about PcG protein complex-mediated regulation of en expression, focusing on mechanisms operating through en PREs. First whether the en and inv PREs are transcribed was investigated, and no evidence of transcription of the PREs was found either by in situ hybridization or by analysis of RNAseq data from the region. It is concluded that transcription of inv or en PREs does not play a role in regulation of en/inv by PcG proteins. Second, using FLAG-tagged PcG proteins expressed in either en or ci cells, it was found that PcG proteins are bound to the en PRE2 in both the 'ON' and 'OFF' transcriptional state in imaginal disks. The data suggest that PcG protein binding to PRE2 is constitutive at the en gene in imaginal disks and that PcG repressive activity must be suppressed or bypassed in the cells that express en (Langlais, 2012).
Transcription through a PRE in a transgene has been shown to inactivate it, and, in the case of the Fab7, bxd, and hedgehog PREs turn them into Trithorax-response elements, where they maintain the active chromatin state. However, is this how PREs work in vivo? Available data suggest that this could be the case for the iab7 PRE. Transcription through the PREs of a few non-HOX PcG target genes, including the en, salm, and tll PREs has been shown by in situ hybridization to embryos. However, in contrast to the robust salm and tll staining, the picture of en stripes using the en PRE probe was very weak and corresponded to a stage where transient invaginations occur that could give the appearance of stripes. Further, there was no hybridization of the en PRE probe to regions of the head, where en is also transcribed at this stage. In situ hybridization experiments with probes to detect transcription of the inv or en PREs did not yield specific staining at any embryonic stage, or in imaginal discs. This finding is confirmed by absence of polyA and non-poly RNA signals in this region at any embryonic or larval stage, upon review of RNA-seq data from ModEncode (Langlais, 2012).
The results show that PcG proteins bind to en PRE2 even in cells where en is actively transcribed. In fact, one member of each of the three major PcG protein complexes, Pho from PhoRC, dRing/Sce from PRC1, and Esc from PRC2, as well as Scm, are constitutively bound to en PRE2 in all cells in imaginal discs. It is noted that dRing/Sce is also present in the PcG complex dRAF, which also includes Psc and the demethylase dKDM2. Further experiments would be necessary to see whether Sce-FLAG is bound to en DNA as part of the PRC1 complex, the dRAF complex, or both (Langlais, 2012).
What are the differences between the 'ON' and 'OFF' transcriptional states? The data suggest that there may be some differences in Pho binding to non-PRE fragments. However, this data has to be interpreted with caution. The en-GAL4 driver is an enhancer trap in the inv intron and contains an en fragment extending from -2.4 kb through the en promoter. Thus, it is possible that the en-GAL4 driver alters Pho binding in the en/inv domain. In fact, the increased Pho-binding to non-PRE probes in the 'ON' versus the 'OFF' state in the FLAG-Sce samples suggests that the presence of the en-GAL4 driver alters Pho binding slightly (Langlais, 2012).
One unexpected result from these experiments was that FLAG-Sce binds to PRE2 but not to PRE1. This is an interesting result that needs to be followed up on. Recent ChIP-Seq data in using imaginal disk/brain larval samples and the anti-Pho antibody show five additional Pho binding peaks between en and tou, which could be five additional PREs. Three of these correspond to known Pho binding peaks. ChIP-seq experiments with the FLAG-tagged proteins expressed in the 'ON' and 'OFF' transcriptional states would be necessary to ask whether the distribution of PcG-proteins is altered at any of the PREs or any other region of the en/inv domain (Langlais, 2012).
In conclusion, the data allows two simple models of PcG-regulation of the en/inv genes to be ruled out. First, the en/inv PREs are not transcribed, so this cannot determine their activity state. Second, PcG proteins bind to at least one of the PREs of the en/inv locus in the 'ON' state, therefore a simple model of PcG-binding determining the activity state of en/inv is not correct. Perhaps the proteins that activate en expression modify the PcG-proteins or the 3D structure of the locus and interfere with PcG-silencing. While FLAG-tagged PcG proteins offer a good tool to study PcG-binding particularly in the 'OFF' state, cell-sorting of en positive and negative cells will be necessary to study the 3D structure and chromatin modification of the en/inv locus (Langlais, 2012).
Covalent modification of histones is important in regulating chromatin dynamics and transcription. One example of such modification is ubiquitination, which mainly occurs on histones H2A and H2B. Although recent studies have uncovered the enzymes involved in histone H2B ubiquitination and a 'cross-talk' between H2B ubiquitination and histone methylation, the responsible enzymes and the functions of H2A ubiquitination are unknown. This study reports the purification and functional characterization of an E3 ubiquitin ligase complex that is specific for histone H2A. The complex, termed hPRC1L (human Polycomb repressive complex 1-like), is composed of several Polycomb-group proteins including Ring1, Ring2, Bmi1 and HPH2. hPRC1L monoubiquitinates nucleosomal histone H2A at lysine 119. Reducing the expression of Ring2 results in a dramatic decrease in the level of ubiquitinated H2A in HeLa cells. Chromatin immunoprecipitation analysis has demonstrated colocalization of dRing with ubiquitinated H2A at the PRE and promoter regions of the Drosophila Ubx gene in wing imaginal discs. Removal of dRing in SL2 tissue culture cells by RNA interference results in loss of H2A ubiquitination concomitant with derepression of Ubx. Thus, these studies identify the H2A ubiquitin ligase, and link H2A ubiquitination to Polycomb silencing (Wang, 2004).
The PRC1 complex contains Psc, Pc, Ph, and Sce proteins. Among these components of the PRC1 complex, it is known that Psc interacts directly with Ph and Pc and that Psc and Ph interact homotypically. Murine and human Ring1/Ring1A and Rnf2/Ring1B interact directly not only with the mammalian homologs of Pc, M33 and Pc2 (Satijn, 1997: Schoorlemmer, 1997), but also with orthologs of Psc such as Bmi1 (Satijn, 1999) and Mel18. In addition, Rnf2/Ring1B interacts with mPH2, a Ph homolog (Hemenway, 1998). To see whether the conservation of the patterns of pairwise interactions between Drosophila PcG protein and their mammalian counterparts also include Sce, its association with Pc, Psc and Ph was studied using an in vitro protein binding assay (Gorfinkiel, 2004).
The complete Sce coding sequence (amino acids 1-435, Sce), and derivatives containing the domains HD1 [Sce amino acids 1-274, Sce(N)] or HD2 and HD3 [amino acids 274-435, Sce(C)] were fused to the glutathione S-transferase (GST) gene, and the resulting hybrid proteins were expressed in Escherichia coli. GST-Sce binds specifically Pc and Psc, but not Ph. Sce(C), but not Sce(N), binds Pc. This shows that Sce binding to Pc occurs through its HD2 and HD3 domains, as previously shown for mammalian Ring1 and Pc proteins. Moreover, the Pc variant lacking the conserved carboxyl domain (PcΔC) does not bind to Sce, a result consistent with previous findings in mammals showing that such domain is responsible for the binding of Pc to Ring. However, binding to Psc occurs preferentially to Sce(N), showing that the interaction between Sce and Psc involves the same domains as the interaction between mammalian Rings and Bmi1 proteins. Sce does not interact with the conserved domain of Ph (amino acids 1297-1576), which mediates homo and heterotypic interactions. Although mouse Rinf2/Ring1B binds Ph (1297-1576), an interaction between Sce and regions in the rest of the Ph protein is still potentially possible. These results indicate that of the interactions among mammalian Ring1/Rnf2 proteins and PcG proteins, at least those between Ring and Pc and Psc are conserved in Drosophila (Gorfinkiel, 2004).
ORD protein is required for accurate chromosome segregation during male and female meiosis in Drosophila melanogaster. Null ord mutations result in random segregation of sister chromatids during both meiotic divisions because cohesion is completely abolished prior to kinetochore capture of microtubules during meiosis I. Previous analyses of mutant ord alleles have led to a proposal that the C-terminal half of the ORD protein mediates protein-protein interactions that are essential for sister-chromatid cohesion. To identify proteins that interact with ORD, a yeast two-hybrid screen was conducted using an ORD bait and isolated dRING, a core subunit of the Drosophila Polycomb repressive complex 1. A missense mutation in ORD completely ablates the two-hybrid interaction with dRING and prevents nuclear retention of the mutant ORD protein in male meiotic cells. Using affinity-purified antibodies generated against full-length recombinant dRING, it is demonstrated that dRING protein is expressed in the male and female gonads and colocalizes extensively with ORD on the chromatin of primary spermatocytes during G2 of meiosis. These results suggest a novel role for the Polycomb group protein dRING and are consistent with the model that interaction of dRING and ORD is required to promote the proper segregation of meiotic chromosomes (Balicky, 2004).
Transcription regulation involves enzyme-mediated changes in chromatin structure. This study describes a novel mode of histone crosstalk during gene silencing, in which histone H2A monoubiquitylation is coupled to the removal of histone H3 Lys 36 dimethylation (H3K36me2). This pathway was uncovered through the identification of dRING-associated factors (dRAF), a novel Polycomb group (PcG) silencing complex harboring the histone H2A ubiquitin ligase dRING, PSC and the F-box protein, and demethylase Lysine (K)-specific demethylase 2 (dKDM2). In vivo, dKDM2 shares many transcriptional targets with Polycomb and counteracts the histone methyltransferases TRX and ASH1. Importantly, cellular depletion and in vitro reconstitution assays revealed that dKDM2 not only mediates H3K36me2 demethylation but is also required for efficient H2A ubiquitylation by dRING/PSC. Thus, dRAF removes an active mark from histone H3 and adds a repressive one to H2A. These findings reveal coordinate trans-histone regulation by a PcG complex to mediate gene repression (Lagarou, 2008).
This study investigated the molecular mechanisms involved in PcG-mediated gene silencing. The major findings of this work are the following. First, a novel PcG silencing complex was idebtufued tat was named dRAF, harboring core subunits dKDM2, dRING, and PSC. Whereas dRING and PSC are also part of PRC1, the other two PRC1 core subunits, PC and PH, are absent from dRAF. In addition, it was found that significant amounts of PSC and PH are not associated with either PRC1 or dRAF, suggesting they might form part of other assemblages. In short, this work suggests a greater diversity among PcG complexes than previously anticipated. Second, genome-wide expression analysis revealed that dKDM2 and PRC1 share a significant number of target genes. Third, it was found that Pc and dkdm2 interact genetically and cooperate in repression of homeotic genes in vivo. Fourth, dKDM2 counteracts homeotic gene activation by the trxG histone methyltransferases TRX and ASH1. Fifth, a novel trans-histone pathway acting during PcG silencing was uncovered. dKDM2 plays a central role by removal of the active H3K36me2 mark and promoting the establishment of the repressive H2Aub mark by dRING/PSC. Finally, the observation that dKDM2 is required for bulk histone H2A ubiquitylation by dRING/PSC, suggests that dRAF rather than PRC1 is the major histone H2A ubiquitylating complex in cells (Lagarou, 2008).
The term trans-histone pathway was first coined to describe that H2B ubiquitylation is required for H3K4 and H3K79 methylation, whereas the reverse is not the case. Recently, it was found that H2Bub determines the binding of Cps35, a key component of the yeast H3K4 methylase COMPASS complex, providing insight in the molecular mechanism by which two positive marks are coupled. This study describes a different type of trans-histone regulation where the removal of the active H3K36me2 mark is directly linked to repressive monoubiquitylation of H2A. A recent study strongly argued that ASH1 mediates H3K36me2. Significantly, the current genetic and biochemical analysis revealed an in vivo antagonism between dKDM2 and ASH1. Thus, dKDM2 appears to reverse the enzymatic activity of trxG protein ASH1 through H3K36 demethylation, whereas it does not affect H3K4 methylation. The observation that chromatin binding of TRX is ASH1 dependent is likely to be part of the explanation of the strong genetic interaction between dkdm2 and trx. The association of the H3K27me2/3 demethylase UTX with the MLL2/3 H3K4 methylase complexes is an example of coupling removal of a repressive mark to the establishment of an active mark (Lagarou, 2008).
This work revealed that the key H2A E3 ubiquitin ligase dRING is part of two distinct complexes, PRC1 and dRAF. A previous study identified the mammalian BCOR corepressor complex, which harbors RING1, NSPC1, and FBXL10 and other proteins, absent from dRAF. These findings suggest that BCOR and dRAF represent a variety of related but distinct silencing complexes. Reduction of dKDM2 caused a dramatic loss of H2Aub levels, which was comparable with that observed after depletion of dRING or PSC. However, knockdown of PRC1 subunits PC or PH had no effect on H2Aub. These observations suggest that dRAF rather than PRC1 is responsible for the majority of H2A ubiquitylation in cells. This notion was reinforced by in vitro reconstitution experiments, suggesting that dRAF is a more potent H2A ubiquitin ligase than PRC1. An unresolved issue remains the molecular mechanisms that underpin the opposing consequences of either H2A or H2B ubiquitylation. It is intriguing that H2Aub appears to be absent in yeast, present but less prominent than H2Bub in Drosophila, and abundant in mammalian cells. An attractive speculation is that H2Aub becomes more important when genome size increases and noncoding regions and transposons need to be silenced (Lagarou, 2008).
In summary, this study identified the PcG complex dRAF, which employs a novel trans-histone pathway to mediate gene silencing. dKDM2 plays a pivotal role by coupling two distinct enzymatic activities, H3K36me2 demethylation and stimulation of H2A ubiquitylation by dRING/PSC. The results indicate that dRAF is required for the majority of H2Aub in the cell. dKDM2 cooperates with PRC1 but counteracts trxG histone methylase ASH1. These findings uncovered a repressive trans-histone mechanism operating during PcG gene silencing (Lagarou, 2008).
The transcriptional status of a gene can be maintained through multiple rounds of cell division during development. This epigenetic effect is believed to reflect heritable changes in chromatin folding and histone modifications or variants at target genes, but little is known about how these chromatin features are inherited through cell division. A particular challenge for maintaining transcription states is DNA replication, which disrupts or dilutes chromatin-associated proteins and histone modifications. PRC1-class Polycomb group protein complexes, consisting of four core PcG subunits, polyhomeotic (Ph), posterior sex combs (PSC), dRING, and Polycomb (Pc), are essential for development and are thought to heritably silence transcription by altering chromatin folding and histone modifications. It is not known whether these complexes and their effects are maintained during DNA replication or subsequently re-established. When PRC1-class Polycomb complex-bound chromatin or DNA is replicated in vitro, Polycomb complexes remain bound to replicated templates. Retention of Polycomb proteins through DNA replication may contribute to maintenance of transcriptional silencing through cell division (Francis, 2009).
The data suggest that PCC is not released into solution during passage of the DNA replication fork. Furthermore, nucleosomes facilitate PCC binding to and retention on templates, but are not essential for either. The finding that PCC can be maintained on either chromatin or naked DNA is interesting in light of the finding that PREs are sites of rapid histone turnover and can be depleted of nucleosomes (Francis, 2009).
One model for the transfer of PCC during DNA replication is that the complex remains in direct contact with DNA during passage of the DNA replication fork. Contacts between PcG proteins and nucleosomes or DNA could be disrupted in front of the replication fork, but replaced by contacts with nucleosomes or DNA behind the replication fork. This mechanism has been proposed for transfers of histone-DNA contacts during replication and transcription in vitro. PCC can likely contact multiple nucleosomes or a long stretch of DNA, which may allow the complex to remain on chromatin when some template contacts are disrupted. A second model is that PCC interacts with the replication machinery, either directly or through intermediary factors. These interactions could retain PCC near DNA during replication, even if direct DNA contacts are disrupted, allowing rapid rebinding of PCC to newly replicated chromatin. Consistent with this idea, several chromatin-modifying proteins can interact with components of the DNA replication machinery (Francis, 2009).
The inhibition of DNA and chromatin replication by PCC in vitro raises the question of how PcG-bound regions are replicated if PRC1-class complexes are indeed continuously bound. If PCC inhibits replication initiation but not elongation, as the results suggest, then PRC1-class complexes would limit replication only if they were bound near replication origins (Francis, 2009).
Intriguingly, targeting of Pc to a replication origin in Drosophila that mediates developmental chorion gene amplification in follicle cells decreased gene amplification (Aggarwal, 2004) and PcG-silenced regions of polytene chromosomes (such as Hox gene clusters) are underreplicated, although this involves additional genes such as Suppressor of DNA Underreplication (Marchetti, 2003; Moshkin, 2001; Francis, 2009 and references therein).
Reduction of PcG protein levels leads to reactivation of their target genes, suggesting that these genes are continuously susceptible to transcriptional activation. It may therefore be important that PRC1-class complexes, which can directly repress transcription, maintain constant association with genes marked for silencing (Francis, 2009).
It was surprising to find that H3K27me3 is not essential for maintaining PRC1-class complexes through DNA replication in vitro. It is possible that retention of parental PRC1-class complexes and recruitment of new complexes are mechanistically distinct because no evidence was found for recruitment of new PCC during replication, and in vivo data suggest that PSC is present on newly replicated chromatin but that additional PSC is recruited after replication. This may be similar to histone proteins in that it is thought that parental histones are transferred randomly to the two daughter strands, followed by deposition of new histones by replication-coupled assembly complexes. In vivo data raise the possibility that recruitment of new PRC1 is not directly coupled to DNA replication; perhaps it involves H3K27me3 (Francis, 2009).
In these experiments, PCC interacts with chromatin through mass action, but in vivo, PRC1-class complexes are specifically targeted to PREs. It is hypothesized that the stable association of PCC with chromatin observed in this study reflects how the complex could behave once it is recruited to a PRE, but it will be important to test this mechanism in a system where PCC is targeted (Francis, 2009).
In conclusion, the ability of parental PCC to be transferred to daughter chromatin may help explain how PcG-mediated repression established by transiently acting factors can be propagated through cell generations. These data also suggest that maintenance of chromatin regulatory proteins through DNA replication might be an important mechanism of epigenetic inheritance (Francis, 2009).
Polycomb group (PcG) proteins are transcriptional repressors that control processes ranging from the maintenance of cell fate decisions and stem cell pluripotency in animals to the control of flowering time in plants. In Drosophila, genetic studies identified more than 15 different PcG proteins that are required to repress homeotic (HOX) and other developmental regulator genes in cells where they must stay inactive. Biochemical analyses established that these PcG proteins exist in distinct multiprotein complexes that bind to and modify chromatin of target genes. Among those, Polycomb repressive complex 1 (PRC1) and the related dRing-associated factors (dRAF) complex contain an E3 ligase activity for monoubiquitination of histone H2A. This study shows that the uncharacterized Drosophila PcG gene calypso encodes the ubiquitin carboxy-terminal hydrolase BAP1. Biochemically purified Calypso exists in a complex with the PcG protein ASX, and this complex, named Polycomb repressive deubiquitinase (PR-DUB), is bound at PcG target genes in Drosophila. Reconstituted recombinant Drosophila and human PR-DUB complexes remove monoubiquitin from H2A but not from H2B in nucleosomes. Drosophila mutants lacking PR-DUB show a strong increase in the levels of monoubiquitinated H2A. A mutation that disrupts the catalytic activity of Calypso, or absence of the ASX subunit abolishes H2A deubiquitination in vitro and HOX gene repression in vivo. Polycomb gene silencing may thus entail a dynamic balance between H2A ubiquitination by PRC1 and dRAF, and H2A deubiquitination by PR-DUB (Scheuermann, 2010; full text of article).
The Drosophila protein Sex Comb on Midleg (Scm) is a member of the Polycomb group (PcG), a set of transcriptional repressors that maintain silencing of homeotic genes during development. Recent findings have identified PcG proteins both as targets for modification by the small ubiquitin-like modifier (SUMO) protein and as catalytic components of the SUMO conjugation pathway. This study found that the SUMO-conjugating enzyme Ubc9 binds to Scm and that this interaction, which requires the Scm C-terminal sterile α motif (SAM) domain, is crucial for the efficient sumoylation of Scm. Scm is associated with the major Polycomb response element (PRE) of the homeotic gene Ultrabithorax (Ubx), and efficient PRE recruitment requires an intact Scm SAM domain. Global reduction of sumoylation augments binding of Scm to the PRE. This is likely to be a direct effect of Scm sumoylation because mutations in the SUMO acceptor sites in Scm enhance its recruitment to the PRE, whereas translational fusion of SUMO to the Scm N terminus interferes with this recruitment. In the metathorax, Ubx expression promotes haltere formation and suppresses wing development. When SUMO levels are reduced, decreased expression of Ubx and partial haltere-to-wing transformation phenotypes were observed. These observations suggest that SUMO negatively regulates Scm function by impeding its recruitment to the Ubx major PRE (Smith, 2011).
Chromatin dependent activation and repression of transcription is regulated by the histone modifying enzymatic activities of the trithorax (trxG) and Polycomb (PcG) proteins. To investigate the mechanisms underlying their mutual antagonistic activities this study analyzed the function of Drosophila Ring and YY1 Binding Protein (dRYBP), a conserved PcG- and trxG-associated protein. dRYBP is ubiquitylated and binds ubiquitylated proteins. Additionally dRYBP was shown to maintain H2A monoubiquitylation, H3K4 monomethylation and H3K36 dimethylation levels and does not affect H3K27 trimethylation levels. Further it was shown that dRYBP interacts with the repressive SCE (Ring) and dKDM2 (Lysine (K)-specific demethylase 2) proteins as well as the activating dBRE1 protein. Analysis of homeotic phenotypes and post-translationally modified histones levels show that dRYBP antagonizes dKDM2 and dBRE1 functions by respectively preventing H3K36me2 demethylation and H2B monoubiquitylation. Interestingly, the results show that inactivation of dBRE1 produces trithorax-like related homeotic transformations, suggesting that dBRE1 functions in the regulation of homeotic genes expression. These findings indicate that dRYBP regulates morphogenesis by counteracting transcriptional repression and activation. Thus, they suggest that dRYBP may participate in the epigenetic plasticity important during normal and pathological development (Fereres, 2014).
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