InteractiveFly: GeneBrief

Su(z)12: Biological Overview | References


Gene name - Su(z)12

Synonyms -

Cytological map position - 76D4-76D4

Function - chromatin protein

Keywords - Polycomb group, polycomb PRC2 complex, histone methyltransferase, responsible for tri-methylation of the lysine 27 residue of histone H3, functions in nucleosome binding of Drosophila PRC2

Symbol - Su(z)12

FlyBase ID: FBgn0020887

Genetic map position - 3L:19,911,337..19,916,500 [-]

Classification - VEFS-Box (VRN2-EMF2-FIS2-Su(z)12) box is the C-terminal region of these proteins, characterised by an acidic cluster and a tryptophan/methionine-rich sequence, the acidic-W/M domain.

Cellular location - nuclear



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Matsuoka, Y., Bando, T., Watanabe, T., Ishimaru, Y., Noji, S., Popadic, A. and Mito, T. (2015) Short germ insects utilize both the ancestral and derived mode of Polycomb group-mediated epigenetic silencing of Hox genes Biol Open. PubMed ID: 25948756
Summary:
In insect species that undergo long germ segmentation, such as Drosophila, all segments are specified simultaneously at the early blastoderm stage. As embryogenesis progresses, the expression boundaries of Hox genes are established by repression of gap genes, which is subsequently replaced by Polycomb group (PcG) silencing. At present, however, it is not known whether patterning occurs this way in a more ancestral (short germ) mode of embryogenesis, where segments are added gradually during posterior elongation. In this study, two members of the PcG family, Enhancer of zeste (E(z)) and Suppressor of zeste 12 (Su(z)12), were analyzed in the short germ cricket, Gryllus bimaculatus. Results suggest that although stepwise negative regulation by gap and PcG genes is present in anterior members of the Hox cluster, it does not account for regulation of two posterior Hox genes, abdominal-A (abd-A) and Abdominal-B (Abd-B). Instead, abd-A and Abd-B are predominantly regulated by PcG genes, which is the mode present in vertebrates. These findings suggest that PcG-mediated silencing of Hox genes may have occurred during animal evolution. The ancestral bilaterian state may have resembled the current vertebrate mode of regulation, where PcG-mediated silencing of Hox genes occurs before their expression is initiated and is responsible for the establishment of individual expression domains. Then, during insect evolution, the repression by transcription factors may have been acquired in anterior Hox genes of short germ insects, while PcG silencing was maintained in posterior Hox genes.

BIOLOGICAL OVERVIEW

Polycomb group (PcG) proteins are required to maintain a stable repression of the homeotic genes during Drosophila development. Mutants in the PcG gene Suppressor of zeste 12 (Su(z)12) exhibit strong homeotic transformations caused by widespread misexpression of several homeotic genes in embryos and larvae. Su(z)12 has also been suggested to be involved in position effect variegation and in regulation of the white gene expression in combination with zeste. To elucidate whether SU(Z)12 has any such direct functions the binding pattern to polytene chromosomes was investigated and the localization to other proteins was compared. SU(Z)12 was found to bind to about 90 specific eukaryotic sites, however, not the white locus. Staining was found at the chromocenter and the nucleolus. The binding along chromosome arms is mostly in interbands and these sites correlate precisely with those of Enhancer-of-zeste and other components of the PRC2 silencing complex. This implies that SU(Z)12 mainly exists in complex with PRC2. Comparisons with other PcG protein-binding patterns reveal extensive overlap. However, SU(Z)12 binding sites and histone 3 trimethylated lysine 27 residues (3meK27 H3) do not correlate that well. Still, it was shown that Su(z)12 is essential for tri-methylation of the lysine 27 residue of histone H3 in vivo, and that overexpression of SU(Z)12 in somatic clones results in higher levels of histone methylation, indicating that SU(Z)12 is rate limiting for the enzymatic activity of PRC2. In addition, the binding pattern of Heterochromatin Protein 1 (HP1) was analyzed and it was found that SU(Z)12 and HP1 do not co-localize (Chen, 2008).

In Drosophila, Polycomb group (PcG) proteins are negative regulators of homeotic gene expression and play an important role in maintaining silenced states during development of the fly. The regulatory regions of homeotic genes, such as Ultrabithorax (Ubx) and Abdominal-B (Abd-B), contain specific cis-regulatory elements needed for PcG complexes to mediate this silencing effect. Several such Polycomb response elements (PREs) have been identified, not only in the Antennapedia (AntpC) and Bithorax complexes (BxC) but also in the regulatory regions of many genes mainly encoding transcription factors. PREs appear to be complex DNA elements targeted by several proteins, not only PcG but also trithorax group (TrxG) proteins, proposed to have an opposing effect to PcG, i.e. to maintain an active state of homeotic genes. Recently, a genome-wide analysis of PcG targets in Drosophila identified over 200 genes that are simultanously bound by the three PcG proteins Polycomb (PC), Enhancer of zeste (E(Z)) and Posterior sex combs (PSC) (Schwartz, 2006). However, none of these proteins bind directly to DNA but are recruited either by DNA-binding proteins bound to PREs or by specifically modified histones. The Frontabdominal-7 (Fab-7) region of the Abd-B locus, e.g., the iab-7 PRE, which is approximately 230 bp in length, contains three DNA motifs that are recognized by five proteins: Zeste (Z), GAGA factor (GAGA), Pipsqueak (PSQ), Pleiohomeotic (PHO) and Pho-like (PHO-L). Interestingly, both PcG repressors and TrxG activators appear to bind at PREs in both the repressed and the active state (Papp, 2006). Instead, transcription of non-coding RNA through the PREs has been shown to be important for initiation of the correct transcriptional status of homeotic genes during development. Earlier work suggested that PRE transcription counteracts PcG-dependent silencing of Hox genes, while more recent work show that non-coding RNA is not present in cells expressing Ubx, indicating that transcription through PREs promotes gene silencing. The silencing ability of a given PRE is, however, also strongly dependent on the genomic context, homologous pairing or proximity to other PRE sequences (Chen, 2008).

The TrxG gene zeste encodes a non-essential transcription factor, which binds to DNA not only within PREs but also in regulatory regions of many genes, e.g. the white locus. The neomorphic zeste 1 (z 1 ) mutation causes an amino acid exchange, which renders the mutated protein extremely sticky. This results in transcriptional repression of paired white + (w + ) genes in z 1 mutants. Thus, in this zeste- white interaction, the X-linked white gene, either paired in trans (e.g., in females) or in cis (e.g. in males carrying a tandem duplication of white), will be repressed, resulting in a yellow eye colour. Whenever unpaired, the white gene is not repressed by z 1 , resulting in wild-type eye pigmentation. The zeste gene is also implicated to cause transvection, i.e., the ability of regulatory elements on one chromosome to affect the expression of the homologous gene in a somatically paired chromosome. A third type of pairing sensitive effects by z 1 is found in transgenic lines, where PRE-induced silencing of a mini-white gene often is pairing dependent. However, homologous pairing is not always required for PRE-induced silencing of w + expression and in such cases the z 1 allele has no influence on the repression (Chen, 2008 and references therein).

The biochemical characterization of Drosophila PcG proteins suggests that there are at least two distinct multi-protein complexes, each containing several PcG proteins. The Polycomb repressive complex 1 (PRC1) contains PC, PSC, polyhomeotic (PH) and Sex combs extra (SCE or dRING) and some additional accessory proteins such as Z, Sex comb on midleg (SCM), and general transcription factors. A second complex (PRC2) consists of E(Z), Extra sex combs (ESC), Suppressor of zeste-12 (SU(Z)12) and NURF55 (Chromatin associated factor-1 subunit, CAF-1), as well as some accessory proteins; Polycomb-like (PCL), RPD3 and SIR2 (reviewed by Schuettengruber, 2007). The core proteins E(Z), ESC and SU(Z)12 are conserved both in mammals and plants. The E(Z) protein contains a SET domain which specifically methylates lysine residues of histone 3. The SU(Z)12 protein contains a zinc finger and a well conserved region called the VEFS box (Birve, 2001), which has been shown to bind to EZH2 protein (the human E(Z) homolog) and Heterochromatin protein 1α (HP1α) in mammalian cells (Yamamoto, 2004). Recently, in vitro binding between mammalian SUZ12 and MEP50 was reported (Furuno, 2006). MEP50 binds selectively to histone H2A and interacts with the arginine methyltransferase PRMT5 and H2A, mediating transcriptional repression of target genes. So far the SU(Z)12 protein has not been ascribed any specific function in the PRC2 complex, apart from increasing the ability of the E(Z) protein to tri-methylate the lysine 27 residue of histone H3 (3meK27 H3) in vitro (Ketel, 2005) and forming, with NURF55, the minimal nucleosome-binding module of PRC2 (Nekrasov, 2005). 3meK27 H3 is a target of the chromodomain of the PC protein in the PRC1 complex. These two main silencing complexes together with their accessory molecules seem to inhibit transcription by preventing nucleosome remodeling and, by binding to promoter regions, block the transcription initiation machinery (Wang, 2004). The precise molecular mechanisms are, however, still poorly understood (Chen, 2008).

The first Su(z)12 mutant allele was first identified as a suppressor of the zeste- white interaction and was also shown to be important for correct maintenance of the silenced state of the Ubx gene during development and to suppress position-effect variegation (PEV) (Birve, 2001). The biochemical studies of the PcG complexes have definitely linked SU(Z)12 to PcG-mediated gene silencing; however, the involvement in position effect variegation and in regulation of the white gene expression in combination with z 1 is less well investigated. To elucidate whether Su(z)12 directly regulates the white gene expression genetic interaction studies were used with Su(z)12 alleles and it could be revealed that the dominant derepression of white expression caused by Su(z)12 mutants is dependent on the repressive action of the z 1 allele, but in order to observe this derepression either the white gene has to be paired (in females) or contain insertions of transposable elements at the white gene (in males). Another way to find out whether SU(Z)12 has other functions, apart from gene silencing via PREs, is to investigate protein binding and nuclear localization and compare with binding patterns of the other PRC2 subunits. Therefore, SU(Z)12 binding to polytene chromosomes was analyzed. It was found that SU(Z)12 binds to at about 90 specific loci, however, not detectably at the white locus. Neither is any increase of 3meK27 H3 binding found at the white locus in repressed strains, indicating that other repressive mechanisms are acting at white. Results with HP1 suggest that heterochromatin silencing may play a role. Moreover, it is concluded that there is a complete co-localization between SU(Z)12 and E(Z) and that the overlap with other PcG proteins is high, indicating that SU(Z)12 in salivary gland tissue always is in complex with PRC2. However, there was a surprisingly low degree of co-localization with 3meK27 H3. In order to rule out the action of other H3-K27 methylases, somatic Su(z)12 knock-out clones were generated and it was concluded that SU(Z)12 is essential for function of the PRC2 complex in tri-methylation of lysine 27 in histone H3 in vivo (Chen, 2008).

Regulation of the white gene by zeste is complex; first, the Z1 mutant protein can form large protein aggregates that cooperatively bind to several ZBS within the regulatory region of white, second, Z protein is involved in recruiting both silencing and activating maintenance complexes (when bound to PREs) and finally the white mutants studied here contain insertions of various mobile elements, which might inflict heterochromatic properties to the white gene or interfere with dosage compensation in males. Using genetic interaction studies, this study found that Su(z)12 + is required for the Z1 mediated repression of white, but no physical binding of SU(Z)12 protein at the white locus is found in polytene chromosomes. Neither is any increased 3meK27 H3 binding there. One explanation for the derepression of white expression by Su(z)12 mutants could be that there are tissue specific differences in PcG silencing, and that in eye discs the white gene is silenced by PcG, but not in salivary glands. This is not likely, since there is no PRE at the white locus, only a set of ZBS, which is not sufficient to recruit silencing complexes. Another alternative is thus that the derepression observed in Su(z)12 mutants are secondary effects. Possibly, lower levels of SU(Z)12 can down-regulate other PcG proteins, which is accompanied by a simultaneous depletion of Z1 proteins, relieving white repression. Alternatively, Z1 can recruit still other silencing mechanisms to the white locus (Chen, 2008).

Su(z)12 mutant derepression is observed in homozygous z 1 females but only in a few white alleles in males. Insertions of mobile elements at the white locus in these mutants might superimpose heterochromatic silencing, repressing transcription further. However, these specific transposon insertions on their own accord do not visibly repress white transcription. Interestingly, a novel weak binding of HP1 at the white locus in polytene chromosomes was found in one of the strains that are derepressed by Su(z)12 mutations, suggesting that the inserted transposable element could recruit heterochromatic silencing proteins, adding to the silencing already present. The loss of one copy of the Su(var)205 gene slightly relieves this silencing. It is concluded that the z 1 repression in conjunction with transposable element insertions results in a yellow eye colour, allowing detection of the derepression caused by Su(z)12 mutants. This shows that a combination of repressive mechanisms is acting at the white gene in the studied mutants, but that PcG and heterochromatin silencing are not the main ones (Chen, 2008).

It was not possible to show any co-localization between SU(Z)12 and HP1 proteins on Drosophila polytene chromosomes (except at telomeres and the chromocenter), which is also confirmed by the binding pattern of HP1 on polytene chromosomes. This is in contrast to the in vitro findings with the mammalian counterparts, where the region between the Zinc finger and the VEFS box in human SU(Z)12 protein directly binds to mammalian HP1α in vitro (Yamamoto, 2004). The human HP1α protein is showing a somewhat higher homology to the Drosophila HP1b protein sequence than to HP1 encoded by Su(var)205. Therefore, Drosophila HP1 may not be the functional homologue of HP1α, which could explain why no co-localization was found with SU(Z)12 and HP1 in Drosophila. It would be interesting to further study the role of HP1b in gene silencing (Chen, 2008).

Müller and coworkers reported that SU(Z)12 is essential for the binding of PRC2 to nucleosomes, and that SU(Z)12 and NURF55 together constitute the minimal nucleosome-binding complex. These two subunits alone show a better binding to nucleosomes in vitro than a complex also containing E(Z) (Nekrasov, 2005). This study has found that SU(Z)12 and E(Z) always co-localize to polytene chromosomes, and that NURF55 is also present at these sites. Since a FLAG-tagged ESC protein also completely co-localizes with E(Z) this indicates that the complete core PRC2 complex is present at these sites and is functional, and that only NURF55 can bind to chromatin without the other subunits. E(Z) and ESC have also been found to bind to the chromocenter so probably the role of SU(Z)12 as suppressor of PEV is still connected to the function of the PRC2 complex. This refutes the hypothesis that SU(Z)12 has functions outside of the PRC2 complex (Chen, 2008).

It was surprising to find that there are SU(Z)12 binding sites that show no or very weak 3meK27 H3 binding on polytene chromosomes and that there are sites that contain high levels of 3meK27 H3 where PRC2 proteins are not present. Furthermore, it was unexpected to find high levels of 3meK27 H3 in puffs, which contain actively transcribing genes. Most 3meK27 H3 is otherwise seen in interbands, which are considered to contain regulatory regions for transcriptional activation of condensed chromatin in adjacent bands. ChIP analyses have revealed that 3meK27 H3 is binding to large domains covering entire genes that are silenced, while PRC2 proteins bind to more restricted regions (Papp, 2006; Schwartz, 2006). In the genome wide analysis of PcG targets performed by Pirrotta and co-workers (Schwartz, 2006), there is a very high co-localization between PcG proteins and 3meK27 H3, and also good correlation between 3meK27 H3 and transcriptional silence. However, 3 of the 149 binding sites reported lack either PcG binding or H3 methylation mark. Probably there are more examples since the report only includes sites where all four or three of the proteins (PC, E(Z), PSC and 3meK27 H3) bind simultaneously with a twofold enrichment. Furthermore, they use cell cultures for their analysis, which might give different results compared to differentiated tissues like salivary glands. The result obtain in the current study could also be caused by physical disruption of sub-nuclear compartments (like PcG bodies) where PRC2 complexes and chromosome regions with high levels of trimethylated histones normally reside. Yet another alternative could be that a dynamic reorganization of chromatin in polytene chromosomes occurs, leading to translocation of PcG proteins. It has been shown that PcG chromosome-association profiles can change during development. Further options could be that there are other yet-unidentified HMTs that can induce 3meK27 H3 formation, or that SU(Z)12, E(Z) and ESC are subunits in larval complexes with other functions than to methylate H3 residues (Chen, 2008).

Somatic knock-out of SU(Z)12 in Wings discs results in a complete abolishment of 3meK27 H3, showing the vital role of SU(Z)12 for the function of the PRC2 complex. This is in agreement with the results reported by Cao (2004) for mammalian cell lines. The Su(z)12 knock-out clones are generally very small in size, indicating a role for SU(Z)12 in cell proliferation. Twin spots show an increase in histone methylation compared to heterozygous mutant cells. Clones over-expressing SU(Z)12+ also exhibit high level of H3 methylation compared to normal cells. This implies that SU(Z)12 is rate limiting and that SU(Z)12 over-expression facilitates assembly of PRC2 subunits, stabilizes existing PRC2 complexes or in some way augments HMT activity of these. It is known that most PcG proteins bind to some PcG genes, e g. Psc/Su(z)2 and ph (Schwartz, 2006) and this study also sees binding of SU(Z)12 at both these loci. Therefore, an alternative explanation could be that increased SU(Z)12 levels induces positive feedback, activating transcription of genes encoding PRC2 subunits. Indeed, examples of such stimulatory effects between PcG genes have been found. esc and E(z) mutants significantly decrease Pc, Psc and ph transcription levels indicating a stimulatory effect. Similarly, ASX, E(PC) and PCL positively regulates Psc transcription, while PH, PC and PSC negatively regulate Psc (Chen, 2008).

In humans the Suz12 locus is a region of frequent translocation in endometrial stromal sarcomas, which generates a fusion protein between JAZF1 and SUZ12. This suggests that over-expression of SUZ12 fusion protein has a causal role in the pathogenesis of this tumour (Koontz, 2001). Furthermore, SU(Z)12 is up-regulated in various other tumours (Kirmizis, 2003). The finding that over-expression of SU(Z)12 increases the HMT activity, possibly by inducing a positive feedback regulation of the other PRC2 genes, emphasize the importance of maintaining a balance between activity and silencing of e.g. tumour suppressor genes (Chen, 2008).

Histone H3 Serine 28 is essential for efficient Polycomb-mediated gene repression in Drosophila

Trimethylation at histone H3K27 is central to the polycomb repression system. Juxtaposed to H3K27 is a widely conserved phosphorylatable serine residue (H3S28) whose function is unclear. To assess the importance of H3S28, a Drosophila H3 histone mutant was generated with a serine-to-alanine mutation at position 28. H3S28A mutant cells lack H3S28ph on mitotic chromosomes but support normal mitosis. Strikingly, all methylation states of H3K27 drop in H3S28A cells, leading to Hox gene derepression and to homeotic transformations in adult tissues. These defects are not caused by active H3K27 demethylation nor by the loss of H3S28ph. Biochemical assays show that H3S28A nucleosomes are a suboptimal substrate for PRC2 (containing Esc, Su(z)12, E(z) and Nurf55), suggesting that the unphosphorylated state of serine 28 is important for assisting in the function of polycomb complexes. Collectively, these data indicate that the conserved H3S28 residue in metazoans has a role in supporting PRC2 catalysis (Yung, 2015).

This report has established a H3S28A histone mutant in Drosophila. In theory, this mutation could have two different effects on the polycomb system. (1) It could be that PcG proteins are not evicted from H3K27me3-binding sites in the absence of H3S28ph, and thus, PcG target genes might become ectopically repressed or (2) the mutation at H3S28 or the absence of H3S28ph could compromise PcG functions, resulting in derepression of PcG target genes. No evidence was found for the first possibility, although it is formally possible that H3S28 is phosphorylated under certain developmental conditions or in response to particular stimuli to counteract polycomb silencing. Instead, the data point to an inhibition of PRC2 activity by the H3S28A mutation. This inhibition is independent of active H3K27 demethylation by dUtx. Besides, RNAi against Aurora B kinase and hence depletion of H3S28ph did not hamper polycomb silencing. On the other hand, H3S28A nucleosomes proved to be a suboptimal substrate for in vitro PRC2 HMT activity. Although a 3D structure of the human Ezh2 SET domain is available, the exact contribution of the hydroxyl group of H3S28 for H3K27 methylation is difficult to deduce from the available data. vSET, the only other protein capable of H3K27 methylation in the absence of PRC2 subunits, does not require H3S28 for catalysis, whereas it does use H3A29 to define substrate specificity. Clearly, more work will be required to determine the exact structural and biochemical role of H3S28 in PRC2 catalysis. Consistent with the in vitro HMT assays, in vivo the H3S28A mutant exhibits defects in H3K27 methylation and shows similar, though milder, Hox derepression profiles and transformation phenotypes to those observed in H3K27R mutant flies (Yung, 2015).

Interestingly, the 'KS' module is frequently found in Ezh2 substrates other than K27S28 of histone H3. These include K26S27 of human histone H1 variant H1b (H1.4), K38S39 of the nuclear orphan receptor RORα, and K180S181 of STAT3. Whether these serine residues act similarly to H3S28 to support methylation of the adjacent lysine residue remains unknown. Of note, some other Ezh2 substrates can be methylated despite the lack of a 'KS' module. These include K26 of mouse histone H1 variant H1e, K49 of STAT3, and K116 of Jarid2, where the lysine residue is followed by an alanine, glutamate, and phenylalanine, respectively. Moreover, the link between peptide sequence and enzymology of Ezh2 was shown to differ in non-histone substrates. Hence, the role of serine following the Ezh2 methylation target amino acid might not be extrapolated to all other Ezh2 substrates and should be tested individually (Yung, 2015).

Previous reports revealed discrepancies in Drosophila PcG protein localization on mitotic chromosomes depending on staining protocols and tissue types. Nonetheless, live imaging of Pc-GFP, Ph-GFP, and E(z)-GFP in early Drosophila embryos has suggested that the majority of these PcG components are dissociated from mitotic chromosomes. Because stress-induced H3S28ph evicts PcG complexes during interphase, one might expect rebinding of PcG proteins on mitotic chromosomes depleted of H3S28ph. Whereas loss of Ph from mitotic chromosomes was observed in WT background, significant Ph association was not observed in H3S28A mutant condition. The reduced levels of H3K27me3 in the H3S28A mutant could contribute to this observation. Alternatively, other mechanisms might operate to dissociate the majority of PcG proteins during mitosis (Yung, 2015).

The establishment of the histone replacement system in Drosophila has proven to be an important tool to complement functional characterization of chromatin modifiers. Whereas depletion of H3K27 methylation, either by mutation of the histone mark writer E(z) or by mutation of the histone itself in the H3K27R mutant, leads to similar loss of polycomb-dependent silencing, other histone mutations revealed different phenotypes than the loss of their corresponding histone mark writers. For example, H3K4R mutations in both H3.2 and H3.3, hence a complete loss of H3K4 methylation, did not hamper active transcription. Also, the loss of H4K20 methylation upon H4K20R mutation unexpectedly supports development and does not phenocopy cell cycle and gene silencing defects reported upon the loss of the H4K20 methylase PR-Set7. In this study, by comparing the phenotype of Aurora B knockdown and H3S28A mutation in vivo, together with in vitro HMT assay, the requirement of the unmodified H3S28 residue is specifically attributed to supporting PRC2 deposition of H3K27 methylation (Yung, 2015).

Whereas the published data suggest that H3S28 phosphorylation might be important for eviction of PcG components for derepression of PcG target genes upon stimulatory cues, the data reveal a so far unacknowledged function of the unphosphorylated state of H3S28. This study shows that serine 28 is required to enable proper methylation of H3K27 by PRC2 and thus to establish polycomb-dependent gene silencing. Serine 28 of histone H3 is universally conserved in species that display canonical PRC2-dependent silencing mechanisms. Given the fact that no major mitotic defects are found upon its mutation, it is proposed that the major role of this residue is to ensure optimal PRC2 function while facilitating the removal of polycomb proteins in response to signals that induce phosphorylation (Yung, 2015).

Regulation of the Drosophila engrailed gene by Polycomb repressor complex 2

Suppressor-of-zeste-12 (Su(z)12) is a core component of the Polycomb repressive complex 2 (PRC2), which has a methyltransferase activity directed towards lysine residues of histone 3. Mutations in Polycomb group (PcG) genes cause de-repression of homeotic genes and subsequent homeotic transformations. Another target for Polycomb silencing is the engrailed gene, which encodes a key regulator of segmentation in the early Drosophila embryo. In close proximity to the en gene is a Polycomb Response Element, but whether en is regulated by Su(z)12 is not known. This report shows that en is not de-repressed in Su(z)12 or Enhancer-of-zeste mutant clones in the anterior compartment of wing discs. Instead, en expression is down-regulated in the posterior portion of wing discs, indicating that the PRC2 complex acts as an activator of en. These results indicate that this is due to secondary effects, probably caused by ectopic expression of Ubx and Abd-B (Chen, 2009).

This investigation did not find any de-repression of en in the anterior pouch compartment of the wing discs when PRC2 function was removed. Somatic loss of PRC1 subunits (Pc and Ph) on the other hand results in de-repression of en in the anterior compartment of the wing discs. The Pcl protein has been shown to be associated with the PRC2 complex in embryos (Tie, 2003; Nekrasov, 2007), but a recent study (Savla, 2008) argues that Pcl is not included in either PRC1 or PRC2 larval complexes. Similarly to Pc and Ph, somatic loss of Pcl results in a de-repression of en in the anterior compartment of wing discs (Nekrasov, 2007). However, the authors state that this de-repression is variable between clones. This study re-analyzed the results in this reference and found that en up-regulation is indeed found in the periphery of the anterior compartment of the wing disc, but not in the pouch region and thus the Pcl results agree well with the PRC2 pattern. Also the PhoRC complex seems to exhibit a similar behaviour since en is mis-expressed in many Sfmbt mutant clones in the anterior compartment of the wing disc, but consistently remains repressed in clones in the pouch compartment (Oktaba, 2008; Chen, 2009).

In the posterior part of the wing disc it was unexpectedly found that en expression is down-regulated in Su(z)12 and E(z) mutant clones. It is also noted that the Su(z)12 mutant clones induced in the posterior compartment of wing discs have a rounded shape commonly observed in cells that have lost their En function. This property of cells to become adhesive and minimize contact with surrounding En-expressing cells is caused by the Hedgehog signaling pathway, that induces Decapentaplegic protein that normally maintains the A/P boundary, aiming at exclusion of cell clones with a more anterior fate (Chen, 2009).

It is hypothesize that the down-regulation of en, occurring when PRC2 function is lost in the posterior compartment of wing discs, is caused by ectopic induction of an en repressor, and it was shown that ectopic expression of Ubx or Abd-B indeed blocks en expression. The PRC2 complex is necessary for silencing of Ubx or Abd-B in wing discs, since de-repression in both compartments is readily seen 48- 96 h after induction of Su(z)12 mutant clones (Birve, 2001). When scrutinizing these results again it was observed that the up-regulation of Ubx and Abd-B is exclusively occurring in the pouch region of wing imaginal discs, for strong as well as weak Su(z)12 alleles. Since en repression was found only within the pouch region in the posterior compartment this further strengthens the hypothesis that Ubx and Abd-B are direct repressors of en. This also poses an explanation for the lack of en mis-expression in the anterior half of the wing pouch; ectopic Ubx and Abd-B, as a consequence of loss of PRC2 silencing, directly repress en and thus override the de-repression expected to occur here. A similar finding was recently published by Jürg Müller and co-workers (Oktaba, 2008), where they had to remove both Scm and BxC (Ubx, Abd-A and Abd-B) gene functions in order to obtain de-repression of the Distal-less gene in the pouch region of wing discs (Chen, 2009).

Ubx and Abd-A are known to function as repressors of several genes in both embryos and larvae. For instance, Ubx is a transcriptional repressor of vestigial (vg) and Serrate (Ser) in larvae and the Ser protein is known to activate wg and cut expression in larval tissue. De-repression of Ubx in Su(z)12 mutant clones could thus explain the findings that wg and cut are down-regulated in their respective expression domains in wing discs (Chen, 2009).

Still, up-regulation of en in Pc and Ph clones does occur in the anterior wing pouch region. Furthermore, clonal loss of Pcl has no effect on en expression in the posterior portion of wing disc (Nekrasov, 2007). This indicates that there is a discrepancy in the impact caused by loss PRC1 and PRC2 silencing functions, respectively. This could be explained by differences in binding and retention of either silencing complex to the PREs in both the Ubx and the en genes (Chen, 2009).

These results emphasize that each gene has to be regarded as a unique entity whose regulation can differ between developmental stages, tissues and between compartments within tissues and that transcriptional regulators together with different types of epigenetic marks collaborate to regulate gene expression. The en gene, that contains a PRE fragment to which PcG complexes bind in embryos, will have further controlling networks for maintenance of its active or silenced status at later stages. Its regulation is further complicated by the fact that the en PRE also possesses an activating function. This activating function might be due to the presence of PRC2 at the PRE, since loss of Su(z)12 results in repression of en expression in the posterior wing blade compartment. However, this possibility was not corroborated in Su(z)12 over-expression experiments, which did not result in a direct activation of en expression. It is concluded that the en gene has a multitude of regulatory elements that control the expression in its various contexts. Further studies are needed to elucidate the role of the different steps in PcG silencing and how these interact with other activating and repressive mechanisms to regulate gene expression (Chen, 2009).

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).

Histone methylation by PRC2 is inhibited by active chromatin marks

The Polycomb repressive complex 2 (PRC2) confers transcriptional repression through histone H3 lysine 27 trimethylation (H3K27me3). This study examined how PRC2 is modulated by histone modifications associated with transcriptionally active chromatin by providing the molecular basis of histone H3 N terminus recognition by the PRC2 Nurf55-Su(z)12 submodule. Binding of H3 is lost if lysine 4 in H3 is trimethylated. It was found that H3K4me3 inhibits PRC2 activity in an allosteric fashion assisted by the Su(z)12 C terminus. In addition to H3K4me3, PRC2 is inhibited by H3K36me2/3 (i.e., both H3K36me2 and H3K36me3). Direct PRC2 inhibition by H3K4me3 and H3K36me2/3 active marks is conserved in humans, mouse, and fly, rendering transcriptionally active chromatin refractory to PRC2 H3K27 trimethylation. While inhibition is present in plant PRC2, it can be modulated through exchange of the Su(z)12 subunit. Inhibition by active chromatin marks, coupled to stimulation by transcriptionally repressive H3K27me3, enables PRC2 to autonomously template repressive H3K27me3 without overwriting active chromatin domains (Schmitges, 2011).

Understanding how histone modification patterns are propagated during cell division is essential for understanding the molecular basis of epigenetic inheritance. Trimethylation of H3K27 by PRC2 has emerged as a key step in generating transcriptionally repressed chromatin in animals and plants. This study investigates how PRC2 recognizes the H3 tail and responds to H3-associated marks of active chromatin. Crystallographic analyses reveal the molecular basis for H31-14 recognition by the Nurf55-Su(z)12 module of PRC2 and demonstrate that H3 tails carrying K4me3 are no longer recognized by Nurf55-Su(z)12. In the context of the whole PRC2 complex, H3K4me3 triggers allosteric inhibition of PRC2, a process that requires H3K4me3 to be present on the same histone molecule containing the substrate Lys27. PRC2 inhibition by H3K36me2/3 was also observed. PRC2 inhibition by active chromatin marks (H3K4me3 and H3K36me2/3) is conserved in PRC2 complexes reconstituted from humans, mouse, flies, and plants (Schmitges, 2011).

Minimal PRC2 complexes lacking Nurf55 retain partial catalytic activity and are inhibited by H3K4me3. H3K4me3, once free of Nurf55, is thereby able to trigger PRC2 inhibition. A model is favored where Nurf55-Su(z)12 serves in sequestration and release of histone H3. It is proposed that the release of the H3 tail from Nurf55-Su(z)12 is required, but not sufficient, to induce H3K4me3 inhibition as it needs to trigger allosteric inhibition in conjunction with Su(z)12 and the E(z) SET domain. Unmodified H3, H3K9me3, or H3R2me-modified tails, on the other hand, remain sequestered and are shielded from other chromatin factors. These sequestered marks are also not expected to interfere with PRC2 regulation. In line with this prediction, it is observed that H3K9me3, which remained bound to Nurf55-Su(z)12, also did not interfere with PRC2 activity in vitro. In vitro, binding of Nurf55 to the N terminus of H3 was not critical for the overall nucleosome binding affinity of PRC2 under the assay conditions. However, small differences in PRC2 affinity amplified by large chromatin arrays could skew PRC2 recruitment toward sites of unmodified H3K4. Additionally, the Nurf55 interaction might play a more subtle role in positioning the complex correctly on nucleosomes (Schmitges, 2011).

The in vitro findings suggest that active chromatin mark inhibition by PRC2 is largely governed through allosteric inhibition of the PRC2 HMTase activity thereby limiting processivity of the enzyme. A minimal trimeric PRC2 subcomplex that retains both activity and H3K4me3/H3K36me2/3 inhibition was defined. This minimal complex consists of ESC, an E(z) fragment that comprises the ESC binding region at the N terminus, the Su(z)12 binding domain in the middle, and the C-terminal catalytic domain, and the Su(z)12 C terminus harboring the VEFS domain. The importance of Su(z)12 is underlined by findings on the Arabidopsis PRC2 complexes that revealed that active mark inhibition is determined by the choice of Su(z)12 subunit (i.e., inhibition with EMF2, but not with VRN2). As the extent of methylation inhibition and the domains required for inhibition were similar for H3K4me3 and H3K36me2/3, it is hypothesized that both peptides function through a related mechanism allosterically affecting E(z) SET domain processivity with the help of Su(z)12. Further structural studies are required to reveal how these active marks are recognized and how this recognition is linked to inhibition of the E(z) SET domain (Schmitges, 2011).

H3K27me3 recognition by PRC2 has been reported to recruit and stimulate PRC2, a mechanism implicated in creating and maintaining the extended H3K27me3 domains at target genes in vivo. Such positive feedback, however, necessitates a boundary element curtailing the expansion of H3K27me3. The results suggest that actively transcribed genes (i.e., marked with H3K4me3 and H3K36me2/3) that flank domains of H3K27me3 chromatin may represent such boundary elements. In conjunction with H3K27me3-mediated stimulation, this provides a model how PRC2 could template domains of H3K27me3 chromatin during replication without expanding H3K27me3 domains into the chromatin of active genes. The inhibitory circuitry present in PRC2, however, does not function as a binary ON/OFF switch. PRC2 is able to integrate opposing H3K4me3 and H3K27me3 modifications into an intermediary H3K27 methylation activity (Schmitges, 2011).

The crosstalk between H3K4me3 and H3K36me2/3 versus H3K27me3 has been extensively studied in vivo. Specifically, HOX genes in developing Drosophila larvae, or in mouse embryos, show mutually exclusive H3K27me3 and H3K4me3 domains that correlate with transcriptional OFF and ON states, respectively. In Drosophila, maintenance of HOX genes in the ON state critically depends on the trxG regulators Trx and Ash1, which methylate H3K4 and H3K36, respectively. At the Ultrabithorax (Ubx) gene, lack of Ash1 results in PRC2-dependent H3K27me3 deposition in the coding region of the normally active gene and the concomitant loss of Ubx transcription. Similarly, in the Arabidopsis Flowering Locus C (FLC), CLF-dependent deposition of H3K27me3 reduces H3K4me3 levels, while deletion of the H3K4me3 demethylase FLD increases H3K4me3 levels and concomitantly diminishes H3K27me3 levels. The results provide a simple mechanistic explanation for these observations in plants and flies. It is proposed that H3K4 and H3K36 modifications in the coding region of active PcG target genes function as barriers that limit H3K27me3 deposition by PRC2 (Schmitges, 2011).

It is noted that a number of HMTase complexes contain histone mark recognition domains that bind the very same mark that is deposited by their catalytic domain. While this positive feedback loop guarantees the processivity of histone mark deposition, it also requires a control mechanism that avoids excessive spreading of marks. The direct inhibition of HMTases by histone marks, as seen for PRC2, may offer a paradigm of how excessive processivity can be counteracted in other HMTases (Schmitges, 2011).

Arabidopsis VRN2 is implicated in the control of the FLC locus after cold shock. FLC is a bivalent locus containing both repressive H3K27me3 and active H3K4me3 marks. In a VRN2-dependent fashion, H3K27me3 levels increase at FLC during vernalization. This study found that while EMF2-containing PRC2 complexes are sensitive to H3K4me3 and H3K36me3, their VRN2-containing counterparts are not. In response to environmental stimuli plant PRC2 H3K4me3/H3K36me3 inhibition can thus be switched OFF (or ON). This offers the possibility that inhibition in animal PRC2 could also be modulated either by posttranslational modification of SUZ12 or by association with accessory factors (Schmitges, 2011).

Quantitative mass spectrometry analyses of posttranslational modifications on the H3 N terminus in HeLa cells found no evidence for significant coexistence of H3K27me3 with H3K4me3 on the same H3 molecule. Similarly, the fraction of H3 carrying both H3K27me3 and H3K36me3 was reported to be extremely low (~0.078%), while H3K27me3 and H3K36me2 coexist on ~1.315% of H3 molecules. However, H3K27me3/H3K4me3 and H3K27me3/H3K36me2/3 bivalent domains have been reported to exist in embryonic stem cells, and they have been implicated to exist on the same nucleosome. Given that PRC2 is inhibited by active methylation marks, how then could such bivalent domains be generated? Two main possibilities are envisioned. First, PRC2 inhibition in vivo could be alleviated by specific posttranslational modifications on PRC2 in embryonic stem cells (see in plants, VRN2). Second, H3K27me3 could be deposited prior to modification of H3K4 or H3K36. According to this view, one would have to postulate that the HMTases depositing H3K4me3 or H3K36me2/3 can work on nucleosomes containing H3K27me3. In support of this view, it was found that H3K36 methylation by an NSD2 catalytic fragment is not inhibited by H3K27me3 marks on a peptide substrate (Schmitges, 2011).

In summary, this study found that mammalian and fly PRC2 complexes are not only activated by H3K27me3, but they are also inhibited by H3K4me3 and H3K36me2/3. PRC2, as a single biochemical entity, can thus integrate the information provided by histone modifications with antagonistic roles in gene regulation. While the biological network overseeing crosstalk between active and repressive chromatin marks in vivo probably extends beyond PRC2, including other chromatin modifiers such as histone demethylases, this identified a regulatory logic switch in PRC2 that intrinsically separates active and repressive chromatin domains. Given the dynamic nature of the nucleosome template that makes up eukaryotic chromosomes, this circuitry probably equips PRC2 with the necessary precision to heritably propagate a repressed chromatin state (Schmitges, 2011).

Elements of the Polycomb repressor SU(Z)12 needed for histone H3-K27 methylation, the interface with E(Z), and in vivo function

Polycomb repressive complex 2 (PRC2) is an essential chromatin-modifying enzyme that implements gene silencing. PRC2 methylates histone H3 on lysine-27 and is conserved from plants to flies to humans. In Drosophila, PRC2 contains four core subunits: E(Z), SU(Z)12, ESC, and NURF55. E(Z) bears a SET domain that houses the enzyme active site. However, PRC2 activity depends upon critical inputs from SU(Z)12 and ESC. The stimulatory mechanisms are not understood. This study presents functional dissection of the SU(Z)12 subunit. SU(Z)12 contains two highly conserved domains: an ∼140 amino acid VEFS domain and a Cys2-His2 zinc finger (ZnF). Analysis of recombinant PRC2 bearing VEFS domain alterations, including some modelled after leukemia mutations, identifies distinct elements needed for SU(Z)12 assembly with E(Z) and stimulation of histone methyltransferase. The results define an extensive VEFS subdomain that organizes the SU(Z)12-E(Z) interface. Although the SU(Z)12 ZnF is not needed for methyltransferase in vitro, genetic rescue assays show that the ZnF is required in vivo. Chromatin immunoprecipitations reveal that this ZnF facilitates PRC2 binding to a genomic target. This study defines functionally critical SU(Z)12 elements, including key determinants of SU(Z)12-E(Z) communication. Together with recent findings, this illuminates PRC2 modulation by conserved inputs from its noncatalytic subunits (Rai, 2013).

Drosophila Polycomb complexes restrict neuroblast competence to generate motoneurons

Similar to mammalian neural progenitors, Drosophila neuroblasts progressively lose competence to make early-born neurons. In neuroblast 7-1 (NB7-1), Kruppel (Kr) specifies the third-born U3 motoneuron and Kr misexpression induces ectopic U3 cells. However, competence to generate U3 cells is limited to early divisions, when the Eve+ U motoneurons are produced, and competence is lost when NB7-1 transitions to making interneurons. This study found that Polycomb repressor complexes (PRCs) are necessary and sufficient to restrict competence in NB7-1. PRC loss of function extends the ability of Kr to induce U3 fates and PRC gain of function causes precocious loss of competence to make motoneurons. PRCs also restrict competence to make HB9+ Islet+ motoneurons in another neuroblast that undergoes a motoneuron-to-interneuron transition, NB3-1. In contrast to the regulation of motoneuron competence, PRC activity does not affect the production of Eve+ interneurons by NB3-3, HB9+ Islet+ interneurons by NB7-3, or Dbx+ interneurons by multiple neuroblasts. These findings support a model in which PRCs establish motoneuron-specific competence windows in neuroblasts that transition from motoneuron to interneuron production (Touma, 2012).

This study used multiple genetic approaches to investigate the timing and specificity of competence restriction by PRCs in Drosophila neuroblasts. The data show that PRCs establish motoneuron competence windows in two distinct neuroblast lineages, regulating the production of both Eve+ and HB9+ Islet+ motoneurons. This provides a mechanistic explanation for the loss of competence that has been previously described in NB7-1 and NB3-1. The experiments manipulating the timing of Pdm and Cas expression show that this mechanism is not limited to fate specification by Kr but is involved in establishing a broad motoneuron competence window. Consistent with this model, there appears to be little restriction of competence in a lineage that produces exclusively interneurons (NB3-3) and, correspondingly, PRC activity does not affect the ability of Kr to alter interneuron fates in this lineage. In addition, whereas Ph gain-of-function is sufficient to inhibit production of HB9+ Islet+ motoneurons by NB3-1, the production of HB9+ Islet+ interneurons by NB7-3 and of Dbx+ interneurons by multiple neuroblasts are unaffected (Touma, 2012).

The initial screen revealed a requirement for a subset of PRC1 and PRC2 genes in the regulation of competence. Lack of a statistically significant phenotype for other genes might be due to dosage: all embryos are heterozygous for the mutant allele and there is maternal contribution of Polycomb group and Trithorax group transcripts. Subsequent studies primarily used the Su(z)123 (null allele) and phd401, ph-p602 (ph-d401 is hypomorphic, ph-p602 is null) mutants. Su(z)12 is a component of PRC2 and Ph is a component of PRC1, allowing assessment the roles of each PRC complex. Su(z)12 loss-of- function extended competence to the end of the NB7-1 lineage. Su(z)12 is an essential co-factor of the E(z) H3K27 methyltransferase and levels of Su(z)12 activity correlate with the extent of H3K27 methylation at target genes. This suggests that the degree of competence restriction is determined by the levels of H3K27 methylation at genes required for motoneuron production. Progressive restriction of competence was still observed in the ph-d401, ph-d602 mutants, which was likely to be due to residual Ph activity. However, competence in these mutants is not completely lost until nearly twice the number of neuroblast divisions have occurred than are normally associated with loss of competence (nine divisions in ph mutants versus five in wild type). It is hypothesized that PRC-induced chromatin modifications accumulate over multiple neuroblast divisions and must reach some threshold for inhibiting motoneuron fates, similar to the accumulation of H3K27 trimethylation at the Neurog1 locus during competence restriction in mammalian cortical progenitors. Without testing additional Polycomb group and Trithorax group genes as homozygous mutants and generating maternal nulls (which in some cases might not survive to the relevant stages of neurogenesis), precisely which Polycomb group proteins are necessary for the restriction of competence cannot be precisely identifed. The core components of PRC1 and PRC2 are likely to be ubiquitously and constitutively expressed throughout neurogenesis, and this has been confirmed for Pc and Ph. However, cell type-specific PRC complexes and developmentally regulated changes in PRC composition have been described previously, suggesting that PRC1 or PRC2 co-factors might regulate the timing of competence restriction. It will be interesting to test the role of co-factors that are known or predicted to recruit PRC2 to specific genes, such as the PhoRC complex, Pipsqueak and Grainy head (Touma, 2012).

The sequential generation of motoneurons followed by interneurons has been observed during nervous system development of many insects. Clonal analysis of Drosophila neuroblasts suggests that motoneurons are always produced first, as demonstrated for NB7-1 and NB3-1, although precise birth order data are lacking for most other lineages. In the mammalian spinal cord, motoneurons and interneurons are produced from spatially segregated populations of progenitors that develop along the dorsal-ventral axis of the neural tube. Drosophila lacks this spatial segregation of motoneuroncommitted or interneuron-committed progenitors. Instead, temporal changes allow single progenitors to produce mixed lineages. PRCs appear to work in parallel to the temporal identity transcription factors by establishing competence windows in which temporal identity factors can specify motoneuron fates. Competence windows might represent a 'quality control' mechanism in which PRCs reinforce the timing of fate specification, similar to the role proposed for miRNAs during Drosophila development. Competence windows might also allow temporal identity factors to be 'redeployed' at later divisions. The majority of neuroblasts express Kr and Cas a second time and this study has confirmed that NB7-1 re-expresses Kr when interneurons are being produced. The function of Kr during later neuroblast divisions remains to be determined. If PRC activity alone were responsible for blocking a Kr-specified motoneuron late in the NB7-1 lineage, at least one ectopic U3 might be expected in ph-d, ph-p hemizygous or Su(z)12 homozygous mutant embryos. However, no altered U motoneuron fates were observed in such mutants. There are at least two potential explanations for this result. First, residual PRC activity in these mutants might allow sufficient changes in chromatin states to block endogenous Kr from specifying a motoneuron. This possibility is supported by data showing a dosage-sensitive relationship between Kr and PRC levels in specifying U3 fates, and the eventual loss of competence in ph mutants subjected to heat shock-induced pulses of Kr. Alternatively, there might be an additional transcription factor (or factors) that specifies interneuron fates in the NB7-1 lineage. This interneuron fate determinant could have a dominant effect, such that even when PRC activity is reduced, interneuron fates (or an Eve- 'hybrid' fate) prevail. Conversion to an Eve+ motoneuron might therefore only occur in a combined PRC loss-of-function and Kr gain-of-function background (Touma, 2012).

In both NB7-1 and NB3-1, later-born motoneuron fates are preferentially inhibited in Ph gain-of-function experiments, supporting a link between the number of neuroblast divisions and the restriction of motoneuron competence. The timing of competence restriction might also be regulated by the temporal identity factors themselves. Previous studies of competence in NB7-1 and NB3-1 have shown that constitutive expression of Hb can maintain neuroblasts in a fully competent state. In addition, precocious Pdm expression can inhibit Kr expression and block U3 fates in NB7-1 and RP3 fates in NB3-1. How Hb or Pdm might interact with Polycomb or Trithorax complexes during the regulation of competence remains to be determined (Touma, 2012).

In an attempt to identify PRC target genes that affect competence, NB7-1 fates were analyzed in embryos with wor-GAL4 driving expression of Kr in combination with the following candidates: the anterior-posterior patterning Hox genes Ultrabithorax, abdominal A, Antennapedia and Abdominal B, the nervous system-expressed Hox gene BarH1, the neuroblast fate determinant gooseberry, and the cell cycle regulator Cyclin A. No extension of competence was detected when these PRC targets are coordinately overexpressed with Kr. It would be technically very challenging and beyond the scope of this work to identify direct PRC targets in NB7-1 or NB3-1. However, clues are provided by previous studies that identified PRC targets in Drosophila embryos). One interesting set of PRC targets is a group of genes involved in motoneuron formation or function: eve, islet, HB9, Nkx6 (HGTX - FlyBase), zfh1 and Lim3. All motoneurons that innervate dorsal muscles express Eve, most motoneurons that innervate ventral muscles express some combination of Lim3, Islet, HB9 and Nkx6, and all somatic motoneurons express Zfh1. None of these genes is sufficient to confer motoneuron fates on their own, and some (eve, HB9, islet) are also expressed in subsets of interneurons. It is possible that PRCs silence the transcription of multiple genes that establish motoneuron fate 'combinatorial codes.' Relevant PRC target genes might be coordinately regulated by the temporal identity transcription factors (as suggested by the ability of high levels of Kr to partially overcome competence restriction) or transcription of these targets might depend on indirect interactions (Touma, 2012).

In mammalian embryonic stem cells, PRCs maintain pluripotency by inhibiting transcription of developmental pathway genes. These genes contain 'bivalent' histone modifications, with PRC-associated H3K27 methylation and Trithorax-associated H3K4 methylation keeping developmental regulators silenced but poised for activation. During differentiation of embryonic stem cells into neural progenitors, neural development genes lose PRC-associated modifications but retain H3K4 methylation, resulting in increased transcription. Although PRC silencing maintains pluripotency in embryonic stem cells, PRCs are likely to have an additional role in restricting fate potential once a progenitor becomes lineage committed. This was recently demonstrated for mouse embryonic endoderm progenitors, which undergo a fate choice for liver or pancreas development. The regulatory elements of liver and pancreas genes have distinct chromatin patterns prior to commitment to either lineage, and EZH2 [an ortholog of Drosophila E(z)] promotes liver development by restricting the expression of pancreatic genes. Similar chromatin 'prepatterns' might exist for motoneuron and interneuron genes in newly formed Drosophila neuroblasts, with subsequent PRC activity selectively silencing motoneuron genes in NB7-1 and NB3-1. PRC activity has also been shown to regulate the timing of terminal differentiation in mouse epidermal progenitors and the transition from neurogenesis to astrogenesis in mouse cortical progenitors. The identification of a related mechanism in Drosophila neuroblasts suggests that temporal restriction of fate potential is a common function of PRCs. Drosophila embryonic neuroblasts will provide a useful system for addressing several outstanding questions regarding PRC regulation of fate potential, including how PRCs are recruited to target genes, the composition of the relevant silencing complexes, and how PRC activity is temporally regulated (Touma, 2012).

Pcl-PRC2 is needed to generate high levels of H3-K27 trimethylation at Polycomb target genes

PcG protein complex PRC2 is thought to be the histone methyltransferase (HMTase) responsible for H3-K27 trimethylation at Polycomb target genes. This study reports the biochemical purification and characterization of a distinct form of Drosophila PRC2 that contains the Polycomb group protein Polycomblike (Pcl). Like PRC2, Pcl-PRC2 is an H3-K27-specific HMTase that mono-, di- and trimethylates H3-K27 in nucleosomes in vitro. Analysis of Drosophila mutants that lack Pcl unexpectedly reveals that Pcl-PRC2 is required to generate high levels of H3-K27 trimethylation at Polycomb target genes but is dispensable for the genome-wide H3-K27 mono- and dimethylation that is generated by PRC2. In Pcl mutants, Polycomb target genes become derepressed even though H3-K27 trimethylation at these genes is only reduced and not abolished, and even though targeting of the Polycomb protein complexes PhoRC and PRC1 to Polycomb response elements is not affected. Pcl-PRC2 is thus the HMTase that generates the high levels of H3-K27 trimethylation in Polycomb target genes that are needed to maintain a Polycomb-repressed chromatin state (Nekrasov, 2007).

Genetic studies using Drosophila first identified Polycomb group (PcG) genes as regulators that are required for the long-term repression of HOX genes during development. To date, 17 different genes in Drosophila are classified as PcG members because mutations in these genes cause misexpression of HOX genes. All Drosophila PcG genes are also conserved in mammals and at least some of them are also conserved in plants. In all these organisms, PcG gene products function as repressors of HOX and/or other regulatory genes that control specific developmental programs. Moreover, recent studies that analyzed genome-wide binding of PcG proteins in Drosophila and in mammalian cells have identified a large number of target sites, and thus a whole new set of genes that potentially is subject to PcG repression (Nekrasov, 2007).

Biochemical purification and characterization of PcG protein complexes has advanced understanding of the PcG system. To date, three distinct PcG protein complexes have been isolated from Drosophila: PhoRC, PRC1 and PRC2. Biochemically purified Drosophila PRC2 contains the three PcG proteins Enhancer of zeste [E(z)], Suppressor of zeste 12 [Su(z)12] and Extra sex combs (Esc) and, in addition, Nurf55 (Caf1), a protein that is present in many different chromatin complexes. Drosophila PRC2 and the homologue mammalian complex are histone methyltransferases (HMTases) that specifically methylate H3-K27 in nucleosomes. Chromatin immunoprecipitation (X-ChIP) analyses in Drosophila showed that PRC2 binds in a localized manner at Polycomb response elements (PREs) of target genes, but that H3-K27 trimethylation is present across the whole upstream control, promoter and coding region of these genes. Studies that compared the inactive and active state of the HOX gene Ubx in developing Drosophila have found that PRC2 is constitutively bound at PREs and, surprisingly, that the whole upstream control region is constitutively trimethylated at H3-K27. However, presence or absence of H3-K27 trimethylation in the Ubx promoter and coding region correlates tightly with the gene being repressed or active, respectively. H3-K27 trimethylation is thus a distinctive mark of PcG-repressed chromatin (Nekrasov, 2007).

Analysis of E(z) mutants suggests that E(z) is also responsible for the genome-wide H3-K27 mono- and dimethylation that has been reported to be present on more than 50% of H3 in Drosophila. However, biochemical analyses showed that E(z) protein alone does not bind to nucleosomes and is virtually inactive as an enzyme; E(z) needs to associate with Su(z)12 and Nurf55 for nucleosome binding and with Esc for enzymatic activity. This implies that the genome-wide H3-K27 mono- and dimethylation is generated by PRC2 or another E(z)-containing complex that is able to interact in a non-targeted manner with nucleosomes across the whole genome. Conversely, this raises the question whether H3-K27 trimethylation at PcG target genes is simply a consequence of PRC2 being targeted to PREs or whether additional features such as post-translational modifications or associated factors are required (Nekrasov, 2007).

Previous studies reported that the PcG protein Polycomblike (Pcl) interacts with E(z) in GST pull-down, yeast two-hybrid and co-immunoprecipitation assays. Like most other PcG proteins, Pcl has also been found to be bound at PREs in Drosophila. However, to date, no Pcl-containing complexes have been purified and the role of Pcl in PcG repression has remained enigmatic. This study reports the biochemical purification of Pcl complexes. Pcl is shown to exist in a stable complex with PRC2. This analyses demonstrate that this Pcl complex plays a critical role in generating high levels of repressive H3-K27 trimethylation at PcG target genes (Nekrasov, 2007).

Biochemically purified Pcl complexes contain Pcl together with the four core subunits of PRC2. In contrast, biochemically purified E(z) complexes contain only substoichiometric amounts of Pcl and the previously described purifications of PRC2 failed to reveal Pcl in the purified material. Moreover, fractionation of crude nuclear extracts by gel filtration indicated that Pcl and PRC2 components Esc, E(z) and Su(z)12 co-fractionate in high-molecular-weight assemblies, but that the bulk of these other PRC2 components is present in lower-molecular-weight fractions that do not contain Pcl. Taken together, these observations suggest that only a fraction of PRC2 is associated with Pcl and that Pcl-PRC2 is a distinct complex (Nekrasov, 2007).

Previous X-ChIP studies showed that Pcl and Su(z)12 colocalize at Ubx and Abd-B PREs. This suggests that Pcl-PRC2 is bound at these PREs. In this study, the analysis of Drosophila mutants that lack Pcl protein and therefore lack Pcl-PRC2, provided insight into the function of this complex. The results provide strong evidence that Pcl-PRC2 is needed to generate high levels of H3-K27 trimethylation in the chromatin of PcG target genes. Unlike in E(z) or Su(z)12 mutants, removal of Pcl in embryos or in imaginal discs only reduces but does not eliminate H3-K27 trimethylation. Nevertheless, repression of several PcG target genes is abolished in Pcl mutants. This suggests that not only the mere presence of H3-K27me3, but presence of high levels of H3-K27me3 is crucial for maintaining these PcG target genes in the repressed state. Previous studies on the Ubx gene suggested that presence of H3-K27 trimethylation in the promoter and coding region is critical for PcG repression. One possibility would be that it is the overall density of H3-K27me3-marked nucleosomes across the promoter and coding region that determines whether a PcG target gene is repressed. Another possibility would be that even though a whole chromatin domain becomes trimethylated at H3-K27, only a few H3-K27me3-marked nucleosomes at a particular position (e.g., around the transcription start site) are actually required for repression, and failure to maintain this trimethylation results in loss of repression (Nekrasov, 2007).

The observation that Su(z)12 binding and H3-K27 trimethylation are reduced but not lost in the absence of Pcl is consistent with the idea that Pcl might help anchoring PRC2 to PREs, but it also suggests that at least some PRC2 must be targeted to PREs independently of Pcl. It seems likely that the residual H3-K27 trimethylation present in Pcl mutant embryos and in Pcl mutant clones in imaginal discs is generated by PRC2 that is bound at PREs independently of Pcl. In this context it is important to note that not only Pcl-PRC2 but also PRC2 is able to trimethylate H3-K27 in recombinant nucleosomes in vitro. Apart from the suggested role in tethering of PRC2 to PREs, it is possible that Pcl also functions in a post-recruitment step to help PRC2 generate high levels of H3-K27 trimethylation at target genes. For example, the tudor domain and PHD fingers of PRE-bound Pcl might interact with modified nucleosomes in the promoter and coding region of target genes to ensure that they become trimethylated at H3-K27 by the associated PRE-tethered PRC2 (Nekrasov, 2007).

Finally, no evidence was found that Pcl-PRC2 would be required for the genome-wide H3-K27 mono- and di-methylation. X-ChIP analyses suggest that H3-K27 mono- and dimethylation across the genome might even slightly increase in the absence of Pcl. In contrast, there is a loss of all H3-K27 methylation in either E(z) or Suz)12 mutants. This suggests that PRC2 or another E(z)-containing complex generates the genome-wide H3-K27 mono- and dimethylation. The experiments in Pcl mutants thus allowed dissection of the role of different H3-K27 methylation states in Drosophila. The selective reduction of H3-K27me3 levels, and the concomitant loss of repression of PcG target genes in Pcl mutants, provides compelling evidence that only the trimethylated state of H3-K27 is functional in PcG repression in Drosophila. Pcl-PRC2 is evidently critically needed to generate the high levels of H3-K27 trimethylation that are required to maintain a Polycomb-repressed chromatin state (Nekrasov, 2007).

Nucleosome binding and histone methyltransferase activity of Drosophila PRC2

The Drosophila Polycomb group protein E(z) is a histone methyltransferase (HMTase) that is essential for maintaining HOX gene silencing during development. E(z) exists in a multiprotein complex called Polycomb repressive complex 2 (PRC2) that also contains Su(z)12, Esc and Nurf55. Reconstituted recombinant PRC2 methylates nucleosomes in vitro, but recombinant E(z) on its own shows only poor HMTase activity on nucleosomes. This study investigated the function of the PRC2 subunits. It was shown that PRC2 binds to nucleosomes in vitro but that individual PRC2 subunits alone do not bind to nucleosomes. By analysing PRC2 subcomplexes, it was shown that Su(z)12-Nurf55 is the minimal nucleosome-binding module of PRC2 and that Esc contributes to high-affinity binding of PRC2 nucleosomes. Nucleosome binding of PRC2 is not sufficient for histone methylation and only complexes that contain Esc protein show robust HMTase activity. These observations suggest that different subunits provide mechanistically distinct functions within the PRC2 HMTase: the nucleosome-binding subunits Su(z)12 and Nurf55 anchor the E(z) enzyme on chromatin substrates, whereas Esc is needed to boost enzymatic activity (Nekrasov, 2005).

Baculovirus expression vectors have been used to reconstitute and purify recombinant tetrameric PRC2 from Sf9 cells. To identify intermolecular interactions within this complex, tests were performed for reconstitution of PRC2 subcomplexes on coexpression of two or more subunits in Sf9 cells. In each case, the Flag-epitope tag present on one of the subunits was used for affinity purification from Sf9 cell extracts. The following stable dimeric complexes could be purfied: Esc-E(z), E(z)-Su(z)12, E(z)-Nurf55 and Su(z)12-Nurf55. In contrast, purification either from cells expressing Flag-Esc and Nurf55 or from cells expressing Flag-Esc and Su(z)12 resulted in the isolation of Flag-Esc protein only, suggesting that these proteins do not bind directly to each other. Purification from Sf9 cells expressing three subunits allowed the isolation of trimeric Esc-E(z)-Su(z)12 and E(z)-Su(z)12-Nurf55 complexes. Finally, tetrameric PRC2 was isolated, using either Flag-E(z) or Flag-Su(z)12 for affinity purification. Importantly, each of these complexes was stable in buffers containing up to 1 M KCl. Taken together, these data suggest that E(z) binds tightly to Su(z)12, Esc and Nurf55 and that Su(z)12 also binds to Nurf55. The failure to isolate dimeric complexes that contain Esc and Su(z)12 or those that contain Esc and Nurf55 indicates that E(z), Su(z)12 and Nurf55 form a trimeric core complex to which Esc binds through interaction with E(z). The observation that E(z) forms stable dimeric complexes with either Esc or Nurf55 in this reconstitution assay is consistent with earlier studies that reported physical interactions between these proteins in glutathione-S-transferase (GST) pull-down assays (Nekrasov, 2005).

It is noted that the molecular architecture of mammalian PRC2 is unclear at present; conflicting data on intermolecular interactions between subunits have been reported. Specifically, it has been reported that human EZH2, SU(Z)12 and RbAp48 all bind to EED, the Esc homologue, and that EZH2 does not interact with SU(Z)12 or RbAp48 in GST pull-down assays; it has been proposed that EED is the core component of the complex and EZH2 associates with other components through EED. It has also been reported that EZH2 binds to SUZ12 in GST pull-down assays, consistent with the finding that Drosophila E(z) and Su(z)12 form a stable complex (Nekrasov, 2005).

The HMTase activity of recombinant E(z) protein is significantly lower than the activity observed with recombinant tetrameric PRC2. A simple mechanistic explanation would be that one or several PRC2 subunits are needed for nucleosome binding to facilitate interaction of the E(z) HMTase with its substrate, the histone H3 tail. Since it is not known whether any of the PRC2 subunits binds to nucleosomes, tests were performed to see whether complex components alone or in combination could form stable complexes with mononucleosomes, in a bandshift assay. To this end, mononucleosomes were reconstituted with recombinant core histones that were expressed in E. coli and a 201 base pairs (bp) long radioactively labelled DNA template that contained a strong nucleosome-binding sequence called '601'. When recombinant tetrameric PRC2 was incubated with such mononucleosomes and the reaction mixture was resolved on a polyacrylamide gel, distinct, slowly migrating complexes were observed that appeared in a concentration-dependent manner. In contrast, when PRC2 was incubated with naked 601 DNA template, it was not possible to resolve specific protein-DNA complexes. Together, these observations suggest that PRC2 binds to mononucleosomes and that these protein-nucleosome complexes remain stably associated under electrophoretic conditions. Individual PRC2 subunits were tested for nucleosome binding, but no formation of protein-nucleosome complexes was detected with any of the four proteins. This suggests that more than one subunit is needed for nucleosome binding and therefore the different di- and trimeric PRC2 subcomplexes were tested. Among the different subcomplexes, only incubation with the Su(z)12-Nurf55 or E(z)-Su(z)12-Nurf55 complexes results in the appearance of distinct, slowly migrating protein-nucleosome complexes. These protein-nucleosome complexes migrate similarly to the complexes observed with tetrameric PRC2, but two- to threefold higher concentrations of the Su(z)12-Nurf55 or E(z)-Su(z)12-Nurf55 complex are needed to shift all of the nucleosome probe. Thus, the presence of Esc in PRC2 increases the affinity of the complex for nucleosomes or allows the complex to bind more stably under the experimental conditions compared with PRC2 subcomplexes that lack Esc. In contrast to the distinct protein-nucleosome complexes observed with the Su(z)12-Nurf55 or E(z)-Su(z)12-Nurf55 complex, no specific protein-nucleosome complexes are formed if nucleosomes are incubated with the E(z)-Su(z)12, E(z)-Nurf55, Esc-E(z) or Esc-E(z)-Su(z)12 complexes. However, incubation with high concentrations of the trimeric Esc-E(z)-Su(z)12 complex also shifts almost all of the nucleosome probe, and much of the probe is retained in the well of the gel. Thus, it seems that the Esc-E(z)-Su(z)12 complex also binds to nucleosomes but that it binds in a manner distinct from the other PRC2 (sub)complexes. Taken together, these binding assays suggest that several subunits need to cooperate for nucleosome binding of PRC2, as follows: (1) Su(z)12 is essential for nucleosome binding because only complexes containing Su(z)12 bind; (2) the minimal nucleosome-binding complex contains Su(z)12 and Nurf55, and only complexes that contain these two proteins give rise to distinct, slowly migrating protein-nucleosome complexes; Su(z)12 and Nurf55 together thus form the minimal nucleosome-binding module of PRC2; (3) as discussed above, Esc also contributes to nucleosome binding because (a.) tetrameric PRC2 binds more strongly than the E(z)-Su(z)12-Nurf55 complex and (b.) in the absence of Nurf55, that is, in the Esc-E(z)-Su(z)12 complex, Esc seems to cooperate with Su(z)12 to cause retention of the nucleosome probe. E(z) is thus the only subunit for which no contribution to nucleosome binding was detected. Note that comparable concentrations of the Su(z)12-Nurf55 or E(z)-Su(z)12-Nurf55 complex are required to shift 50% of the nucleosome probe (Nekrasov, 2005).

Nurf55 and Esc are both WD40 repeat proteins. RbAp46 and RbAp48, the mammalian homologues of Nurf55, have been reported to bind directly to helix 1 of histone H4, a portion of H4 that is thought to be inaccessible within the nucleosome, and, consistent with this, RbAp46 and RbAp48 are unable to bind to H4 in nucleosomal templates. As shown in this study, Drosophila Nurf55 or Esc alone are not able to bind to mononucleosomes but they bind in combination with Su(z)12, which, by itself, also does not bind to nucleosomes. It is possible that the combination of Su(z)12 and Nurf55 or Esc is needed to create the necessary surface for stable nucleosome binding. Alternatively, it could be that these proteins act in a cooperative manner to disrupt histone-DNA contacts locally to expose the histone core (i.e. H4) for binding by Nurf55 or Esc (Nekrasov, 2005).

These results suggest that Su(z)12 together with Nurf55 or Esc tethers the complex to nucleosomes, whereas E(z), the catalytic subunit of the complex, contributes little to nucleosome binding. Next the HMTase activity of the different PRC2 (sub)complexes were analyzed. As substrates for these reactions, non-radiolabelled mononucleosomes were used identical to those used in the bandshift assay. Recombinant tetrameric PRC2 methylates H3 in mononucleosomes. In contrast, E(z) protein alone, the dimeric Esc-E(z), and the Su(z)12-E(z) or E(z)-Nurf55 complexes do not detectably methylate mononucleosomes. Strikingly, the Su(z)12-E(z)-Nurf55 complex also shows no detectable HMTase activity, whereas the Esc-E(z)-Su(z)12 complex methylates H3 in mononucleosomes with efficacy similar to tetrameric PRC2. Thus, no straightforward correlation is observed between nucleosome binding in bandshift assays and HMTase activity. In particular, the Su(z)12-E(z)-Nurf55 complex seems to bind to nucleosomes with only two- to threefold lower affinity than tetrameric PRC2, but shows markedly reduced HMTase activity. In contrast, the Esc-E(z)-Su(z)12 complex, which shows almost no nucleosome binding at the concentration used in the HMTase assay, shows HMTase activity comparable with PRC2. Together, these data suggest that nucleosome binding is not sufficient for HMTase activity and that Esc has a crucial role in boosting the enzymatic activity of E(z). It is possible that Esc is required to dock the complex in a specific orientation on the nucleosome that presents the H3 tail in a particularly favourable position to the E(z) enzyme. Alternatively, Esc could directly increase the catalytic activity of E(z) by inducing a conformational change in the enzyme. However, it is important to note that the Esc-E(z) complex shows no detectable HMTase activity on mononucleosomes and the presence of Su(z)12 is thus essential for HMTase activity of the Esc-E(z)-Su(z)12 complex. The bandshift data show that Su(z)12 is strictly needed for nucleosome binding of PRC2. At the low complex concentrations used in the HMTase assay, there is probably very little nucleosome binding of the Esc-E(z)-Su(z)12 complex; nevertheless, it seems probable that the nucleosome interactions that Su(z)12 shows in cooperation with Esc contribute to the HMTase activity of the Esc-E(z)-Su(z)12 complex. Finally, it is puzzling that the absence of Nurf55 from the complex (i.e. in the Esc-E(z)-Su(z)12 complex) does not seem to diminish HMTase activity because Nurf55 is important for the formation of stable PRC2-nucleosome complexes in bandshift assays. It is possible that Nurf55 is not needed for HMTase activity under the assay conditions that were used but that it is important for methylation of chromatin in vivo (Nekrasov, 2005).

Recent studies have shown that SU(Z)12 is crucial for HMTase activity of mammalian PRC2 in vitro, but the molecular basis for this requirement has remained unclear. This study reports two main findings: (1) Su(z)12 has a crucial role in nucleosome binding of Drosophila PRC2; (2) PRC2 subcomplexes that bind to nucleosomes but lack Esc are poorly active; Esc thus has an important role in boosting HMTase activity of the complex (Nekrasov, 2005).

The requirement of Su(z)12 for HMTase activity can for the most part be explained by its ability to tether PRC2 to nucleosomes, and the data suggest that nucleosome binding requires Su(z)12 in combination with either Nurf55 or Esc. Although both Nurf55 and Esc contribute to nucleosome binding of the complex, only inclusion of Esc leads to an increase in HMTase activity. The contribution of Esc to HMTase activity thus goes beyond the activity that one would expect if Esc were required only for nucleosome binding. In summary, these data suggest that the Su(z)12, Nurf55 and Esc subunits all contribute to nucleosome binding of PRC2 but that these three subunits make distinct contributions to the activation of the E(z) HMTase (Nekrasov, 2005).

The findings reported here imply that E(z) HMTase activity in vivo could be regulated at the level of chromatin binding and/or enzyme activity by modulating the abundance or activity of different PRC2 subunits. It is important to discuss the results reported here in the context of the in vivo requirement for different PRC2 subunits. Genetic studies have shown that Su(z)12 and E(z) are required throughout development to maintain silencing of HOX genes in Drosophila and that this process requires the enzymatic activity of E(z). In contrast, Esc protein is required early in development and then becomes to a large extent, although not completely, dispensable for maintenance of HOX gene silencing during postembryonic development. There are two possible explanations for the paradoxical observation that Esc is required for strong HMTase activity in vitro but that the protein seems to be largely dispensable for the HMTase activity of E(z) that is needed to maintain HOX gene silencing during larval development. (1) It is possible that strong HMTase activity of PRC2 is required primarily early in embryogenesis and that, once it is established, H3-K27 methylation can be maintained with a catalytically less active form of the complex (i.e., lacking Esc). (2) It is possible that another protein substitutes for Esc at later developmental stages. It is noted that the Drosophila genome encodes a second Esc-like protein (CG5202) but, at present, it is not known whether this protein is required for HOX gene silencing (Nekrasov, 2005).

Epigenetic blocking of an enhancer region controls irradiation-induced proapoptotic gene expression in Drosophila embryos

Drosophila embryos are highly sensitive to gamma-ray-induced apoptosis at early but not later, more differentiated stages during development. Two proapoptotic genes, reaper and hid, are upregulated rapidly following irradiation. However, in post-stage-12 embryos, in which most cells have begun differentiation, neither proapoptotic gene can be induced by high doses of irradiation. The sensitive-to-resistant transition is due to epigenetic blocking of the irradiation-responsive enhancer region (IRER), which is located upstream of reaper but is also required for the induction of hid in response to irradiation. This IRER, but not the transcribed regions of reaper/hid, becomes enriched for trimethylated H3K27/H3K9 and forms a heterochromatin-like structure during the sensitive-to-resistant transition. The functions of histone-modifying enzymes Hdac1(Rpd3) and Su(var)3-9 and PcG proteins Su(z)12 and Polycomb are required for this process. Thus, direct epigenetic regulation of two proapoptotic genes controls cellular sensitivity to cytotoxic stimuli (Zhang, 2008).

Irradiation responsiveness appears to be a highly conserved feature of reaper-like IAP antagonists. A recently identified functional ortholog of reaper in mosquito genomes, michelob_x (mx), is also responsive to irradiation. These results highlighted that stress responsiveness is an essential aspect of functional regulation of upstream proapoptotic genes such as reaper/hid. It is also worth mentioning that several mammalian BH3 domain-only proteins, the upstream proapoptotic regulators of the Bcl-2/Ced-9 pathway, are also regulated at the transcriptional level (Zhang, 2008).

This study shows that the irradiation responsiveness of reaper and hid is subject to epigenetic regulation during development. The epigenetic regulation of the IRER is fundamentally different from the silencing of homeotic genes in that the change of DNA accessibility is limited to the enhancer region while the promoter of the proapoptotic genes remains open. Thus, it seems more appropriate to refer this as the 'blocking' of the enhancer region instead of the 'silencing' of the gene. This region, containing the putative P53RE and other essential enhancer elements, is required for mediating irradiation responsiveness. ChIP analysis indicates that histones in this enhancer region are quickly trimethylated at both H3K9 and H3K27 at the sensitive-to-resistant transition period, accompanied by a significant decrease in DNA accessibility. DNA accessibility in the putative P53RE locus (18,368k), when measured by the DNase I sensitivity assay, did not show significant decrease until sometime after the transition period. It is possible that other enhancer elements, in the core of IRER_left, are also required for radiation responsiveness. An alternative explanation is that the strong and rapid trimethylation of H3K27 and association of PRC1 at 18,366,000-18, 368,000 are sufficient to disrupt DmP53 binding and/or interaction with the Pol II complex even though the region remains relatively sensitive to DNase I. Eventually, the whole IRER is closed with the exception of an open island around 18,387,000 (Zhang, 2008).

The finding that epigenetic regulation of the enhancer region of proapoptotic genes controls sensitivity to irradiation-induced cell death may have implications in clinical applications involving ionizing irradiation. It suggests that applying drugs that modulate epigenetic silencing may help increase the efficacy of radiation therapy. It also remains to be seen whether the hypersensitivity of some tumors to irradiation is due to the dedifferentiation and reversal of epigenetic blocking in cancer cells. In contrast, loss of proper stress response to cellular damage is implicated in tumorigenesis. The fact that the formation of heterochromatin in the sensitizing enhancer region of proapoptotic genes is sufficient to convey resistance to stress-induced cell death suggests it could contribute to tumorigenesis. In addition, it could also be the underlying mechanism of tumor cells' evading irradiation-induced cell death. This is a likely scenario given that it has been well documented that oncogenes such as Rb and PML-RAR fusion protein cause the formation of heterochromatin through recruiting of a human ortholog of Su(v)3-9. In this regard, the reaper locus, especially the IRER, provides an excellent genetic model system for understanding the cis- and trans-acting mechanisms controlling the formation of heterochromatin associated with cellular differentiation and tumorigenesis (Zhang, 2008).

The developmental consequence of epigenetic regulation of the IRER is the tuning down (off) of the responsiveness of the proapoptotic genes, thus decreasing cellular sensitivity to stresses such as DNA damage. Epigenetic blocking of the IRER corresponds to the end of major mitotic waves when most cells begin to differentiate. Similar transitions were noticed in mammalian systems. For instance, proliferating neural precursor cells are extremely sensitive to irradiation-induced cell death while differentiating/differentiated neurons become resistant to γ-ray irradiation, even though the same level of DNA damage was inflicted by the irradiation. These findings here suggest that such a dramatic transition of radiation sensitivity could be achieved by epigenetic blocking of sensitizing enhancers (Zhang, 2008).

Later in Drosophila development, around the time of pupae formation, the organism becomes sensitive to irradiation again, with LD50 values similar to what was observed for the 4–7 hr AEL embryos. Interestingly, it has also been found that during this period, the highly proliferative imaginal discs are sensitive to irradiation-induced apoptosis, which is mediated by the induction of reaper and hid through P53 and Chk2. However, it remains to be studied whether the reemergence of sensitive tissue is due to reversal of the epigenetic blocking in the IRER or the proliferation of undifferentiated stem cells that have an unblocked IRER (Zhang, 2008).

The blocking of the IRER differs fundamentally from the silencing of homeotic genes in several aspects. (1) The change of DNA accessibility and histone modification is largely limited to the enhancer region. The promoter regions of reaper (and hid) remain open, allowing the gene to be responsive to other stimuli. Indeed, there are a few cells in the central nervous system that could be detected as expressing reaper long after the sensitive-to-resistant transition. Even more cells in the late-stage embryo can be found having hid expression. Yet, the irradiation responsiveness of the two genes is completely suppressed in most if not all cells, transforming the tissues into a radiation-resistant state (Zhang, 2008).

(2) The histone modification of the IRER has a mixture of features associated with pericentromeric heterochromatin formation and canonic PcG-mediated silencing. Both H3K9 and H3K27 are trimethylated in the IRER. Both HP1, the signature binding protein of the pericentromeric heterochromatin, and PRC1 are bound to the IRER. As demonstrated by genetic analysis, the functions of both Su(var)3-9 and Su(z)12/Pc are required for the silencing. Preliminary attempts to verify specific binding of PRC2 proteins to this region were unsuccessful. The fact that none of the mutants tested could completely block the transition seems to suggest that there is a redundancy of the two pathways in modifying/blocking the IRER. It is also possible that the genes tested are not the key regulators of IRER blocking but only have participatory roles in the process (Zhang, 2008).

(3) Within the IRER, there is a small region around 18,386,000 to 18,188,000 that remains relatively open until the end of embryogenesis. Interestingly, this open region is flanked by two putative noncoding RNA transcripts represented by EST sequences. If they are indeed transcribed in the embryo as suggested by the mRNA source of the cDNA library, then the 'open island' within the closed IRER will likely be their shared enhancer/promoter region. Sequences of both cDNAs revealed that there is no intron or reputable open reading frame in either sequence. Despite repeated efforts, their expression was not confirmed via ISH or northern analysis. Overexpression of either cDNA using an expression construct also failed to show any effect on reaper/hid-induced cell death in S2 cells. Yet, sections of the two noncoding RNAs are strongly conserved in divergent Drosophila genomes. The potential role of these two noncoding RNAs in mediating reaper/hid expression and/or blocking of the IRER remains to be studied (Zhang, 2008).

Association of Polycomb group SUZ12 with WD-repeat protein MEP50 that binds to histone H2A selectively in vitro

SUZ12 is a Polycomb group protein that forms Polycomb repressive complexes (PRC2/3) together with EED and histone methyltransferase EZH2. Although the essential role of SUZ12 in regulating the activity of the PRC2/3 complexes has been demonstrated, additional function of this protein was suggested. This study shows that SUZ12 interacts with WD-repeat protein MEP50 in vitro and in vivo. MEP50 binds histone H2A selectively among core histones, and mediates transcriptional repression of protein arginine methyltransferase PRMT5, which is known to methylate H2A and H4. These results suggest that SUZ12 might have a role in transcriptional regulation through physical interaction with MEP50 that can be an adaptor between PRMT5 and its substrate H2A (Furuno, 2006).

Subunit contributions to histone methyltransferase activities of fly and worm polycomb group complexes

The ESC-E(Z) complex of Drosophila Polycomb group (PcG) repressors is a histone H3 methyltransferase (HMTase). This complex silences fly Hox genes, and related HMTases control germ line development in worms, flowering in plants, and X inactivation in mammals. The fly complex contains a catalytic SET domain subunit, E(Z), plus three noncatalytic subunits, SU(Z)12, ESC, and NURF-55/CAF-1. The four-subunit complex is >1,000-fold more active than E(Z) alone. ESC and SU(Z)12 play key roles in potentiating E(Z) HMTase activity. Loss of ESC disrupts global methylation of histone H3-lysine 27 in fly embryos. Subunit mutations identify domains required for catalytic activity and/or binding to specific partners. Missense mutations are described in surface loops of ESC, in the CXC domain of E(Z), and in the conserved VEFS domain of SU(Z)12, which each disrupt HMTase activity but preserve complex assembly. Thus, the E(Z) SET domain requires multiple partner inputs to produce active HMTase. A recombinant worm complex containing the E(Z) homolog, MES-2, has robust HMTase activity, which depends upon both MES-6, an ESC homolog, and MES-3, a pioneer protein. Thus, although the fly and mammalian PcG complexes absolutely require SU(Z)12, the worm complex generates HMTase activity from a distinct partner set (Ketel, 2005).

In vitro and in vivo data indicate that the noncatalytic ESC subunit makes a critical contribution to HMTase function of the ESC-E(Z) complex. In particular, since global levels of H3-K27 methylation are similarly reduced by genetic loss of ESC or E(Z), ESC appears to be an obligate functional partner for E(Z) HMTase activity (Ketel, 2005).

Two main molecular explanations are envisioned for the ESC requirement. (1) ESC could potentiate HMTase activity through direct interaction with E(Z). ESC binding could trigger a conformational change in E(Z) that improves catalytic efficiency, and/or ESC residues could directly interact with and influence the E(Z) active site. (2) Alternatively, the main role of ESC could be to bind nucleosomes. In this scenario, ESC would boost HMTase activity by facilitating interaction of the enzyme complex with its substrate. Based on several lines of evidence, a mechanism is favored that works through direct ESC-E(Z) contact. (1) The ESC M236K and V289M mutations, which significantly reduce HMTase activity, are located in surface loops previously shown to mediate direct ESC contact with E(Z). Furthermore, M236K displays dominant-negative properties in vivo. This genetic behavior is consistent with an enzyme complex that assembles normally but is compromised in catalytic function. (2) A recent report documents that ESC lacks nucleosome-binding activity on its own and that addition of ESC to a trimeric NURF-55/SU(Z)12/E(Z) complex has little additive effect on ability to bind nucleosomes. (3) ESC potentiation through direct E(Z) binding is supported by evolutionary considerations. Every organism examined that has an E(Z) homolog, ranging from plants to worms, flies, and humans, has at least one ESC homolog. In addition, 28 residues within the ESC surface loops implicated in E(Z) binding are identical from flies to humans. This conservation may reflect a tight functional requirement wherein direct ESC-E(Z) partnership, combining to produce HMTase activity, is maintained by evolutionary pressure. Future studies will be needed to define the precise biochemical mechanism by which ESC potentiates HMTase activity, including tests for binding-induced conformational changes in E(Z) (Ketel, 2005).

Studies on the mammalian homolog of ESC, called EED, have also highlighted its important role as a regulatory subunit. In human cells, multiple EED isoforms are expressed, which differ in the extents of their N-terminal tails. These isoforms are generated by alternative start codon usage of the same EED mRNA. Intriguingly, incorporation of particular EED isoforms into EZH2 complexes can shift the enzyme specificity so that K26 of histone H1 is methylated in addition to H3-K27. Thus, it appears that the ESC/EED subunit can influence both catalytic efficiency and lysine substrate preference. In the fly system, ESC isoforms produced from the same mRNA have not been detected. Instead, alternative ESC isoforms could be supplied by an esc-related gene (CG5202) located about 150 kb proximal to esc. Since mutations in this second esc gene, called esc-like (escl), have not yet been reported, its in vivo contributions remain to be assessed. However, since genetic loss of ESC alone dramatically reduces global methylation of H3-K27 in fly embryos, it is concluded that ESC is the predominant functional E(Z) partner during embryonic stages (Ketel, 2005).

Studies on recombinant complexes show that fly SU(Z)12 is absolutely required for HMTase activity of the ESC-E(Z) complex. A key requirement for SU(Z)12 in mammalian EZH2 complexes has also been established based upon in vitro tests and loss-of-function studies in vivo. How does SU(Z)12 contribute molecularly to HMTase activity? Again, two main possibilities are envisioned: influence through direct contact with E(Z) or by mediating nucleosome binding. To address this, it is instructive to consider the SU(Z)12 mutants affecting the conserved VEFS domain. Deletion of the entire VEFS domain eliminates assembly of the fly complex by disrupting SU(Z)12-E(Z) binding. Pairwise binding assays with mammalian SU(Z)12 have similarly shown that the VEFS domain is needed for binding to EZH2 in vitro. Thus, a conserved function of this domain is to contact E(Z). However, missense mutations within the VEFS domain, D546A and E550A, preserve full complex assembly yet have reduced levels of HMTase. Taken together, these results implicate the VEFS domain in both binding to E(Z) and potentiating its enzyme activity, which suggests a connection between these two functions. In contrast, a recent report provides evidence that SU(Z)12 contributes to affinity for nucleosomes. Although SU(Z)12 cannot bind to nucleosomes by itself, the SU(Z)12/NURF-55 dimer has nucleosome-binding properties that are similar to those of the four-subunit complex. Thus, as also suggested for the human PRC2 complex, at least one role of SU(Z)12 is to mediate nucleosome binding. Further work will be needed to define the SU(Z)12 functional domains required for interactions with NURF-55 and with nucleosomes. Based on the available data, SU(Z)12 potentiation of E(Z) HMTase activity may involve both direct E(Z) contact and facilitated binding to nucleosome substrate (Ketel, 2005).

Although the SET domain is the most well-characterized functional domain of E(Z), the adjacent cysteine-rich CXC domain is also remarkably conserved from flies to humans. To address CXC function, in vitro properties of two missense mutants, C545Y and C603Y were analyzed. Both mutations correspond to E(z) loss-of-function alleles; in particular, in vivo effects of the E(z)61 mutation (C603Y) have been well documented. This mutation disrupts global H3-K27 methylation in embryos and causes loss of methyl-H3-K27 from a Hox target gene in imaginal discs. Mutant complexes bearing E(Z)-C603Y can assemble normally but show an approximately 10-fold reduction in HMTase levels. C545Y causes a more modest HMTase reduction, which parallels results obtained with the analogous substitution (C588Y) in human EZH2. These results suggest that the CXC domain interfaces with the SET domain to produce robust HMTase activity. In this regard, the CXC domain could be considered similar to cysteine-rich "preSET" domains required for robust HMTase activity in other SET domain proteins. Another effect of these CXC mutations in vivo is that they dislodge E(Z) from target sites in chromatin. Although the molecular basis for this dissociation is not known, in vitro assembly results suggest that it is not due to wholesale destabilization of the ESC-E(Z) complex. The dissociation may reflect another proposed role for the CXC domain, which is to interact with the PcG targeting factor PHO (Ketel, 2005).

In vitro tests were performed to investigate the role of E(Z) domain II. Both the complete domain deletion and the C363Y missense mutation show that domain II is required for stable association of E(Z) with SU(Z)12. Thus, the composite domain organization of E(Z) reflects division of labor among catalytic functions and requirements for complex assembly. In addition, it appears that none of the E(Z) domains are specifically built for nucleosome interactions; E(Z) plays little or no role itself in stable binding of the complex to nucleosomes (Ketel, 2005).

The NURF-55 subunit is distinct from the other three subunits in several ways. (1) It makes only minimal contributions to in vitro HMTase activity in both the fly and mammalian complexes. (2) Whereas the other three subunits appear dedicated to PcG function, NURF-55 is present in diverse chromatin-modifying complexes, including NURF, chromatin assembly factor 1 (CAF-1), and histone deacetylase complexes. The ability of the mammalian NURF-55 homologs RbAp46 and RbAp48 to bind to free histone H4 has led to the suggestion that NURF-55 may help chromatin complexes interact with substrate. Indeed, the absence of a NURF-55-related protein from the trimeric worm MES complex could help explain its inability to methylate free histones. The free histone-binding property of NURF-55 has also prompted the intriguing suggestion that silencing by the ESC-E(Z) complex in vivo could involve methylation of histones prior to nucleosome assembly. Since NURF-55 loss-of-function alleles have not been described in flies, many questions about roles of NURF-55 remain to be addressed. Even with alleles available, the multiplicity of NURF-55-containing complexes will likely complicate in vivo dissection of its PcG functions (Ketel, 2005).

The basic enzymatic function of the ESC-E(Z) complex, to methylate H3-K27, is shared between the worm, fly, and mammalian versions. Another similarity revealed from this study of recombinant worm complexes is that robust HMTase activity depends critically upon the two noncatalytic subunits. Although MES-6 and MES-3 can each individually bind to the catalytic subunit, MES-2, all three subunits are required together to produce enzyme activity. Since worm MES-6 is a WD repeat protein related to fly ESC, it seems likely that MES-6 and ESC potentiate HMTase activity through similar mechanisms. It is suggested that this mechanism entails direct subunit interactions rather than an influence upon affinity for nucleosomes. However, a major puzzle is presented by the dissimilarity between worm MES-3 and fly SU(Z)12. Though each is required for HMTase activity in their respective complexes, no relatedness was recognized between these two proteins in primary sequence or predicted secondary structure arrangement. From an evolutionary standpoint, it appears that SU(Z)12 represents the more ancient partner, since it is functionally conserved across plant and animal kingdoms. MES-3, a novel protein, may have evolved more recently to replace SU(Z)12 in the worm complex. Since a molecular role attributed to SU(Z)12 in the fly complex is nucleosome binding, it is speculated that MES-3 may supply this function for the worm complex. There are many strategies for building nucleosome contact domains, as represented among divergent chromatin proteins, so MES-3 could have acquired functional similarity without overt sequence similarity to SU(Z)12. In this view, MES-3 function in the worm complex would require, at minimum, affinity for nucleosomes and ability to bind MES-2. In this regard, it is interesting that MES-2 appears to lack domain II, which is needed in E(Z) for stable binding to SU(Z)12. Presumably, MES-2 has instead acquired a site for stable MES-3 interaction. In summary, it is suggested that the E(Z)/ESC and MES-2/MES-6 dimers have been conserved as core subunits of the HMTase complex, whereas the additional required partners in each complex, SU(Z)12 and MES-3, have been allowed to diverge. Future studies will be needed, including functional tests of chimeric worm and fly proteins, to address such a model (Ketel, 2005).

AEBP2 as a potential targeting protein for Polycomb Repression Complex PRC2

AEBP2 (Drosophila homolog: Jing) is a zinc finger protein that has been shown to interact with the mammalian Polycomb Repression Complex 2 (PRC2). This study characterized this unknown protein and tested its potential targeting roles for the PRC2. AEBP2 is an evolutionarily well-conserved gene that is found in the animals ranging from flying insects to mammals. The transcription of mammalian AEBP2 is driven by two alternative promoters and produces at least two isoforms of the protein. These isoforms show developmental stage-specific expression patterns: the adult-specific larger form (51 kDa) and the embryo-specific smaller form (32 kDa). The AEBP2 protein binds to a DNA-binding motif with an unusual bipartite structure, CTT(N)15-23cagGCC with lower-case being less critical. A large fraction of AEBP2's target loci also map closely to the known target loci of the PRC2. In fact, many of these loci are co-occupied by the two proteins, AEBP2 and SUZ12. This suggests that AEBP2 is most likely a targeting protein for the mammalian PRC2 complex (Kim, 2009).

SUZ12 is required for both the histone methyltransferase activity and the silencing function of the EED-EZH2 complex

Recent studies have revealed the intrinsic histone methyltransferase (HMTase) activity of the EED-EZH2 complex and its role in Hox gene silencing, X inactivation, and cancer metastasis. This study focuses on the function of individual components. It was found that the HMTase activity requires a minimum of three components -- EZH2, EED, and SUZ12 -- while AEBP2 is required for optimal enzymatic activity. Using a stable SUZ12 knockdown cell line, it has been shown that SUZ12 knockdown results in cell growth defects, which correlate with genome-wide alteration on H3-K27 methylation as well as upregulation of a number of Hox genes. Chromatin immunoprecipitation (ChIP) assay identified a 500 bp region located 4 kb upstream of the HoxA9 transcription initiation site as a SUZ12 binding site, which responds to SUZ12 knockdown and might play an important role in regulating HoxA9 expression. Thus, this study establishes a critical role for SUZ12 in H3-lysine 27 methylation and Hox gene silencing (Cao, 2004).

Silencing of human polycomb target genes is associated with methylation of histone H3 Lys 27

Polycomb group complexes 2 and 3 are involved in transcriptional silencing. These complexes contain a histone lysine methyltransferase (HKMT) activity that targets different lysine residues on histones H1 or H3 in vitro. However, it is not known if these histones are methylation targets in vivo because the human PRC2/3 complexes have not been studied in the context of a natural promoter because of the lack of known target genes. RNA expression arrays and CpG-island DNA arrays were used to identify and characterize human PRC2/3 target genes. Using oligonucleotide arrays, a cohort of genes were identified whose expression changes upon siRNA-mediated removal of Suz12 [Drosophila homolog Su(z)12], a core component of PRC2/3, from colon cancer cells. To determine which of the putative target genes are directly bound by Suz12 and to precisely map the binding of Suz12 to those promoters, a high-resolution chromatin immunoprecipitation (ChIP) analysis was combined with custom oligonucleotide promoter arrays. Additional putative Suz12 target genes were identified by using ChIP coupled to CpG-island microarrays. HKMT-Ezh2 and Eed, two other components of the PRC2/3 complexes, colocalize to the target promoters with Suz12. Importantly, recruitment of Suz12, Ezh2 and Eed to target promoters coincides with methylation of histone H3 on Lys 27 (Kirmizis, 2004).

Identification of mammalian PcG target genes has remained elusive for two main reasons. First, the majority of the previous PcG studies focused mostly on the biochemical purification and in vitro characterization of the activities of the PcG complexes and second, the lack of DNA-binding domains within PcG proteins makes the search for their target loci difficult. In this present study, the first known direct target genes of mammalian PcG complexes has been identified. To do so, RNAi was first used to identify genes deregulated by the loss of Suz12 protein in colon cancer cells. Next, Suz12 was shown to bind directly to the promoter of one of these genes (MYT1). Other members of the PRC2/3 complexes were shown to colocalize with Suz12 at the MYT1 promoter. Most importantly, recruitment of Suz12, Ezh2, and Eed to the MYT1 promoter was shown to correlate with methylation of H3-K27. To demonstrate that this silencing mechanism is not unique to MYT1, other Suz12 target genes were identified using a ChIP assay coupled to a CpG island microarray. Similarly to MYT1, the other target promoters of Suz12 are bound by the PRC2/3 components and are characterized by H3-K27 methylation. Thus, the first identified human PcG target genes all appear to be regulated by the histone methylase activity of the PRC complexes (Kirmizis, 2004).

The Suz12 target gene MYT1 was originally cloned from a human brain cDNA library on the basis of its ability to bind cis-regulatory elements of the glia-specific myelin proteolipid protein (PLP) gene and is suggested to be the prototype of the C2HC-type zinc finger protein family. More recently, the Xenopus ortholog of MYT1 (X-MYT1) was identified as a transcriptional activator because it could induce expression of an N-tubulin promoter reporter construct in transient transfection assays. Dominant-negative forms of X-MYT1 inhibited normal neurogenesis, suggesting that X-MYT1 is essential for inducing neuronal differentiation. Intriguingly, a recent report shows that the Xenopus ortholog of Ezh2 (XEZ) is expressed exclusively in the anterior neural plate during early Xenopus embryogenesis, and it was postulated that XEZ might be involved in delaying anterior neuronal differentiation (Barnett, 2001). Based on the current findings, it is possible that Ezh2 delays neuronal differentiation, via the PRC2/3 complexes, by repressing the activity of the MYT1 gene. In addition to MYT1, four additional promoters were identified as being robustly bound by components of the PRC2/3 complexes; each of these promoters is also characterized by high levels of H3-K27. Although a link between components of the PRC2/3 complexes and Wnt1, the cannabinoid receptor (CNR1), or the potassium channel KCNA1 have not been previously reported, it is intriguing to note that these mRNAs are expressed at very low levels in most human tissues, suggesting that they may be generally silenced by the PRC complexes. In support of this hypothesis, some of these target genes were shown to be bound by PRC2/3 components in other cell lines, such as the human MCF7 and mouse F9 (Kirmizis, 2004).

Histone methyltransferase activity associated with a human multiprotein complex containing the Enhancer of Zeste protein

Enhancer of Zeste [E(z)] is a Polycomb-group transcriptional repressor and one of the founding members of the family of SET domain-containing proteins. Several SET-domain proteins possess intrinsic histone methyltransferase (HMT) activity. However, recombinant E(z) protein was found to be inactive in a HMT assay. A multiprotein E(z) complex has been isolated from humans that contains extra sex combs, suppressor of zeste-12 [Su(z)12], and the histone binding proteins RbAp46/RbAp48 (see Caf1). This complex, which has been termed Polycomb repressive complex (PRC) 2, possesses HMT activity with specificity for Lys 9 (K9) and Lys 27 (K27) of histone H3. The HMT activity of PRC2 is dependent on an intact SET domain in the E(z) protein. It is hypothesized that transcriptional repression by the E(z) protein involves methylation-dependent recruitment of PRC1. The presence of Su(z)12, a strong suppressor of position effect variegation, in PRC2 suggests that PRC2 may play a widespread role in heterochromatin-mediated silencing (Kuzmichev, 2002).

The polypeptide composition of the PRC2, specifically the presence of Su(z)12, suggests that PRC2 plays a more general role in transcriptional silencing outside of the repression of HOX genes. Su(z)12 is a protein with dual PcG and Su(var) functions, and this, therefore, suggests that PRC2 has functions other than homeotic gene repression and, in fact, may play a more general role in heterochromatin-mediated silencing. The observation that human E(z) can function as an inducer of silencing in yeast and as an enhancer of PEV in Drosophila supports this notion. It is speculated that the requirement for E(z) and ESC during early embryonic development reflects its function in general transcriptional silencing. The multifunctional nature of both the E(z) and Su(z)12 proteins suggests that they may also display biochemical heterogeneity. For example, the heterogeneous elution profile of E(z) on various columns suggests that E(z) exists in several distinct complexes (Kuzmichev, 2002).

Purified PRC2 displayed specificity for K9 and K27 of the histone H3 tail. The complex, under the conditions of the assays used, displayed a strong preference for K27. However, when the H3-tail was used as a GST-fusion protein, PRC2 displayed apparently equal specificity for K9 and K27. Analyses of the amino acid sequence in which these lysines are embedded shows a great deal of conservation. K9 is present within the sequence QTARK9STG, whereas K27 is present within the sequence KAARK27SAP. Therefore, at least two different possibilities can be postulated to account for the specificity observed. In one case, the specificity of PRC2 is relaxed in vitro, under the assay conditions used, and the methylation of K9 is nonspecific because of the sequence similarity of the residues within which K9 resides. An apparently similar situation was observed in studies analyzing the specificity of the histone methyltransferase G9a, which biochemically behaves as a H3-histone methyltransferase that preferentially targets K9 and, to much lower levels, K27. In vivo, however, G9a clearly targets H3-K9: whether or not the extent of H3-K27 methylation is decreased in G9a-null cells is unknown. A second possibility is that E(z) targets both K9 and K27, but that this is a regulated process such that methylation of K9 and/or K27 is modulated by factors that associate with E(z) and/or by other modifications existing in the nucleosome. This second possibility is favored based on the following observations. First, the E(z) protein can be considered to be a PcG as well as a TrxG. Not surprisingly, the analyses demonstrate that E(z) is present in distinct complexes. One of the complexes containing E(z) is PRC2; however; this complex also includes Su(z)12. Su(z)12 is a polypeptide that has been found in genetic analyses to regulate the expression of the HOX genes, but loss of function of Su(z)12 suppresses PEV. Therefore, the presence of Su(z)12 in PRC2 may regulate the methylation sites within the histone H3 tail (Kuzmichev, 2002).

Methylation of histone H3-K9 was shown to be an essential step in the establishment of inactive X chromosome. H3-Lys 9 methylation of the inactive X chromosome is not mediated by Suv39 or by G9a. Studies have demonstrated that the imprinted inactivation of the X chromosome in females is lost in mutant mice lacking eed (the mammalian homolog of ESC). Moreover, studies have also demonstrated that during imprinted X inactivation, the mammalian ESC-E(z) complex is localized to the inactive X chromosome in a mitotically stable manner. It is speculated, in light of the accumulated data, that H3-K9 methylation of the inactive X chromosome might be mediated by E(z) within PRC2 or a PRC2-like complex. Importantly, however, the function of methylation of histone H3 at K27 has not been analyzed in the establishment and/or maintenance of the inactive X chromosome. In light of the results discussed above, it is postulated that methylation of H3-K27 may also be important in the process of X inactivation (Kuzmichev, 2002).

It is proposed that the role of E(z) HMT activity in the repression of homeotic gene expression is to establish a binding site for other PcG proteins. It is suggested that PRC2 is recruited to the HOX gene cluster by a transiently acting repressor, for example, through an EED-YY1/Pho interaction or an RbAp46/48-HDAC/dMi2/Hb interaction. Once recruited, PRC2 methylates K27 on histone H3, and this mark recruits PC1. The PC1 protein can convert this mark into a permanently repressed state through methylation of K9 through the recruitment of the Su(var)3-9 H3-K9-specific histone methyltransferase and/or the recruitment of PRC1. Alternatively and/or additionally, PC1 may stimulate the H3-K9 HMT activity of PRC2. This hypothesis is supported by studies demonstrating that trimethylation of K27 is necessary for binding of PC1 to an H3 tail peptide. These findings are in full agreement with studies demonstrating loss of chromosome binding for several PRC1 components upon inactivation of E(z). Interestingly, immunolocalization experiments using antibodies specific for methylated H3-K9 suggest that almost all of the H3-K9 methylation is concentrated in the chromocenter of Drosophila polytene chromosomes, with almost no staining detectable on the chromosomal arms. In contrast, E(z) and other PcG proteins, with subnuclear localization that is regulated by E(z), bind only to discrete bands along the arms of polytene chromosomes. These observations suggest that methylation at K27, rather than methylation at K9, is more likely to establish a binding site for the PC1 protein. This may explain why methylation of K9 alone was not sufficient to allow PC1 to recognize specifically the H3 tail in vitro. The observed PC1 binding was independent of DNA. However, repression of HOX genes in vivo is dependent on PRE. From these results, it must be concluded that although methylation of the H3 tail is important in creating a recognition site for PC1 binding, stable and specific binding must require additional factors and or modifications. A likely candidate is a nucleosome on the PRE with the histone H3-tail methylated at position 27 (Kuzmichev, 2002).

The presence of the RbAp46 and RbAp48 proteins in the ESC-E(z) complex may be important for several reasons. First, these histone-binding proteins are often found in complexes with enzymes involved in the covalent modification of histones. For example, RbAp46 is essential for substrate recognition by, and enzymatic activity of, the histone acetyltransferase enzyme Hat1. Therefore, it is speculated that the inability to detect HMT activity in preparations of recombinant E(z) protein is owing, in part, to the lack of the RbAp46/RbAp48. Another implication of the presence of RbAp proteins in the E(z) complex is that they might facilitate interaction with HDACs. During development, GAP proteins facilitate repression of the HOX genes. GAP proteins, such as Hunchback, are short-lived. Hunchback represses HOX genes by recruiting the Drosophila homolog of human Mi-2 protein, a constituent of the NuRD complex which also contains HDACs 1 and 2 and RbAp46/RpAp48. An interesting possibility is that HDACs or RbAp proteins initially recruited by Hunchback can later recruit PRC2 containing HMT activity via interaction with E(z). This may constitute a switch from short-term to long-term repression (Kuzmichev, 2002).

In support of this hypothesis, PRC2 contains E(z) and RbAp proteins. In addition, there is strong experimental evidence for an interaction between HDACs and E(z). One function of the E(z)-HDAC interaction is to deacetylate histones so that the E(z)-containing complex can methylate them. A similar mechanism was found to operate in yeast, in which methylation of H3-K9 by Clr4 requires deacetylation of H3-K9 and Lys 14 (K14) by Clr6 and Clr3, respectively. A similar mechanism is likely to operate in higher eukaryotes because acetylation and methylation are mutually exclusive marks, and methylation of H3-K9 by Suv39h1 requires deacetylation of this residue. The findings demonstrating two distinct ESC-E(z) complexes, one of which coelutes with HDAC1, raises the possibility that the PRC2 can transiently associate with an HDAC complex. This observation raises the possibility that PRC2 HDAC1 may be a highly-specialized complex dedicated to the methylation of H3-K27, which apparently is not acetylated in vivo in higher eukaryotes. Therefore, it is possible that these two different ESC-E(z) multiprotein complexes establish different marks on the histone H3 tail (Kuzmichev, 2002).

Su(z)12, a novel Drosophila Polycomb group gene that is conserved in vertebrates and plants

In both Drosophila and vertebrates, spatially restricted expression of HOX genes is controlled by the Polycomb group (PcG) repressors. Mutants of a novel Drosophila PcG gene, Suppressor of zeste 12 [Su(z)12], exhibit very strong homeotic transformations. Su(z)12 function is required throughout development to maintain the repressed state of HOX genes. Unlike most other PcG mutations, Su(z)12 mutations are strong suppressors of position-effect variegation (PEV), suggesting that Su(z)12 also functions in heterochromatin-mediated repression. Furthermore, Su(z)12 function is required for germ cell development. The Su(z)12 protein is highly conserved in vertebrates and is related to the Arabidopsis proteins EMF2, FIS2 and VRN2. Notably, EMF2 is a repressor of floral homeotic genes. These results suggest that at least some of the regulatory machinery that controls homeotic gene expression is conserved between animals and plants (Birve, 2001).

Su(z)12 hemizygous mutant embryos derived from Su(z)12 mutant germ cells already show very extensive misexpression of Ubx at the extended germ band stage. These animals showed severe homeotic phenotypes with all abdominal, thoracic and several head segments transformed into copies of the eight abdominal segment. This phenotype is consistent with Abd-B being misexpressed in all segments. The strong PcG phenotype of these Su(z)12 mutant embryos is comparable to that of embryos lacking esc or Pc function. Zygotically provided Su(z)12 function is sufficient to prevent the inappropriate activation of HOX genes; Su(z)12 -/+ heterozygotes obtained as the progeny of Su(z)12 mutant germ cells and a wild-type sperm develop into wild-type-looking adults (Birve, 2001).

The requirement for Su(z)12 at later developmental stages was tested by generating Su(z)12 mutant clones in imaginal discs. Assays were performed for HOX gene silencing in such clones by monitoring the expression of the HOX genes Ubx and Abd-B in the imaginal wing disc (where they are normally stably repressed) using antisera against their protein products. In these experiments, the Su(z)12 mutant cells were identified by the absence of a GFP-expressing marker gene. In addition, the Minute technique was used to generate Su(z)12-/Su(z)12-clones that carry two copies of a wild-type Minute allele [i.e., Su(z)12- M+/ Su(z)12- M+], which gives them a growth advantage relative to their Su(z)12- M+/ Su(z)12+ M- neighbors (Birve, 2001).

Cell clones of the different Su(z)12 alleles were examined 96 hours after clone induction. Su(z)12 mutant clones show strong misexpression of both Ubx and Abd-B in most mutant cells. In summary, the PcG phenotypes observed with several Su(z)12 alleles suggest that Su(z)12 is needed throughout development to keep HOX genes repressed. Moreover, these results support the allele classification obtained by the analysis of germ-line clones; namely, that Su(z)122 and Su(z)125 are hypomorphic alleles whereas Su(z)121 and Su(z)124 appear to be stronger alleles (Birve, 2001).

Database searches show that the Su(z)12 protein is highly conserved in vertebrates and, strikingly, that Su(z)12-related proteins also exist in plants. In contrast, the worm and yeast genomes do not seem to encode Su(z)12-related proteins. The function of the highly conserved human homolog of Su(z)12, HsSU(Z)12, is not known but EMF2, FIS2 and VRN2, the three Su(z)12-related proteins in Arabidopsis, have been identified as regulators in plant development. One characteristic feature of all these proteins is a single classical C2H2 zinc finger similar to the fingers found in sequence-specific DNA-binding proteins. Attempts to show any DNA-binding activity of a polypeptide containing the Su(z)12 zinc finger have failed so far. A second stretch of amino acids that is conserved between Su(z)12, HsSU(Z)12, EMF2, VRN2 and FIS2 is located C-terminal to the zinc finger. This part of the protein has been termed the VEFS box [VRN2-EMF2-FIS2-Su(z)12 box]. The predicted protein products encoded by Su(z)123 and Su(z)124 lack both the zinc finger and the VEFS box, whereas the protein encoded by Su(z)121 is predicted to contain the zinc finger but lacks the VEFS box (Birve, 2001).

Jarid2/Jumonji coordinates control of PRC2 enzymatic activity and target gene occupancy in pluripotent cells

Polycomb Repressive Complex 2 (PRC2) regulates key developmental genes in embryonic stem (ES) cells and during development. Jarid2/Jumonji, a protein enriched in pluripotent cells and a founding member of the Jumonji C (JmjC) domain protein family, is a PRC2 subunit in ES cells. Genome-wide ChIP-seq analyses of Jarid2, Ezh2, and Suz12 binding reveal that Jarid2 and PRC2 occupy the same genomic regions. Jarid2 promotes PRC2 recruitment to the target genes while inhibiting PRC2 histone methyltransferase activity, suggesting that it acts as a 'molecular rheostat' that finely calibrates PRC2 functions at developmental genes. Using Xenopus laevis as a model, Jarid2 knockdown was shown to impair the induction of gastrulation genes in blastula embryos and results in failure of differentiation. These findings illuminate a mechanism of histone methylation regulation in pluripotent cells and during early cell-fate transitions (Peng, 2009).

Jarid2 and Jarid1a regions responsible for Suz12 binding do not overlap with any discernible structural domains and display low similarity, with the exception of a highly homologous short sequence 'GSGFP.' It is hypothesized that this motif may play a role in Suz12 recognition. Indeed, mutations of GSGFP to GAGAA diminished binding of Jarid2 and Jarid1a fragments to full-length Suz12. This motif is conserved in all vertebrate Jarid2 proteins, as well as in C. elegans Jarid2, whereas D. melanogaster and other Drosophila species contain a non-conservative substitution within the motif (GYGFP). The GSGFP motif is also conserved in all four Jarid1 family proteins: Jarid1a/RBP2, Jarid1b/PLU-1, Jarid1c/SMCX and Jarid1d/SMCY, as well as in the single Jarid1 homolog in Drosophila, Lid. The presence of the GSGFP motif in metazoan Jarid proteins suggests that the association with Suz12 may be a common feature of Jarid family members. However, the possibility that additional molecular interactions control Jarid-PRC2 complex formation in vivo cannot be excluded (Peng, 2009).

Polycomb repressive complex 2-dependent and -independent functions of Jarid2 in transcriptional regulation in Drosophila

Jarid2 was recently identified as an important component of the mammalian Polycomb repressive complex 2 (PRC2), where it has a major effect on PRC2 recruitment in mouse embryonic stem cells. Although Jarid2 is conserved in Drosophila, it has not previously been implicated in Polycomb (Pc) regulation. Therefore, Drosophila Jarid2 and its associated proteins were purified, and it was found that Jarid2 associates with all of the known canonical PRC2 components, demonstrating a conserved physical interaction with PRC2 in flies and mammals. Furthermore, in vivo studies with Jarid2 mutants in flies demonstrate that among several histone modifications tested, only methylation of histone 3 at K27 (H3K27), the mark implemented by PRC2, was affected. Genome-wide profiling of Jarid2, Su(z)12 (Suppressor of zeste 12), and H3K27me3 occupancy by chromatin immunoprecipitation with sequencing (ChIP-seq) indicates that Jarid2 and Su(z)12 have very similar distribution patterns on chromatin. However, Jarid2 and Su(z)12 occupancy levels at some genes are significantly different, with Jarid2 being present at relatively low levels at many Pc response elements (PREs) of certain Homeobox (Hox) genes, providing a rationale for why Jarid2 was never identified in Pc screens. Gene expression analyses show that Jarid2 and E(z) (Enhancer of zeste, a canonical PRC2 component) are not only required for transcriptional repression but might also function in active transcription. Identification of Jarid2 as a conserved PRC2 interactor in flies provides an opportunity to begin to probe some of its novel functions in Drosophila development (Herz, 2012).

Different and distinct gene expression patterns are established during development, which need to be maintained and regulated. This is important to allow for the integrity of cell identity and thus the functional preservation of tissues and organs. However, at the same time, transcribed loci must be equipped with an intrinsic flexibility to regulate these expression patterns and initiate changes if necessary. The core components that are required for the maintenance of gene expression or gene repression have been characterized quite extensively to date . Trithorax and Polycomb group genes play antagonistic roles in determining whether a gene is transcriptionally turned on or off, respectively. In Drosophila, so far four distinct complexes, pleiohomeotic repressive complex (PhoRC), Polycomb repressive complex 2 (PRC2), Polycomb repressive complex 1 (PRC1), and recently Polycomb repressive deubiquitinase (PR-DUB) have been described to play a role in Polycomb group-mediated gene repression. However, little is known about the factors involved in controlling recruitment and activity of these complexes on chromatin or about the mechanisms that drive such changes. It should be expected that quite a significant number of proteins would convey Polycomb group-mediated transcriptional changes in order to allow an uncoupling of individual gene activity from that of a group of Polycomb group-controlled genes. Functional redundancy might account for part of the problem to discover such candidates. Furthermore, biochemical approaches might be hindered by the fact that such context-specific and more gene-specific recruiters are contained in only a minor fraction of Polycomb repressive complexes (Herz, 2012).

Recently, Jarid2 the founding member of the JmjC domain-containing protein family, which plays important developmental roles in mice and Drosophila, has been characterized as a component of PRC2 in embryonic stem (ES) cells. The consensus indicates that in ES cells, PRC2 recruitment to many of its targets requires Jarid2. However, levels of bulk histone 3 trimethylated at K27 (H3K27me3) in ES cells depleted of Jarid2 were reported to be only slightly changed at best. This also holds true when individual PRC2 target genes are analyzed. Even though core components of the PRC2 complex were lost from chromatin in the absence of Jarid2, H3K27me3 was not reproducibly affected to a similar degree. Additionally, gene expression analyses in Jarid2-/- ES cells did not confirm a genome-wide derepression of PRC2 target genes as would be expected for any core component of PRC2 (Landeira, 2010; Herz, 2012 and references therein).

To further address whether Jarid2 constitutes a core PRC2 component, is involved in recruitment of PRC2 to chromatin, and regulates H3K27 methylation in Drosophila, a Jarid2 complex was purified from flies and a global in vivo analysis of was performed of Suppressor of zeste 12 [Su(z)12] and H3K27me3 occupancy in Jarid2 mutant animals. The data confirm that Drosophila Jarid2 purifies with the core members of the PRC2 complex. In imaginal discs, global H3K27me3 levels are only weakly but reproducibly affected under Jarid2 mutant and Jarid2-overexpressing conditions. These genome-wide studies suggest that in Drosophila, under physiological conditions, Jarid2 does not appear to be a canonical component of the PRC2 complex as PRC2 recruitment is not altered on most target genes in Jarid2 mutant animals. Interestingly, overexpression of Jarid2 results in reduced Su(z)12 binding and changed chromatin compaction on polytene chromosomes, highlighting a possible role for Jarid2 in altering chromatin architecture. Genome-wide, Jarid2 and Su(z)12 binding correlate very well. However, certain loci, such as Homeobox (Hox) genes, differ significantly from this pattern. Here, Jarid2 occupancy on Polycomb response elements (PREs) is often very low where usually the highest enrichment for Su(z)12 can be observed. Gene expression analyses suggest a PRC2-dependent and -independent role for Jarid2 in transcriptional regulation. Jarid2 appears to be involved in the regulation of a certain number of PRC2 target genes and also transcriptionally controls a subset of genes independently of PRC2. These data not only imply a function for Jarid2 and PRC2 in transcriptional repression but also support a possible role for both Jarid2 and PRC2 in active transcription on genes that are occupied by these factors (Herz, 2012).

This study describes the purification of a Jarid2 complex in Drosophila. Consistent with previous results in mammalian systems, Jarid2 was found to be a component of PRC2. Evidence is provided that in imaginal discs and on polytene chromosomes, Jarid2 is required to fine-tune global H3K27me3 levels. Jarid2 might accomplish this by modulating the activity of the core complex [E(z), Su(z)12, Esc, and Caf1]. The data indicate that Jarid2 could play an inhibitory role in the implementation of H3K27me3 as Jarid2 mutant imaginal disc clones display a global increase and as overexpression of Jarid2 results in a reduction in H3K27me3. Despite having a JmjC domain, Jarid2 has been predicted and reported to be catalytically inactive as a histone demethylase. Therefore, it is unlikely but not impossible that it could function in this manner toward H3K27me3, thereby counteracting PRC2 activity. Even if Jarid2 would be inactive as a histone demethylase, it might still be able to bind to chromatin and prevent spreading of the H3K27me3 mark, such as opposing a possible positive spreading effect of Esc (EED in mammals) (Herz, 2012).

Furthermore, even though Jarid2 could be purified with the PRC2 core members and its occupancy generally correlates very well with canonical PRC2 components such as Su(z)12, it does not appear to play a significant role in regulating PRC2 recruitment in a physiological context, as assessed by Jarid2 mutant animal studies. Apparent differences with published mammalian studies, which imply a major role for Jarid2 in recruitment of PRC2, could be explained by variation in the mechanisms employed or by the fact that the recruitment of PRC2 in ES cells generally differs from that in differentiated tissues. For example, PREs have been known to be highly effective in recruiting PRC2 to target sites in Drosophila. In mammals, attempts have been made to identify functionally analogous sequences but with only limited success. Indeed, it seems more likely that the recruitment of PRC2 in mammals not only requires specific sequences but is also more dependent on additional factors (proteins and RNA), which might explain why PRC2 recruitment is more strongly affected in Jarid2-depleted cells and why PRC1 recruitment in some instances appears to be dependent on PRC2 (H3K27me3). However, the data in Drosophila salivary glands suggest that recruitment of PRC2 (and methylation of H3K27) is not a prerequisite for targeting of PRC1, and the generality of this mechanism is also increasingly questioned in the mammalian system. Nonetheless, when Jarid2 is overexpressed in Drosophila, changes in chromosome compaction can be observed. Under these conditions, Jarid2 extensively occupies the chromosomes, and Su(z)12 localization and H3K27me3 are negatively affected). It is possible that increasing Jarid2 levels beyond a certain physiological level might interfere with PRC2 integrity. Larger amounts of Jarid2 might alter the stoichiometry of the PRC2 subunits, resulting in destabilization of the PRC2 complex on chromatin (Herz, 2012).

Jarid2 also behaves differently from other canonical PRC2 members in Drosophila, as is evident from its binding pattern on certain Hox genes. At Hox genes, occupancy of PRE sites by canonical PRC2 members is one of the highest in the whole genome. In contrast, Jarid2 displays relatively low occupancy on many of these loci, implying a minor or different function for Jarid2 in controlling transcription of these well-described PRC2 targets. It is also possible that at these loci Jarid2 has a more transient association or even that it is less accessible to interact with the antibodies that were have generated. However, these findings are also in agreement with modifier screens that have been performed in Drosophila to identify major regulators of Polycomb group-mediated phenotypes but that were unable to capture Jarid2 (Herz, 2012).

Additionally, the data suggest that Jarid2 appears to control PRC2-dependent transcription, although not necessarily in the same way as expected for canonical PRC2 members. For example, in contrast to the mammalian findings, this study observed that PRC2-mediated transcriptional regulation by Jarid2 in Drosophila is generally independent of changes in Su(z)12 occupancy and does not correlate with changes in H3K27me3 enrichment. However, it needs to be stressed that most Jarid2/PRC2 cobound genes with altered expression patterns in Jarid2 mutants and E(z)-RNAi larvae contain no or low levels of H3K27me3, which is in contrast to the mammalian system where PRC2 components are usually found only at genes with high H3K27me3 enrichment. Nonetheless, in Drosophila, genes with high H3K27me3 enrichment exist that change in transcription in Jarid2 mutants and E(z)-RNAi animals, demonstrating that H3K27me3 is not necessarily instructive for transcriptional repression per se. To date most of the evidence ascribing to H3K27me3 the role of a repressive mark is based on correlation from the observation that PRC2 components colocalize with H3K27me3 and that the respective genes seem to be transcriptionally silenced. The data imply that this might generally be the case but that there are also exceptions to the rule. That certain H3K27me3 patterns can also be connected to transcriptionally active genes in mammals has just recently been reported (Young, 2011; Herz, 2012 and references therein).

Finally, the results imply that Jarid2 and PRC2 are not only involved in maintenance of gene repression but could also function in active transcriptional processes such as transcriptional activation of elongation. This is in agreement with previous reports and demonstrates that PRC2 has cellular functions that extend beyond what was learned from its role at Hox genes. Importantly, the current studies also suggest that despite a very good correlation of Jarid2 and Su(z)12 occupancies, Jarid2 might function in transcriptional repression and activation independently of the canonical PRC2 complex [E(z)] and vice versa. This distinction in target genes between Jarid2 and canonical PRC2 components [E(z)] provides additional confirmation that Jarid2 in some respects behaves fundamentally differently than the canonical PRC2 complex. Together with the varied functions proposed for Jarid2 in mammals, these studies highlight the diverse aspects of Jarid2 function in PRC2-mediated gene regulation (Herz, 2012).

A novel human polycomb binding site acts as a functional polycomb response element in Drosophila

Polycomb group (PcG) proteins are key chromatin regulators implicated in multiple processes including embryonic development, tissue homeostasis, genomic imprinting, X-chromosome inactivation, and germ cell differentiation. The PcG proteins recognize target genomic loci through cis DNA sequences known as Polycomb Response Elements (PREs), which are well characterized in Drosophila. However, mammalian PREs have been elusive until two groups reported putative mammalian PREs recently. Consistent with the existence of mammalian PREs, this study reports the identification and characterization of a potential PRE from human T cells. The putative human PRE has enriched binding of PcG proteins, and such binding is dependent on a key PcG component SUZ12. The putative human PRE carries both genetic and molecular features of Drosophila PRE in transgenic flies, implying that not only the trans PcG proteins but also certain features of the cis PREs are conserved between mammals and Drosophila (Cuddapah, 2012).

A dimeric state for PRC2

Polycomb repressive complex-2 (PRC2) is a histone methyltransferase required for epigenetic silencing during development and cancer. EZH2 is the catalytic subunit of PRC2, and SUZ12 is an essential regulatory subunit. EED is a histone-binding subunit that binds H3K27me3-modified histone tails, resulting in increased affinity to nucleosomes and stimulation of the catalytic activity of PRC2. Long non-coding RNAs (lncRNAs) can recruit PRC2 to chromatin. Previous studies identified PRC2 subunits in a complex with the apparent molecular weight of a dimer, which might be accounted for by the incorporation of additional protein subunits or RNA rather than PRC2 dimerization. This study shows that reconstituted human PRC2 is in fact a dimer, using multiple independent approaches including analytical size exclusion chromatography (SEC), SEC combined with multi-angle light scattering and co-immunoprecipitation of differentially tagged subunits. Even though it contains at least two RNA-binding subunits, each PRC2 dimer binds only one RNA molecule. Yet, multiple PRC2 dimers bind a single RNA molecule cooperatively. These observations suggest a model in which the first RNA binding event promotes the recruitment of multiple PRC2 complexes to chromatin, thereby nucleating repression (Davidovich, 2014).


REFERENCES

Search PubMed for articles about Drosophila Su(z)12

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Cuddapah, S., Roh, T. Y., Cui, K., Jose, C. C., Fuller, M. T., Zhao, K. and Chen, X. (2012). A novel human polycomb binding site acts as a functional polycomb response element in Drosophila. PLoS One 7: e36365. PubMed ID: 22570707

Davidovich, C., Goodrich, K. J., Gooding, A. R. and Cech, T. R. (2014). A dimeric state for PRC2. Nucleic Acids Res 42(14): 9236-48. PubMed ID: 24992961

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date revised: 15 July 2015

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