Sex combs on midleg

Polycomb group proteins bind an engrailed PRE in both the 'ON' and 'OFF' transcriptional states of engrailed

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).

Protein Interactions

The Polycomb group (PcG) genes are required for maintenance of homeotic gene repression during development. Mutations in these genes can be suppressed by mutations in genes of the SWI/SNF family. A complex, termed PRC1 (Polycomb repressive complex 1), has been purified that contains the products of the PcG genes Polycomb, Posterior sex combs, polyhomeotic, Sex combs on midleg, and several other proteins. Preincubation of PRC1 with nucleosomal arrays blocks the ability of these arrays to be remodeled by SWI/SNF. Addition of PRC1 to arrays at the same time as SWI/SNF does not block remodeling. Thus, PRC1 and SWI/SNF might compete with each other for the nucleosomal template. Several different types of repressive complexes, including deacetylases, interact with histone tails. In contrast, PRC1 is active on nucleosomal arrays formed with tailless histones (Shao, 1999).

It is apparent from the composition of PRC1 that there must be other PcG complexes in addition to PRC1. PRC1 purified via either tagged PH or PSC contains Pc, Psc, Ph-p, Ph-d, and Scm, as well as several other proteins. PRC1 does not contain Pcl and E(z). Previous studies using immunoprecipitation, in vitro binding, and/or yeast two-hybrid analysis have shown that Pc, Psc, and Ph interact with each other, and that Scm interacts with Ph. E(z) and Esc have been shown to interact with each other by similar approaches, and E(z) separates from PRC1 during chromatography. Similarly, mammalian homologs of PcG can also be separated into roughly two complexes, one containing homologs to Pc, Psc, and Ph, and the other containing homologs to E(z) and Esc. Another argument that E(z) and Esc form a separate complex with a distinct function is based on the observation that homologs to these genes are found in the C. elegans genome, whereas homologs to Pc, Ph, or Psc are not. The activities of PRC1 suggest that it may be directly involved in creating the repressed state, and that it may require other complexes for targeting. Through screens for homeotic derepression, 14 PcG genes have been well characterized genetically. It is possible that a subset of these genes are required for direct repression, while other PcG proteins function in targeting, regulation of repression activity, or maintenance of the repressed state through mitosis. How PcG proteins are recruited to their targets is still unknown, but several proteins have been suggested as candidates for this function, such as Esc and E(z), and sequence-specific DNA-binding proteins Pho, Trithorax-like, Hunchback (Hb), and the Hb interacting protein dMi-2. PRC1 does not contain E(z) and Trithorax-like. Using antibodies against a region of human YY1 that is conserved in Pho, it was found that Pho is unlikely to be in PRC1; an antibody made specifically against Pho is needed to verify this result. Due to the lack of antibodies, whether PRC1 contains Esc, Hb, or dMi-2 was not tested (Shao, 1999 and references).

A tethering assay was developed to study the effects of Polycomb group (PcG) proteins on gene expression in vivo. This system employed the Su(Hw) DNA-binding domain (ZnF) to direct PcG proteins to transposons that carried the white and yellow reporter genes. These reporters constituted naive sensors of PcG effects, since bona fide PcG response elements (PREs) were absent from the constructs. To assess the effects of different genomic environments, reporter transposons integrated at nearly 40 chromosomal sites were analyzed. Three PcG fusion proteins, ZnF-PC, ZnF-SCM, and ZnF-ESC, were studied, since biochemical analyses place these PcG proteins in distinct complexes. Tethered ZnF-PcG proteins repress white and yellow expression at the majority of sites tested, with each fusion protein displaying a characteristic degree of silencing. Repression by ZnF-PC is stronger than ZnF-SCM, which is stronger than ZnF-ESC, as judged by the percentage of insertion lines affected and the magnitude of the conferred repression. ZnF-PcG repression is more effective at centric and telomeric reporter insertion sites, as compared to euchromatic sites. ZnF-PcG proteins tethered as far as 3.0 kb away from the target promoter produce silencing, indicating that these effects are long range. Repression by ZnF-SCM requires a protein interaction domain, the SPM domain, which suggests that this domain is not primarily used to direct SCM to chromosomal loci. This targeting system is useful for studying protein domains and mechanisms involved in PcG repression in vivo (Roseman, 2001).

Biochemical studies indicate that PcG repression involves multiple, distinct PcG complexes. Thus, an underlying assumption of the assay system that was used is that gene silencing by the tethered ZnF-PcG protein involves assembly with endogenous PcG proteins at the reporter site. This hypothesis leads to the prediction that repression by a tethered ZnF-PcG protein should be compromised by loss of function for an endogenous PcG partner. It was difficult to test this prediction for the comprehensive set of endogenous PcG proteins because the basic assay system involved generating a very complex genotype. Nevertheless, the requirement was tested for endogenous PH protein, which is encoded by an X-linked gene and for which a hemizygous viable allele is available (Roseman, 2001).

A reporter integration site was identified that normally lacks PH binding, as scored on polytene chromosomes: it was repressed by all three ZnF-PcG proteins. Genetic tests show that reduction in PH dosage relieves tether-based repression by PC and SCM at this site. These results can be reconciled with the known PC-PH and SCM-PH molecular interactions. Surprisingly, ZnF-ESC repression is sensitive to PH dosage. This result was not expected since ESC-PH interactions have not been reported and there is evidence that ESC and PH are in separate complexes in embryos (Roseman, 2001).

Several explanations may account for the effect of PH dosage upon ZnF-ESC repression. (1) Since only a single reporter site was investigated, the PH dependency at this site may not be a general property at other genomic sites. It is noted, however, that this reporter site was chosen for analysis because polytene chromosome immunostaining studies indicate that it is not pre-equipped with endogenous PH. (2) Alternatively, it is possible that the functions of biochemically separable PcG complexes are interdependent in vivo, at least at certain loci. This could also explain the basic observation that PC and ESC are both required for repression at homeotic loci even though they sort into distinct complexes. An excellent example of the interplay between distinct chromatin complexes at a single locus is provided by regulation of the HO gene in yeast. Both the SWI/SNF nucleosome remodeling complex and the SAGA histone acetyltransferase complex are required for HO activation in vivo. These complexes cooperate in an ordered series of events, wherein SWI/SNF action is a prerequisite for SAGA activity upon HO chromatin. (3) Similarly, loci that require multiple PcG complexes for transcriptional repression may use a multistep mechanism where one PcG complex alters the chromatin template to 'pave the way' for binding or action of another PcG complex. Indeed, this type of interplay could explain the observation that E(Z) function is required for association of the PRC1 components PSC and PH at many chromosomal sites (Roseman, 2001).

The carboxyl-terminal SPM domain of SCM is highly conserved in mammalian SCM homologs. Analyses of Scm mutant alleles that remove the SPM domain, together with site-directed mutational analysis, have shown that the SPM domain is required for SCM function in vivo. Although in vitro studies indicate that the SPM domain is a protein interaction module, the functional contribution of this domain to SCM repression in vivo is not known. One possible role for the SPM domain would be to recruit SCM to target sites in chromatin, by analogy to the role that the conserved chromodomain plays in chromatin targeting of PC. Deletion of the SPM domain in ZnF-SCM^DeltaSPM abolishes silencing in the tethering system. Although ZnF-SCM^DeltaSPM accumulates to a level similar to that of wild-type ZnF-SCM, its repression activity is indistinguishable from ZnF alone. These results imply that the SPM domain does not solely provide interactions that target SCM to chromosomes, since the Su(Hw)-binding domain circumvents this targeting function. Instead, it is suggested that the SPM domain is more directly involved in the repression mechanism or in maintaining integrity of SCM complexes (Roseman, 2001).

Requirement for sex comb on midleg protein interactions in Drosophila polycomb group repression

The Drosophila Sex comb on midleg (Scm) protein is a transcriptional repressor of the Polycomb group (PcG). Although genetic studies establish Scm as a crucial PcG member, its molecular role is not known. To investigate how Scm might link to PcG complexes, the in vivo role of a conserved protein interaction module, the SPM domain was analyzed. This domain is found in Scm and in another PcG protein, Polyhomeotic (Ph), which is a core component of Polycomb repressive complex 1 (PRC1). Scm-Ph interactions in vitro are mediated by their respective SPM domains. Yeast two-hybrid and in vitro binding assays were used to isolate and characterize greater than 30 missense mutations in the SPM domain of Scm. Genetic rescue assays show that Scm repressor function in vivo is disrupted by mutations that impair SPM domain interactions in vitro. Furthermore, overexpression of an isolated, wild-type SPM domain produced PcG loss-of-function phenotypes in flies. Coassembly of Scm with a reconstituted PRC1 core complex shows that Scm can partner with PRC1. However, gel filtration chromatography showed that the bulk of Scm is biochemically separable from Ph in embryo nuclear extracts. These results suggest that Scm, although not a core component of PRC1, interacts and functions with PRC1 in gene silencing (Peterson, 2004).

Purifications of nuclear complexes and in vitro studies have identified eight proteins that are core components of two distinct fly PcG complexes: Esc, E(z), Su(z)12, and NURF-55 in the ESC-E(Z) complex plus Pc, Ph, Psc, and dRING1 in PRC1. One function of the Esc-E(z) complex is histone H3 methylation on K27. Further studies are needed to address whether the Esc-E(z) complex has additional functions. The molecular mechanism of PRC1 is not yet known. Studies to date suggest that it represses transcription through a noncatalytic mechanism that restricts template access, but it is not yet clear how PRC1 molecularly affects nucleosome array organization and/or packaging of the chromatin fiber. Since genetic studies in Drosophila identify at least 15 genes involved in PcG repression, many additional components need to be fit into the framework of PcG complexes and functions. In addition to identifying the players, analyses of loss of function for individual PcG genes distinguishes those repressors with central PcG roles from those that are more peripheral. In good agreement with the biochemical studies, loss of function for core subunits of either PcG complex produces severe homeotic defects. These mutants show robust Hox misexpression and die as embryos with most segments transformed into copies of the eighth abdominal segment. By these criteria, Scm is clearly a central player in the PcG repression system. In contrast, other repressors such as Asx and Pcl appear more peripheral since their complete loss from embryos yields significantly weaker homeotic defects (Peterson, 2004).

In this work, a combination of in vivo and in vitro approaches are presented to address Scm molecular function. Mutational analysis shows that Scm function absolutely depends upon an intact SPM protein interaction domain. There is a strong correlation between disruption of protein interactions in vitro and failure of Scm function in vivo. These results agree with the finding that Scm repressor function in an in vivo tethering assay requires its SPM domain. The importance of SPM domain interactions is also revealed by PcG loss-of-function phenotypes produced by overexpression of an isolated SPM domain. It is suggested that this dominant negative reflects SPM domain interactions critical for PcG repression that are disrupted by this avidly binding but otherwise nonfunctional competitor. The embryonic lethality of SPM domain mutants, together with embryonic and imaginal defects seen with SPM overexpression, indicate that SPM interactions contribute to PcG repression at both embryonic and postembryonic times. Thus, these interactions appear required for long-term maintenance of PcG silencing in vivo (Peterson, 2004).

The biochemical properties of the SPM domain suggest three potential types of Scm interactions in vivo: (1) binding to PRC1, (2) binding to other fly SPM domain proteins, or (3) binding to itself. Although the data do not rule out contributions from any of these, several lines of evidence favor Scm interaction and function with PRC1. (1) in vivo evidence derives from studies showing that Scm can repress reporter genes when tethered by fusion to a DNA-binding domain. Since this repression depends upon Ph function, Scm cannot repress on its own but rather requires PRC1 to repress in this context. (2) Substoichiometric quantities of Scm consistently copurify with tagged PRC1 complexes from both fly and mammalian extracts. Although the majority of Scm appears to not be stably bound, the conserved association of some Scm with purified PRC1 likely reflects in vivo interactions. (3) No stably associated partner proteins have been detected that copurify when FLAG-Scm is affinity purified from embryo extracts. Thus, there is no evidence for a heteromeric Scm-containing complex that could repress independently of PRC1 (Peterson, 2004).

If Scm does work with PRC1, then what might explain its substoichiometric association with purified PRC1? One possibility is that Scm assembles only into a subset of PRC1 complexes, perhaps restricted to certain tissues or times of development. Such a model has been proposed to explain how Asx contributes to PcG repression in the embryonic epidermis but not in the central nervous system. This explanation for Scm, however, is not favored because its requirement in PcG repression is widespread in both embryonic and imaginal tissues. Another possibility is that Scm interaction with PRC1 is robust in chromatin but is not fully preserved during preparation of soluble nuclear extracts used in purification. In this view, nucleosome arrays might provide a platform that promotes Scm-PRC1 binding. Indeed, both PRC1 and Scm have affinity in vitro for nucleosome arrays. Additional in vitro studies will be needed to address the nature of Scm-PRC1 interactions in the context of chromatin templates. It is noted that the GAGA factor provides an example of a protein that is not stably associated with PRC1 in embryo extracts but can nevertheless help recruit PRC1 to nucleosomal templates in vitro (Peterson, 2004).

At present, the evidence favors a noncatalytic role for PRC1 in PcG repression. How might Scm, which also lacks recognizable catalytic domains, contribute to PRC1 mechanism? Recent in vitro studies show that mouse PRC1 bound to a single nucleosome array can recruit a second chromatin template that then also becomes repressed. These bridging interactions between repressed templates in vitro may reflect the PcG-dependent chromosome-pairing and chromosome-chromosome interactions frequently observed in vivo. Thus, one role of PRC1 may be to promote higher-order chromosome interactions that spread or stabilize repression. Intriguingly, among the core PRC1 components, the mouse Ph protein was found most critical for in vitro bridging activity. Since Ph is the key subunit that mediates Scm interaction with PRC1, the possibility is raised that Scm could facilitate PRC1-mediated long-distance chromatin interactions. In this view, Scm might work by helping to anchor PRE-promoter and/or PRE-PRE interactions needed for PcG repression in vivo (Peterson, 2004).

A second type of potential Scm-PRC1 partnership in chromatin has been proposed on the basis of structural properties of the SPM domain. The SPM domain of fly Ph, determined by X-ray crystallography, is a five-helix bundle that has the special property of forming helical self-polymers in vitro. The possibility of an extended protein polymer that could bind alongside nucleosome arrays has prompted speculation that SPM proteins might organize higher-order chromatin arrangements. In such a model, SPM domain-containing proteins or complexes form a core helical polymer around which the chromatin fiber could be wrapped. This model, although speculative, is appealing since it brings structural data to bear upon the long-standing hypothesis that PcG proteins create extended tracts of repressed chromatin. Intriguingly, when mixed together, the SPM domains of Ph and Scm can also form copolymers in vitro. Thus, PH and Scm could collaborate in forming the proposed higher-order chromatin structures. In this context, the dominant-negative properties of overexpressed SPM domain could reflect disruption of contacts needed to produce PH-Scm chromatin polymers. To evaluate this model, it will be necessary to test if full-length PcG proteins or their intact complexes can form polymers in vitro like those seen for their isolated SPM domains. If so, then further studies would need to address the existence and roles of such polymers in vivo (Peterson, 2004).

Structural organization of a Sex-comb-on-midleg/Polyhomeotic copolymer

The polycomb group proteins are required for the stable maintenance of gene repression patterns established during development. They function as part of large multiprotein complexes created via a multitude of protein-protein interaction domains. This study examines the interaction between the SAM (sterile alpha motif) domains of the polycomb group proteins polyhomeotic (Ph) and Sex-comb-on-midleg (Scm). Ph-SAM polymerizes as a helical structure. Scm-SAM also polymerizes, and a crystal structure reveals an architecture similar to the Ph-SAM polymer. These results suggest that Ph-SAM and Scm-SAM form a copolymer. Binding affinity measurements between Scm-SAM and Ph-SAM subunits in different orientations indicate a preference for the formation of a single junction copolymer. To provide a model of the copolymer, the structure of the Ph-SAM/Scm-SAM junction was determined. Similar binding modes are observed in both homo- and hetero-complex formation with minimal change in helix axis direction at the polymer joint. The copolymer model suggests that polymeric Scm complexes could extend beyond the local domains of polymeric Ph complexes on chromatin, possibly playing a role in long range repression (Kim, 2005).

The results clearly demonstrate that Scm-SAM can form a polymer in vitro, and there is considerable evidence that the same polymer is an important aspect of the biological function of Scm. First, the high affinity of the intersubunit interaction is a strong indication that polymerization is a normal function of Scm-SAM in vivo. Second, it is hard to see how polymerization could be an in vitro artifact, because similar polymer architectures have now been seen for SAM domains from three divergent transcriptional repressors (TEL, Ph, and Scm). Moreover, polymer blocking mutations in TEL render the protein unable to repress transcription, suggesting that polymerization is required for repressive function in TEL. Third overexpression of an isolated Scm-SAM generates an Scm defect in vivo. It is easy to envision that an overabundance of the isolated SAM domain could infiltrate endogenous Scm polymers. Finally, a set Scm-SAM mutants have been identified; these mutant domains fail to self-associate. When these mutants were introduced into the full-length Scm protein, they failed to complement Scm mutants in Drosophila (Kim, 2005).

The SAM domain mutants that fail to self-associate and cannot rescue Scm mutant flies are readily rationalized by this polymer structure. Five mutants were found to be defective both in vitro and in vivo: I45T, G47D, M59Delta, M62R, and K71E. The sites of the mutations are localized to the interface seen in the Scm polymer structure. Ile-45 and Gly-47 are found in the mid-loop (ML) binding surface. Both residues are highly buried in the monomer structure (Ile-45 is 91% buried, and Gly-47 is 100% buried). The I45T and G47D mutations are therefore likely to distort the structure of this critical binding surface. Met-59 and Met-62 are both located on helix 4, which contributes to both the ML and end-helix (EH) binding surfaces. Thus, helix 4 is a particularly important region for polymer formation. Moreover, Met-59 is an important hydrophobic residue in the interface. Deletion of Met-59 would therefore remove an important contribution to the interface and would necessarily distort the local structure. Met-62 is 98% buried in the monomer, making it difficult to accommodate the M62R substitution without some sort of structural distortion. Finally, Lys-71 is located on the EH interface and makes a salt bridge across the polymer interface to Asp-46. Thus a K71E mutation would eliminate this interaction and introduce unfavorable electrostatic repulsion. Overall, the results argue that the observed polymer structure is biologically relevant (Kim, 2005).

SAM domain polymerization must be regulated in some fashion to facilitate complex assembly and disassembly. Yan, a member of the Ets family of transcription factors, contains a SAM domain that polymerizes in the same fashion as Ph- and Scm-SAM as well as the closely related ortholog of Yan, TEL-SAM. Yan-SAM can be depolymerized via its interaction with the SAM domain of its regulator, Mae. Mae-SAM binds to a polymerization interface of Yan-SAM with 1000-fold greater binding energy than Yan-SAM has with itself thereby effectively competing away Yan-SAM self-association and ultimately leading to the down-regulation of Yan activity. Regulation of either Ph- or Scm-SAM polymer by each other in the same fashion as Yan/Mae appears unlikely because Ph/Scm-SAM lack the large disparity in binding affinities. It is possible that polymerization is regulated by some still unidentified Mae-like protein that can cap Scm and Ph polymers, or that SAM polymerization is regulated internally by another domain with Scm and Ph (Kim, 2005).

Work on TEL raises the intriguing possibility that polymerization is regulated by covalent modification with small ubiquitin-like modifier (SUMO). TEL is sumoylated at a lysine residue at the edge of the polymeric binding interface. Examination of the site of modification in the context of the TEL polymer structure strongly suggests that polymer formation and sumoylation are mutually incompatible. Thus, it would be expected that SUMO would disrupt TEL-SAM polymers. In this light, it is interesting to note that the polycomb group protein, Pc2, is a SUMO-ligating enzyme, indicating that sumoylation plays an important role in PcG function. Moreover, sumoylation of the Caenorhabditis elegans PcG protein SOP-2 is essential for Hox gene repression. Both Drosophila Ph- and Scm-SAM possess potential sumoylation sites, but there is still no evidence for sumoylation of these proteins (Kim, 2005)

Ph and Scm are known to bind to each other and cooperate in their repressive functions. The findings that Scm-SAM and Ph-SAM both form polymers argues that they must interact in the form of a copolymer. Measurements of binding affinities in different orientations demonstrate that one of the possible joints between the two polymers is strongly preferred. This suggests that PcG complexes involving Ph and Scm would tend to form separate domains on chromatin, by means of a single joint copolymer. If so, a domain of Ph complexes would be invisioned; these complexes are known to localize within a few kilobases around a polycomb response element, extended by Scm complexes. This hypothesis is consistent with previously reported observations. (1) The repressive function of Scm artificially tethered to a DNA binding site depends on the presence of the SAM domain and is enhanced by the presence of Ph. (2) In a Drosophila PRC1 complex, Scm co-purified with the other members including Ph and was thus originally identified as a member of the complex. Subsequent experiments, however, showed smaller amounts of Scm compared with the other members, Pc, Psc, dRING1, and Ph. From the copolymer model, variable amounts of Scm associated with a core domain of Ph complexes would be expected. (3) An analysis of regulatory DNA elements suggested the site of action of Scm is adjacent to the site of action of Psc, a member of the PRC1 complex along with Ph. Scm function extending over an adjacent site is exactly what is expected from the single junction copolymer model (Kim, 2005).

The results strongly argue that polymerization plays an important role in Ph and Scm function. The biological implications of these findings require further investigation, but it is reasonable to suggest that polymerization facilitates the spreading of PcG complexes along the chromosome. Although Ph is found localized around polycomb response elements, the location of Scm in repressed genes is not known. Scm is capable of long range repression, and the results suggest that Scm could be utilized for the extension of repression outside the immediate region of the polycomb response element (Kim, 2005).

Comparative analysis of chromatin binding by Sex Comb on Midleg (SCM) and other polycomb group repressors at a Drosophila Hox gene

Sex Comb on Midleg (SCM) is a transcriptional repressor in the Polycomb group (PcG), but its molecular role in PcG silencing is not known. Although SCM can interact with Polycomb repressive complex 1 (PRC1) in vitro, biochemical studies have indicated that SCM is not a core constituent of PRC1 or PRC2. Nevertheless, SCM is just as critical for Drosophila Hox gene silencing as canonical subunits of these well-characterized PcG complexes. To address functional relationships between SCM and other PcG components, chromatin immunoprecipitation studies were performed using cultured Drosophila Schneider line 2 (S2) cells and larval imaginal discs. It was found that SCM associates with a Polycomb response element (PRE) upstream of the Ubx gene which also binds PRC1, PRC2, and the DNA-binding PcG protein Pleiohomeotic (PHO). However, SCM is retained at this Ubx PRE despite genetic disruption or knockdown of PHO, PRC1, or PRC2, suggesting that SCM chromatin targeting does not require prior association of these other PcG components. Chromatin immunoprecipitations (IPs) to test the consequences of SCM genetic disruption or knockdown revealed that PHO association is unaffected, but reduced levels of PRE-bound PRC2 and PRC1 were observed. These results are discussed in light of current models for recruitment of PcG complexes to chromatin targets (Wang, 2010).

How might SCM fit in molecularly with the other PcG components Although in vitro associations of SCM with PRC1 subunits have been described, the ChIP analyses here indicate that SCM can associate with the Ubx PRE despite the loss of PRC1. Similarly, although SCM can bind to the PHO-RC subunit SFMBT in a pairwise assay, SCM localization at the PRE does not appear to be dependent on PHO. Taken together, these ChIP results are consistent with biochemical studies that reveal SCM separability from PHO-RC, PRC1, and PRC2 in fly embryo extracts (Wang, 2010).

An intriguing finding from the matrix of molecular epistasis tests is that SCM exhibits recruitment properties very similar to those of PHO. Specifically, both SCM and PHO can localize to the Ubx PRE independent of all other PcG components tested, and loss of either SCM or PHO diminishes PRC2 and PRC1 association with the PRE. This similarity suggests that SCM may function, like PHO-RC, at an early step in PcG recruitment. In this context, it is worth emphasizing the striking overall similarities between SCM and the PHO-RC subunit SFMBT. Perhaps SCM partners with a yet-to-be identified PcG DNA-binding protein, akin to the functional partnership of SFMBT with PHO. Indeed, since PHO-binding sites are insufficient for PRE function in vivo and many other DNA-binding proteins have been implicated in Drosophila PcG silencing, there is abundant evidence that PRE recognition involves more than just PHO-RC. The common view is that many PREs contain a composite of PHO sites plus additional types of factor-binding motifs. At present, little is known about the nature of SCM-containing complexes beyond the detection of an approximately 500-kDa moiety in fly embryo extracts. It will be informative to characterize stably associated SCM partner proteins and evaluate their potential roles in binding to PRE DNA (Wang, 2010).

Although the ChIP assays presented in this study emphasize SCM separability from other PcG components, SCM must still integrate with its PcG cohorts to achieve gene silencing. This interdependence is highlighted by in vivo assays where robust silencing of a miniwhite reporter by a tethered form of SCM is disrupted if the PRC1 subunit PH is compromised by mutation. Despite advances in understanding biochemical activities of individual PcG complexes, it is not yet clear how their multiple functions are integrated to achieve gene silencing. Further studies will be needed to determine how SCM functions in concert with other PcG components at target chromatin (Wang, 2010).

Ultimately, a precise understanding of SCM function requires deciphering the mechanistic contributions of each of its three identified domains. SCM contains a C-terminal SPM domain, two mbt repeats, and two Cys₂-Cys₂ zinc fingers. Strikingly, each of these domains is also present in SFMBT, suggesting that the overall biochemical roles of these two PcG components may be very similar. Indeed, a recent study provides evidence of functional synergy between SCM and SFMBT (Grimm, 2009). In addition, the PH PcG protein possesses two of these three homology domains. This presents the curious situation of three different PcG proteins related by shared domains yet with none appearing to reside in a stable common complex in nuclear extracts (Wang, 2010).

There are currently in vitro and in vivo data on roles of the SPM domain and mbt repeats but little knowledge yet about the zinc fingers. The SPM domain is a subtype within the broader category of SAM domains that mediate protein interactions. The SCM version of this domain is capable of robust self-binding and cross-binding to the PH version in vitro. The importance of SPM domain interactions in vivo is emphasized by PcG phenotypes observed after overexpressing a dominant-negative isolated SPM domain in developing flies (Peterson, 2004). However, it remains unclear precisely what SPM interactions contribute to the PcG silencing mechanism. The simple idea that they constitutively glue PcG complex subunits together is at odds with the biochemical separabilities in embryo extracts. Perhaps SPM interactions function primarily directly at chromatin targets, where they could sponsor contacts among different PcG complexes rather than among subunits within the same complex. Such chromatin-specific interactions could contribute to intralocus loops, which have been hypothesized to exist at PcG silenced loci (Wang, 2010).

The functional significance of the SCM mbt repeats is reflected by partial loss-of-function alleles that alter the first repeat and by Hox gene silencing defects observed after disruption of the second repeat. Structural determinations and in vitro binding studies have revealed that mbt repeats are modules for binding to methylated lysines. Since trimethylated H3-K27 is a prominent feature of PcG-silenced chromatin, the mbt repeats could, at first glance, play a role akin to that of the PC chromodomain. However, there are important differences between the substrate-binding properties of these mbt repeats and the PC chromodomain. First, the mbt repeats prefer mono-and dimethylated lysines, whereas the chromodomain prefers the trimethylated form. An intriguing hypothesis is that this mono/di preference could reflect a 'grappling hook' function whereby hypomethylated nucleosomes are recognized and brought into proximity for trimethylation by PRC2. Another distinction is that the binding mode of mbt repeats is not much influenced by peptide sequence context, whereas chromodomain binding features extensive contact with residues flanking the methylated lysine. Consistent with this, the SCM mbt repeats lack binding preference for any particular histone tail lysines. Thus, mbt repeats provide a pocket for methyl-lysine binding, but it is not yet clear if the relevant substrate for SCM is a particular methylated histone residue or even a nonhistone protein. Certainly, the in vitro binding preferences could be modified by additional associated factors in vivo (Wang, 2010).

A sequence alignment of the Cys₂-Cys₂ fingers present in SCM, SFMBT and PH shows that this zinc finger is a distinct subtype that adheres to the consensus sequence CXXCG-X_n-K/R-X-F/Y-CSXXC. These fingers do not appear to function by binding DNA, since sequence-specific binding is not observed in vitro for any of them. Thus, their molecular role is unknown, but their common inclusion in these related fly PcG proteins suggests some key contribution to PcG chromatin function. Curiously, both the SCM and SFMBT human homologs appear to have lost their Cys₂-Cys₂ fingers, whereas all three human PH homologs have retained them. Thus, if these zinc fingers are critical in PcG silencing, then they apparently can be supplied from different combinations of PcG proteins in flies and in mammals. It will be important to test the genetic requirement for the SCM zinc fingers in Drosophila and to further define the mechanistic contributions of all three SCM functional domains to PcG chromatin silencing (Wang, 2010).

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