Enhancer of zeste
E(z) acts to repress knirps.
Anteroposterior polarity of the Drosophila embryo is initiated by the localized activities of maternal genes bicoid and nanos. They establish a gradient of the Hunchback (HB) morphogen. nanos determines the distribution of the maternal HB protein by inhibiting its translation. To identify further components of this pathway, suppressors of nanos mutations have been isolated. In the absence of nanos, high levels of HB protein repress the abdomen-specific genes knirps and giant. In suppressor-of-nanos mutants, knirps and giant are expressed in spite of high HB levels. The suppressors are alleles of Enhancer of zeste . A small region of the knirps
promoter mediates the regulation by E(Z) and HB (Pelegri, 1994).
The requirements for the multi sex combs (mxc) gene during development have been examined to gain further insight into the mechanisms and developmental processes that depend on the important trans-regulators forming the Polycomb group (PcG) in Drosophila. Although mxc has not yet been cloned, it is known to be allelic with the tumor suppressor locus lethal (1) malignant blood neoplasm [l(1)mbn]. The mxc product is dramatically needed in most tissues because its loss leads to cell death after a few divisions. mxc also has a strong maternal effect. Hypomorphic mxc mutations are found to enhance other PcG gene mutant phenotypes and cause ectopic expression of homeotic genes, confirming that PcG products are cooperatively involved in repression of selector genes outside their normal expression domains. The mxc product is needed for imaginal head specification, through regulation of the ANT-C gene Deformed. This analysis reveals that mxc is involved in the maternal control of early zygotic gap gene expression known to involve some other PcG genes and suggests that the mechanism of this early PcG function could be different from the PcG-mediated regulation of homeotic selector genes later in development (Saget, 1998).
Induction of uncontrolled growth and deregulation of Hox genes are linked in mammals, where Hox products can induce leukemia. In Drosophila, modification of homeotic gene expression causes homeosis, sometimes associated with increased proliferation but not with uncontrolled tumorous growth, possibly because
the identity of each segment is specified by a combination of HOM products. Loss or gain of one HOM gene will likely lead to a new combination that is found elsewhere in wild type, and cells expressing this combination could be expected to follow the corresponding developmental pathway and give rise to homeotic transformations. However, because each cellular identity apparently corresponds to a given proliferation rate, loss or ambiguity of identity due to deregulation of several selector genes in a single cell, such as mxc mutations apparently induce, could lead to loss of proliferation control. Identification of mxc partners and targets, as well as of the molecular nature of the mxc product, may help throw light on the genes and mechanisms involved in this process (Saget, 1998).
It has been proposed that certain PcG genes are required for the maintenance of the expression domains of knirps and giant, through a mechanism similar to the regulation of homeotic genes. The regionalization of the Drosophila embryo depends on the maternally supplied products of bicoid (bcd), hunchback (hb), and nanos (nos). Nos represses the translation of the maternal HB mRNA in the posterior embryonic region. This permits the expression of the zygotic gap genes knirps (kni) and giant (gt), which specify posterior identities. These genes would otherwise be repressed by Hb. Embryos from nos/nos mothers form no abdominal segments, but this phenotype can be rescued by a total lack of hb in the maternal germline. It can also be dominantly rescued by the mutation of maternally supplied regulator molecules that normally repress kni and gt in the zygote. Pelegri and Lehmann (1994) have shown that certain mutant products of the PcG genes E(z), Psc, and pleiohomeotic can partially rescue nos by such a maternal effect. To determine if mutation of mxc also affects this regulation, the cuticles of embryos were examined from mxc/+;hb nos/nos mothers that were heterozygous for different mxc mutations. This genetic background was used because a decrease in the amount of maternal hb product can partially rescue the nos phenotype in F1 embryos. Such embryos can differentiate a few abdominal denticle belts and form an adequate background to evaluate increased rescue of nos. Thus loss-of-function PcG mutations should have a strong effect on rescue, and the embryos from hb nos/nos mothers that have two PcG mutations in their genetic background should permit increased rescue of the nos phenotype (Saget, 1998).
Any of three E(z)son (suppressor of nanos) alleles or a hypomorphic pleiohomeotic allele partially rescue the phenotypes of hb nos/nos progeny by a maternal effect; deficiencies covering E(z) or the Psc/Su(z)2 complex also allow some maternal rescue of hb nos/nos progeny, yet the strongest effect is observed with the gain-of-function E(z)son alleles. The EMS-induced allele mxcG48 rescues the hb nos/nos progeny phenotype, whereas a deficiency of mxc does not. Some rescue with the Psc/Su(z)2 complex deletion Df(2)vgB is also observed and strong rescue (consistently >50%) is observed with an EMS-induced pleiohomeotic allele phob, described as amorphic. This suggests that phob and mxcG48 are probably not amorphic alleles, and that maternal rescue of hb nos/nos progeny by a PcG gene is most efficient with a non-null mutation (Saget, 1998).
Segmentation of embryos from transheterozygous mothers was also examined. Because neither a reduction of wild-type PcG product nor two PcG mutations in trans in the hb nos/nos mothers increases nos rescue, these data strongly suggest that, whatever the mechanism of gap gene regulation by these PcG mutations may be, it does not function like the PcG-mediated maintenance of homeotic gene expression in embryos and in imaginal discs. The strong rescue provided by several non-null EMS-induced mutations, which may produce mutant proteins, leads to a proposal that modified PcG proteins are poisoning a normal process. How this process depends on wild-type regulation by PcG products has yet to be established (Saget, 1998).
Polycomb group (PcG) proteins maintain the transcriptional silence of target genes through many cycles of cell division. This study provides evidence for the sequential binding of PcG proteins at a Polycomb response element (PRE) in proliferating cells in which the sequence-specific DNA binding Pho and Phol proteins directly recruit E(z)-containing complexes, which in turn methylate histone H3 at lysine 27 (H3mK27). This provides a tag that facilitates binding by a Pc-containing complex. In wing imaginal discs, these PcG proteins also are present at discrete locations at or downstream of the promoter of a silenced target gene, Ubx. E(z)-dependent H3mK27 is also present near the Ubx promoter and is needed for Pc binding. The location of E(z)- and Pc-containing complexes downstream of the Ubx transcription start site suggests that they may inhibit transcription by interfering with assembly of the preinitiation complex or by blocking transcription initiation or elongation (Wang, 2004; full text of article).
Polycomb group (PcG) complexes are multiprotein assemblages that bind to chromatin and establish chromatin states leading to epigenetic silencing PcG proteins regulate homeotic genes in flies and vertebrates, but little is known about other PcG targets and the role of the PcG in development, differentiation and disease. This study determined the distribution of the PcG proteins PC, E(Z) and PSC and of trimethylation of histone H3 Lys27 (me3K27) in the Drosophila genome
using chromatin immunoprecipitation (ChIP) coupled with analysis of immunoprecipitated DNA with a high-density genomic tiling microarray. At more than 200 PcG target genes, binding sites for the three PcG proteins colocalize to presumptive Polycomb response elements (PREs). In contrast, H3 me3K27 forms broad domains including the entire transcription unit and regulatory regions. PcG targets are highly enriched in genes encoding transcription factors, but they also include genes coding for receptors, signaling proteins, morphogens and regulators representing all major developmental pathways (Schwartz, 2006).
The components of PcG complexes are products of PcG genes, first discovered as crucial regulators of homeotic genes in Drosophila. Immunostaining of Drosophila polytene chromosomes, however, showed PcG proteins at about 100 cytological loci, implying a much larger number of target genes. Functional analysis has identified PREs as DNA sequences able to recruit PcG proteins and establish PcG silencing of neighboring genes. Two types of PcG complexes bind to PREs. PRC1-type complexes include a core quartet of proteins: PC, PSC, PH and dRing. PRC2-type complexes include E(Z), which methylates histone H3 Lys27. Mono- and dimethylated Lys27 is widely distributed in the genome, but PcG sites characteristically contain trimethylated Lys27 (me3K27). The activity of the E(Z) complex is essential for stable silencing, and it has been proposed that H3 me3K27 recruits the PRC1 complex through the specific affinity of the PC chromodomain for me3K27. But the relationships between PRC1 and PRC2 complexes, between their binding sites and histone methylation, and between binding, methylation and gene expression are not well understood and remain the subject of debate. The genomic distribution of three PcG proteins [PC, PSC and E(Z)] and of histone H3 me3K27 was examined using using chromatin immunoprecipitation (ChIP). Since PcG target genes may be repressed in some tissues and active in others, a cultured cell line was used to minimize heterogeneity (Schwartz, 2006).
Viewed at the scale of a chromosome arm, the distributions of PC, PSC, E(Z) and me3K27 coincide at a number of distinct binding peaks (which are referred to as 'PcG sites') that correspond to 70% of the bands reported in salivary gland polytene chromosomes stained with the corresponding antibodies. To minimize false positives, the analysis focussed on the PcG sites that showed simultaneous binding of two or more proteins, each above twofold enrichment. Of the 149 PcG sites detected (see the supplemental figure), 95 showed strong binding of all four proteins ('strong' PcG sites), whereas in 54 sites the binding was lower and below threshold for one of the proteins ('weak' PcG sites). At higher resolution, most PcG sites involve two or more genes, often sharing structural or functional similarities. Thus, PcG sites involve the following: engrailed (en) and invected (inv); the PcG genes ph-p and ph-d; the Dorsocross T-box gene cluster; the muscle NK homeobox gene cluster; the wingless cluster; and the two homeotic complexes ANT-C and BX-C (Schwartz, 2006).
The Bithorax complex (BX-C) is a cluster of three homeotic genes (Ubx, abd-A and Abd-B) responsible for segmental identity in the abdomen and posterior thorax. The most prominent features are two sharp binding peaks for all three PcG proteins at the sites of the bx and bxd PREs that control Ubx. No peak was detected over the Ubx proximal promoter, although the entire gene shows a low but significant level of PC. A series of lower peaks emerged in the abd-A region and part of the Abd-B gene. Some of these correspond to the known PREs iab-2. In contrast, the distribution of H3 me3K27 oscillated rapidly above a high plateau that covers Ubx and abd-A but not Abd-B. RT-PCR was used to determine the mRNA levels corresponding to these three genes. Transcription of Ubx and abd-A in these cells was very low but distinctly above background. Abd-B was highly transcribed, at levels 300 times higher than Ubx. This pattern of activity was reflected by the distribution of both PcG proteins and me3K27. It is noted that in the Abd-B regulatory region, the previously characterized Fab-7 and Fab-8 PREs neither bound PcG proteins nor were methylated in these cells. The Abd-B gene has five distinct promoters. A sharp resurgence of both methylation and PcG protein binding in the region of the most upstream Abd-B promoter suggests that, in contrast to the other four promoters, this one might be repressed in the cultured cells. RT-PCR analysis using primers specific for mRNAs initiating from each promoter confirmed that the most upstream promoter is silent and that the other four are active. These results support the view that binding of PcG proteins to PREs is associated with transcriptional quiescence, whereas robust transcriptional activity is accompanied by lack of binding to the PREs and lack of Lys27 methylation over the transcription unit (Schwartz, 2006).
Strong genomic sites bind all three PcG proteins. The PSC and E(Z) peaks generally rise sharply and are contained within less than 2 kb, whereas PC frequently forms a broader peak that may include shoulders or subsidiary peaks absent for E(Z) and PSC and subsides to background more gradually. These peak binding regions are thought of as corresponding to PREs, which they in fact do in the cases where these are known. Additional binding peaks may be found within or downstream of the transcription unit. In contrast, distribution of H3 me3K27 at each site is very broad, forming a domain of tens or even hundreds of kilobases encompassing the transcription unit and regulatory regions of one or more genes but, rather than a level plateau, it consists of a series of deep oscillations (Schwartz, 2006).
The strong binding peaks or putative PREs are often associated with low values or troughs in the methylation profile and at secondary peaks the PC distribution frequently echoes methylation peaks. Overall, their relationship does not support the idea that methylation of Lys27 suffices to recruit binding of PC. It is proposed instead that PC bound to the strong binding peaks, the presumptive PREs, is recruited by proteins that bind specifically to those sequences. The weaker PC binding peaks and tails that mirror the methylation profile near PREs may represent a second mode of PC binding mediated by the interaction of the chromodomain with H3 me3K27 (Schwartz, 2006).
It is supposed that methylation domains initiated by a PRE might spread bidirectionally until they encounter 'active' chromatin, characterized by histone acetylation or methylation of H3 Lys4, marks typical of transcriptionally active genes. Alternatively, specific features might shape the methylation domain either positively, by attracting the methyltransferase complex, or negatively, by blocking productive interactions with the PRE. As in the case of the Abd-B gene or of CG7922 and CG7956 genes, sudden drops in levels of me3K27 are generally associated with transcriptional activity. Are insulators involved in protecting CG7922 and CG7956 from silencing, or is the activity of these two genes simply epigenetically maintained from the time the cell line was originally established? Further work is required to answer this question (Schwartz, 2006).
In many cases, the presumptive PRE lies between divergently transcribed genes such as dco and Sox100B. Which of the two is the PRE target? As PREs can act at distances of 20-30 kb, the proximity of PcG peaks to a promoter is not a reliable guide. It is proposed that the methylation domain is the clue to the target of PcG regulation. A PcG peak is not considered to regulate a promoter if the gene is not included in the methylation domain. When multiple genes are included in the methylation domain, it is likely that they are all affected by PcG regulation. However, this study distinguishes between genes that contain methylation as well as one or more PcG proteins and genes that contain only methylation (Schwartz, 2006).
The 95 'strong' binding sites in the genome encompass a total of 392 genes. Of these 392 genes, 186 contain both PcG binding and methylation, and the remainder are found within broad methylation domains associated with PcG proteins binding but do not bind PcG proteins over their own promoter or transcription unit. They may represent genes not directly targeted but affected by the spread of methylation. An analysis of their ontology indicates that these two classes are in fact very different. Transcription regulators constitute 64.5% of the first set, compared to 4.3% for the full annotation set. Instead they constitute only 4.0% of those genes that contain only me3K27. These comparisons strongly suggest that (1) genes that regulate transcription are preferred PcG targets, and (2) genes that only include the tails of a methylation domain are probably not primary targets of PcG regulation. A similar preference is also seen among the 'weak' binding sites. These include a total of 74 genes containing both PcG proteins and methylation, 28.4% of which encode transcription regulators. Flanking genes containing only methylation include only 5.7% transcription regulators. Although transcription regulators are preferred PcG targets, secreted proteins, growth factors or their receptors, and signaling proteins are also targeted. PcG target genes include components of all the major differentiation and morphogenetic pathways in Drosophila (Schwartz, 2006).
The major features of PcG binding shown by this work are that, although the proteins themselves are highly localized at presumptive PREs, the domain of histone methylation they produce is much broader. If the E(Z) methyltransferase is localized at the PRE, how is the extensive methylation domain produced? A looping mechanism is proposed in which interaction of PRE-bound complexes with flanking chromatin is mediated by the PC chromodomain. The observed broader distribution of PC might result from crosslinking of the chromodomain to methylated H3, reflecting this mechanism (Schwartz, 2006).
Are PREs defined by characteristic sequence motifs? Although the analysis of the sequences underlying the binding peaks will be presented elsewhere, it is noted that Ringrose (2003) devised an algorithm based on GAGA factor, PHO and Zeste binding motifs to identify sequences likely to represent PREs. This algorithm correctly predicts a number of the strong PcG binding sites (27%) and a few of the weaker sites (7%), overall 20%; however, it does not predict the majority of the PcG sites. The reverse is also true: only 22% of the PREs predicted by Ringrose bind PcG proteins in these experiments. Together, these data suggest that additional criteria are necessary to predict most PREs reliably (Schwartz, 2006).
As expected, PcG proteins and me3K27 are associated with transcriptional quiescence, but the data suggest that this is not an absolute condition. Low but significant transcription levels are detected even for the repressed Ubx and abd-A genes. Two target sites, polyhomeotic and the Psc-Su(z)2 site, contain PcG genes, which must be active to ensure the functioning of the PcG mechanism. The polyhomeotic locus is one of two sites in the entire genome that bind PC but lack appreciable levels of E(Z) and of Lys27 methylation. Instead, the Psc-Su(z)2 region is well methylated and binds both PC and E(Z) at multiple peaks. It is concluded that PcG mechanisms do not invariably lead to transcriptional silencing and are compatible with moderate levels of transcription (Schwartz, 2006).
Another point of interest is the number and kind of genes that are PcG targets. Considering the developmental difference between salivary gland cells and the embryo-derived tissue culture cells, the substantial number of shared PcG sites suggests that a majority of target sites are occupied in a large percent of cells. Target genes are in fact predominantly regulatory genes that control major differentiation and morphogenetic pathways. These pathways and their genes are highly conserved, and recent work shows that they are also regulated by PcG in mammals. It might be expected that in a given cell type most alternative genomic programs would be repressed save the subset required in that cell type. The emerging picture from these studies is that PcG regulation is a key mechanism in genomic programming (Schwartz, 2006).
The ability of a chimeric HP1-Polycomb (PC) protein to bind both to heterochromatin and to euchromatic sites of PC protein binding was exploited to detect stable protein-protein interactions in vivo. Endogenous PC protein
is recruited to ectopic heterochromatic binding sites by the chimeric protein. Posterior sex combs (PSC) protein also is recruited to heterochromatin by the chimeric protein, demonstrating that PSC protein participates in direct protein-protein interaction with PC protein or PC-associated proteins. In flies carrying temperature-sensitive alleles of Enhancer of zeste[E(z)] the general decondensation of polytene chromosomes that occurs at the restrictive temperature is associated with loss of binding of endogenous PC and chimeric HP1-Polycomb protein to euchromatin, but binding of HP1 and chimeric HP1-Polycomb protein to the heterochromatin is maintained. The E(z) mutation also results in the loss of chimera-dependent binding to heterochromatin by endogenous PC and PSC proteins at the restrictive temperature, suggesting that interaction of these proteins is mediated by E(Z) protein. A myc-tagged full-length Suppressor 2 of zeste [SU(Z)2] protein interacts poorly or not at all with ectopic Pc-G complexes, but a truncated SU(Z)2 protein is strongly recruited to all sites of chimeric protein binding. Trithorax protein is not recruited to the heterochromatin by the chimeric HP1-Polycomb protein, suggesting either that this protein does not interact directly with Pc-G complexes or that such interactions are regulated. Ectopic binding of chimeric chromosomal proteins provides a useful tool for distinguishing specific protein-protein interactions from specific protein-DNA interactions important for complex assembly in vivo (Platero, 1996).
The in vivo distribution of the E(Z) protein shows it to be ubiquitously present in embryonic and larval nuclei. In salivary gland polytenized nuclei, the identifiable E(Z) chromosome binding sites are a subset of those described for other Polycomb-group proteins, suggesting that E(Z) may also participate in Polycomb-group complexes. E(Z) binds to chromosomes in a DNA sequence-dependent manner, as illustrated by the creation of a new E(Z)-binding site at the location of a P element reporter construct that contains a Polycomb response element (PRE). This P element contains a 14.5 kb segment from the bxd/pbx Ubx regulatory region. The sequences of one null and three temperature-sensitive E(z) alleles are presented. These mutations diminish all chromosome binding of E(Z) protein. It is suggested that a Cys-rich region altered in these mutations functions as a DNA binding domain. E(Z) binding is noticibly weaker in trx mutants. A reduced level of E(Z) chromosome binding may be due to alteration in the expression of one or more other proteins that are involved in E(Z) binding and does not necessarily imply a direct interaction between E(Z) and TRX (Carrington, 1997).
The Extra sex combs (Esc) and Enhancer of zeste [E(z)] proteins, members of the Polycomb group (PcG) of transcriptional
repressors, interact directly and are coassociated in fly embryos. These two proteins are components of a 600-kDa
complex in embryos. Using gel filtration and affinity chromatography, it has been shown that this complex is biochemically distinct from previously
described complexes containing the PcG proteins Polyhomeotic, Polycomb, and Sex comb on midleg. In addition, evidence is presented
that Esc is phosphorylated in vivo and that this modified Esc is preferentially associated in the complex with E(z). Modified Esc
accumulates between 2 and 6 h of embryogenesis, which is the developmental time when esc function is first required. Mutations in E(z) reduce the ratio
of modified to unmodified Esc in vivo. Germ line transformants were generated that express Esc proteins bearing site-directed mutations that disrupt
Esc-E(z) binding in vitro. These mutant Esc proteins fail to provide esc function, show reduced levels of modification in vivo, and are still assembled into
complexes. Taken together, these results suggest that Esc phosphorylation normally occurs after assembly into Esc-E(z) complexes and that Esc contributes to the
function or regulation of these complexes (Ng, 2000).
ESC mRNA is expressed primarily during early development, with the highest levels
being found before 4 h of embryogenesis. This early expression has prompted the hypothesis that esc functions in the transition
between initiation of homeotic gene repression by gap proteins, such as hunchback, and maintenance of this repression by PcG proteins. This transition occurs at about 4 h, when gap gene products decay. Esc protein is
expressed at peak levels at 6 to 12 h, after ESC mRNA has decayed to low levels. In addition, Esc is detected until the end of
embryogenesis. The presence of substantial levels of Esc in mid- to late-stage embryos suggests that Esc may play a greater role than simply in the transition
between gap protein and PcG protein repression. In addition, a second peak of Esc protein is detected during larval and pupal stages, consistent with its
nonessential function in imaginal discs (Ng, 2000).
The predicted Esc structure identifies two surface-accessible regions likely to contain phosphorylation sites: the highly charged N terminus and the surface
loops of the ß-propeller. The Esc phosphorylation sites have not been mapped.
However, it is predicted that Esc is serine/threonine phosphorylated, because many of the Ser and Thr residues are surface accessible. In particular, the N-terminal tail
is very rich in Ser and Thr residues (35%), a feature which has been conserved in Esc during evolution. A scan of the accessible Esc regions for
consensus kinase recognition motifs identifies numerous possible modification sites and is therefore not particularly instructive (Ng, 2000).
Gel filtration experiments also show that modified Esc is found preferentially in the Esc-E(Z) complex while unmodified Esc behaves predominantly as
unassociated monomer. Interestingly, mutant Esc proteins with reduced levels of modification also associate in complexes with the same apparent molecular mass as
the wild-type complex. This suggests that Esc modification is not required for its stable association in complexes. Consistent with this idea,
low levels of unmodified wild-type Esc are reproducibly detected in the 600-kDa complex. Based on these data, a model is favored in which Esc modification contributes to function
rather than to assembly of the complex. The finding that E(z) function is required for wild-type levels of Esc modification further suggests that this
modification occurs after Esc has complexed with its partners (Ng, 2000).
The mutant Esc proteins described in this study show reduced Esc-E(z) binding in vitro. Therefore, it was surprising to find that these mutants assemble into
complexes of apparently wild-type size. It is suggested that Esc may bind to multiple protein partners in the Esc-E(z) complex, such that specific disruption of
Esc-E(z) interaction still allows complex assembly. In support of this idea, ß-propeller proteins have been shown to make simultaneous contacts with
multiple partners (Ng, 2000).
The PcG proteins Pc and Ph are associated in a complex estimated to be 2 MDa. In addition, Ph and Ph
coimmunoprecipitate and interact with another PcG protein, Psc. The Esc-E(Z) complex is biochemically distinct from
complexes containing Ph. In agreement with this, a Ph-Pc-Psc complex does not contain E(z). Taken
together, these results support a model in which there are at least two distinct PcG complexes in vivo, one containing Esc and E(z) and the other containing
Ph, Pc, and Psc. Consistent with this idea, the mammalian Esc and E(z) homologs, EED and EZH2, fail to coimmunoprecipitate with the mammalian Ph, Psc,
and Pc homologs. In addition, EED and EZH2 do not colocalize with mammalian PH, PSC, and PC within nuclei of osteosarcoma cells.
Furthermore, the patterns of pairwise interactions among Drosophila PcG proteins are reiterated among their mammalian counterparts, which suggests that this division of labor in the PcG has been conserved in evolution (Ng, 2000 and references therein).
Although the existence of at least two different PcG complexes has been established, the complete spectrum of PcG protein interactions has not yet been elucidated.
There appears to be further division among Ph-Pc-Psc complexes, which have different compositions at different target genes. In addition, multiple complexes
containing the mammalian Ph, Pc, and Psc proteins have been detected. Moreover, there are additional PcG proteins, such as Asx, Pcl, and Pho, whose
in vivo associations have yet to be described. Some of these proteins may correspond to as yet unidentified components of Esc-E(z) or Ph-Pc-Psc complexes, or they may sort into additional distinct complexes. In particular, complexes containing Pho, the only known DNA-binding member of the PcG, may be
important for targeting other PcG complexes to sites of action. It is noted that Pho is not detected as a stable member of either the Esc-E(z) or
Ph-Pc-Psc complexes (Ng, 2000).
Despite the presence of biochemically separable PcG complexes, the similar mutant phenotypes and genetic interactions of PcG genes indicate that they work
together at some level. Any model for PcG repression must therefore accommodate both the biochemical separability and functional synergy of PcG complexes. One
possibility is that repression requires multiple chromatin-modifying events by the different PcG complexes. This would be similar to the in vivo synergy between the
chromatin-modifying SWI-SNF and SAGA complexes, which are both required for maintenance of HO expression in yeast. An alternative possibility is that
one PcG complex directly modifies chromatin while the other complex counteracts trithorax group activation by inhibiting the chromatin-remodeling activity of the
brahma complex. Indeed, the first evidence that a PcG complex may covalently modify chromatin is provided by the recent report of histone deacetylase
activity associated with mammalian homologs of Esc and E(z) (Ng, 2000 and references therein).
These mechanisms are inconsistent with an esc role limited to the transition from gap repressors to PcG repressors. Instead, it is suggested that Esc is
more globally involved in chromatin regulation and that this involvement is most critical early in fly development. Consistent with a global role, EED mRNA is
expressed in many tissues during mouse development. Furthermore, the C. elegans homolog of Esc, MES-6, is a transcriptional repressor that functions in
germ line development. MES-6 in worms therefore plays a distinct developmental role from Esc in flies. This suggests that Esc participates in a general
repression mechanism that has been adapted for use in different cell lineages, rather than in the specific transition between gap protein and PcG protein repression (Ng, 2000 and references therein).
If Esc-E(z) complexes function as general chromatin regulators, the early requirement for Esc in Drosophila must be reconciled with the need for long-term PcG
repression during development. One possibility is that another protein replaces Esc in the Esc-E(z) complex at late developmental stages, when Esc is no longer
critically required. Alternatively, E(z) may associate with a completely different set of PcG proteins to supply the biochemical function provided by Esc-E(z)
complexes during embryogenesis. To address these possibilities, the nature of E(z) complexes at postembryonic stages will have to be investigated (Ng, 2000).
The products of Polycomb group (PcG) genes are required for the epigenetic repression of a number of important developmental regulatory genes, including homeotic genes. Enhancer of zeste [E(Z)] is a Drosophila PcG protein that binds directly to another PcG protein, Extra Sex Combs (ESC), and is present along with ESC in a 600-kDa complex in Drosophila embryos. Using yeast two-hybrid and in vitro binding assays, it was shown that E(Z) binds directly to another PcG protein, Polycomblike (PCL). PCL.E(Z) interaction is shown to be mediated by the plant homeodomain (PHD) fingers domain of PCL, providing evidence that this motif can act as an independent protein interaction domain. An association was also observed between PHF1 and EZH2, human homologs of PCL and E(Z), respectively, demonstrating the evolutionary conservation of this interaction. E(Z) was found to not interact with the PHD domains of three Drosophila trithorax group (trxG) proteins, which function to maintain the transcriptional activity of homeotic genes, providing evidence for the specificity of the interaction of E(Z) with the PCL PHD domain. Coimmunoprecipitation and gel filtration experiments demonstrate in vivo association of PCL with E(Z) and ESC in Drosophila embryos. The implications of PCL association with ESC.E(Z) complexes is discussed and the possibility that PCL may either be a subunit of a subset of ESC.E(Z) complexes or a subunit of a separate complex that interacts with ESC.E(Z) complexes (O'Connell, 2001; full text of article).
The Drosophila Polycomb Group (PcG) proteins are
required for stable long term transcriptional silencing of
the homeotic genes. Among PcG genes, esc is unique
in being critically required for establishment of PcG-mediated
silencing during early embryogenesis, but not for
its subsequent maintenance throughout development. Esc has been shown to be physically associated
with the PcG protein E(Z). Esc,
together with E(z), is present in a 600 kDa complex that is
distinct from complexes containing other PcG proteins. This Esc complex has been purified and it also
contains the histone deacetylase Rpd3 and the histone-binding
protein p55 (Chromatin assembly factor 1 subunit), which is also a component of the
chromatin remodeling complex NURF and the chromatin
assembly complex CAF-1. The association of Esc and
E(z) with p55 and Rpd3 is conserved in mammals. Rpd3 is required for silencing mediated by a
Polycomb response element (PRE) in vivo and E(z)
and Rpd3 are bound to the Ubx PRE in vivo, suggesting
that they act directly at the PRE. It is proposed that histone
deacetylation by this complex is a prerequisite for
establishment of stable long-term silencing by other
continuously required PcG complexes (Tie, 2001).
To test whether the association of Esc and E(z) with p55/Caf1 and
Rpd3 has been conserved in mammals, the
human complex containing the Esc homolog (EED) was examined for the
presence of Rpd3 and p55 homologs. Database searches
reveal that Drosophila Rpd3 is most closely related to
two human histone deacetylases, HDAC1 and HDAC2
(77% and 75% identical to Rpd3). Similarly, there are
two closely related p55 homologs in mammals,
RbAp48 and RbAp46 (91% and 86% identical to p55: Drosophila homolog Caf1).
RbAp48 and RbAp46 have also been found together in
the SIN3 and Mi-2 deacetylase complexes, as have
HDAC1 and HDAC2. A test was performed to see
whether all four proteins are associated with the
human EED complex. A GST-ESC fusion
protein encoding full-length Esc can pull down full-length
in vitro translated Esc and a GST-ESC1-60 fusion
protein encoding just the N-terminal 60 residues of Esc
is sufficient to pull down full-length in vitro translated
Esc. Similarly, GST-EED1-81, which
contains the corresponding N-terminal region of EED, binds directly to in vitro
translated EED. In addition to FLAG-Esc, GST-ESC1-60 also
pulls down p55 and Rpd3 from Drosophila embryo
nuclear extract. This strongly suggests that GST-ESC1-
60 specifically pulls down the Esc complex. GST-EED1-81 pulls down HDAC1, HDAC2 and RbAp48
from HeLa cell nuclear extract.
RbAp46 has also been detected. Thus, the association of ESC with
p55 and Rpd3 is mirrored in the conserved association of
mammalian EED with RbAp48, RbAp46 and HDAC1 and
HDAC2. These results confirm the previously reported
association of EED with HDAC1 and HDAC2 (Tie, 2001).
The presence of p55 in the ESC complex provides a direct
molecular link to chromatin. The highly conserved mammalian
p55 homologs, RbAp48 and RbAp46, have been shown to bind
directly to histone H4 and possibly H2A, but not H2B or H3. The N- and C-terminal regions of RbAp48 that mediate
binding to histone H4 are virtually identical to the
corresponding regions of Drosophila p55,
strongly suggesting that p55 has the same histone-binding
specificity (Tie, 2001).
What, then, is the role of p55 in the Esc complex? It is
unlikely that p55 is responsible for the selective recruitment or
targeting of Esc and E(Z) to the ~100 specific chromosomal sites
at which they co-localize. The histone-binding
activity of p55 does not, by itself, suggest a mechanism for such
specificity and p55 binds to many more sites on the polytene
chromosomes than Esc and E(Z),
presumably reflecting its distribution in other complexes, such as
CAF1 and NURF. It seems more likely that p55 acts after the
Esc complex is recruited and serves to direct the deacetylase
activity of Rpd3 to local histone substrates. This is analogous to
the role proposed for RbAp46 in the heterodimeric HAT1
complex. RbAp46 greatly stimulates the acetyltransferase
activity of the non-histone-binding HAT1 catalytic subunit,
presumably by tethering it to its substrate via its histone-binding
activity. Similarly,
although recombinant Rpd3 can deacetylate histone H4 in vitro,
free Rpd3 does not bind to H4 when the two are co-expressed
in vivo and is unlikely to be able to
deacetylate nucleosomal histones. This suggests that p55 may
play a similar essential role in the Esc complex by targeting
Rpd3 to histone substrates for deacetylation (Tie, 2001).
The presence of Rpd3 in the Esc complex suggests that
histone deacetylation is an intrinsic activity of the Esc
complex and that Rpd3 is required for PRE-mediated
silencing. The related mammalian EED complex has been
shown to contain the Rpd3 homologs HDAC1 and HDAC2,
and immunoprecipitates containing this complex can
deacetylate a histone H4 tail-peptide in vitro. In yeast, Rpd3-dependent repression in vivo has
been shown to be associated with deacetylation of histones H4
and H3.
Which nucleosomes would be deacetylated by the Esc
complex? Histone deacetylation by yeast Rpd3 appears to be
highly localized, extending only one or two nucleosomes from
a site to which it is recruited. Since components of the Esc complex
are physically associated with the Ubx PRE in vivo, Esc-mediated
deacetylation may be restricted to nucleosomes
comprising and immediately adjacent to PREs. Nucleosomes
outside the PRE might also be targeted if the PRE has long-range
interactions with the promoter or if the Esc complex
itself also binds to the promoter or other regions outside the Ubx
PRE, a possibility that the data presented here do not rule out.
Although an effect is observed of several Rpd3 mutations on
silencing of a PRE-mini-white reporter, which is an extremely
sensitive assay, PcG phenotypes have not been reported for
Rpd3 mutants. A hypomorphic Rpd3 allele associated with the
insertion of a P-element transposon in the noncoding 5'
untranslated region has been analyzed in the most detail.
Homozygous mutant embryos derived from germline clones of
this allele do not exhibit PcG phenotypes, but have a pair-rule
phenotype similar to that of ftz mutants. Abundant ubiquitously
distributed Rpd3 RNA and protein of maternal origin are
detectable in early (0-2 hour) wild-type embryos, but are
reduced no more than fivefold in these Rpd3 mutant embryos
derived from germline clones. By stage 9-10, the level of
maternally derived Rpd3 RNA and protein is greatly
diminished. Localized zygotic expression of Rpd3 becomes
detectable in the brain and ventral nervous system of wild-type
embryos, but is not detectable in these mutant embryos, suggesting that this
Rpd3 allele may have a stronger effect on zygotic expression
than maternal expression. If Rpd3 protein derived from
maternally synthesized RNA is sufficient to promote
development of a normal cuticular phenotype, then it remains
possible these mutant embryos may contain sufficient
maternally derived protein to do so and that germline clones of
a true null Rpd3 allele would display PcG phenotypes.
Alternatively, it is possible that the function of Rpd3 in the
Esc complex is not absolutely essential for Esc-dependent
silencing or is redundant, i.e. when eliminated, it can be
compensated by another histone deacetylase, either one
normally associated with the Esc complex or a related one that
can associate with the complex in the absence of Rpd3. A
number of other histone deacetylases have been identified in
Drosophila and at least two are reported to be ubiquitously
distributed in the early embryo (Tie, 2001).
However, unlike mammals, which have two very closely
related Rpd3 orthologs (HDAC1 and HDAC2), both of which
are associated with mouse EED, the Drosophila genome
contains no equally closely related homolog of Rpd3. The next
most closely related Drosophila HDAC is an unequivocal
ortholog of mammalian HDAC3, which
is a class I HDAC like Rpd3. Interestingly, mouse HDAC3 has
been reported to interact with the mouse Esc homolog EED
in a yeast two-hybrid assay,
consistent with the possibility that Rpd3 function in the Esc
complex might be at least partially redundant. Further genetic
analysis of Rpd3 should help to clarify its role in the Esc
complex (Tie, 2001).
The 600 kDa Esc complex is distinct from complexes
containing PC and other PcG proteins. This suggests that the
Esc complex and other PcG complexes are likely to have
separate functions. Furthermore, in embryos lacking any
functional Esc protein, some weak residual Pc-dependent
silencing activity is still detected, also
supporting separate, if interdependent, functions. Similar
conclusions have been drawn for the homologous mammalian
PcG complexes, which have been reported to be expressed in temporally distinct stages of B cell
differentiation, further suggesting they have distinct functions. In Drosophila, derepression of
homeotic genes is detected slightly earlier in Esc mutants than
in other PcG mutants, raising the possibility
that Esc complex function might be required earlier than other
PcG complexes. However, unlike the apparent temporal
separation of the homologous complexes during mammalian B
cell development, both Esc- and PC-containing complexes are
present together throughout most of embryogenesis, before
Esc disappears, and E(z), like other PcG proteins, is required
continuously throughout development. The phenotypic
similarities between Esc, E(z) and other PcG mutants, the
genetic interactions among them and their common association
with PREs, suggests that their functions, however distinct at
the biochemical level, are interdependent (Tie, 2001).
What role might Esc-mediated histone deacetylation play
in PcG silencing? Given the critical early requirement for Esc,
Esc-mediated deacetylation of PRE-associated nucleosomes
might be an essential prerequisite for the initial binding of one
or more components of PRC1 or other PcG complexes to
PREs. A schematic model is presented for such a function
of the Esc complex in which Esc complex-mediated
deacetylation of PRE associated histones is a critical step in
establishing stable long-term PcG silencing. Alternatively, the
Esc complex may be required for events subsequent to the
initial binding of other PcG proteins to a PRE, perhaps for their
assembly into active silencing complexes or for interaction of
PRE-bound PcG complexes with the promoter. Indeed,
repression of a reporter gene by a tethered GAL4-Pc fusion
protein remains dependent on endogenous Esc and E(z) as
well as other PcG proteins. This indicates that,
at least for PC, constitutive binding to DNA does not bypass
the requirement for Esc and E(z). This also suggests that
while the biochemical evidence reveals no stable direct association of the
Esc complex with other PcG complexes, it remains possible
that there is a transient or less stable association in vivo that is
essential for establishing PcG silencing (Tie, 2001).
The association of mammalian EED with the two closely
related HDACs and two histone-binding proteins could reflect
the existence of two separate EED complexes or some different
functionality of the EED complex compared with the Esc
complex. Consistent with this latter possibility, EED has
recently been shown to be required after embryogenesis for
aspects of adult hematopoietic development. Interestingly, analysis of the
complete Drosophila genome sequence
using the BLASTP and TBLASTN algorithms reveals that p55 has no other
closely related Drosophila homologs, strongly suggesting that
it is the functional counterpart of both RbAp48 and RbAp46
in Drosophila. Likewise, Rpd3 is the only Drosophila
counterpart of mammalian HDAC1 and HDAC2. Given the
remarkably high degree of similarity between RbAp48 and
RbAp46 and HDAC1 and HDAC2, it is not yet clear whether
each of these proteins has a distinct or redundant role in the
EED complex. Perhaps this situation reflects a greater degree
of functional specialization or versatility within the
mammalian EED complexes. Since HDAC1 and HDAC2 have
also been found together with RbAp48 and RbAp46 in other
co-repressor complexes, it is also possible that the EED and
Esc complexes represent specialized relatives of these
complexes, perhaps more dedicated to a specific subset of
genes (Tie, 2001).
The Drosophila Enhancer of zeste [E(z)] gene encodes a member of the Polycomb group of transcriptional repressors. This study provides evidence for direct physical interaction between E(Z) and dSAP18, which previously has been shown to interact with Drosophila GAGA factor and Bicoid proteins. dSAP18 shares extensive sequence similarity with a human polypeptide originally identified as a subunit of the SIN3A-HDAC (switch-independent 3-histone deacetylase) co-repressor complex. Yeast two-hybrid and in vitro binding assays demonstrate direct E(Z)-dSAP18 interaction and show that dSAP18 is capable of interacting with itself. Co-immunoprecipitation experiments provide evidence for in vivo association of E(Z) and dSAP18. Gel filtration analysis of embryo nuclear extracts shows that dSAP18 is present in native protein complexes ranging from approximately 1100 to approximately 450 kDa in molecular mass. These studies provide support for a model in which dSAP18 contributes to the activities of multiple protein complexes, and potentially may mediate interactions between distinct proteins and/or protein complexes (Wang, 2002).
In Drosophila, PcG complexes provide heritable transcriptional silencing of target genes. Among them, the ESC/E(Z) complex is thought to play a role in the initiation of silencing whereas other complexes such as the PRC1 complex are thought to maintain it. PcG complexes are thought to be recruited to DNA through interaction with DNA binding proteins such as the GAGA factor, but no direct interactions between the constituents of PcG complexes and the GAGA factor have been reported so far. The Drosophila corto gene interacts with E(z) as well as with genes encoding members of maintenance complexes, suggesting that it could play a role in the transition between the initiation and maintenance of PcG silencing. Moreover, corto also interacts genetically with Trl, which encodes the GAGA factor, suggesting that it may serve as a mediator in recruiting PcG complexes. Corto bears a chromo domain, and evidence is provided for in vivo association of Corto with ESC and with PC in embryos. Moreover, GST pull-down and two-hybrid experiments show that that Corto binds to E(Z), ESC, PH, SCM and GAGA and co-localizes with these proteins on a few sites on polytene chromosomes. These results reinforce the idea that Corto plays a role in PcG silencing, perhaps by confering target specificity (Salvaing, 2003).
Polycomb group (PcG) proteins maintain transcriptional repression during development, likely by creating repressive chromatin states. The Extra Sex Combs (Esc) and Enhancer of Zeste [E(z)] proteins are partners in
an essential PcG complex, but its full composition and biochemical activities are not known. A SET domain in E(z) suggests this complex might methylate histones. An Esc-E(z) complex has been purified from Drosophila
embryos and four major subunits were found: Esc, E(z), NURF-55, and the PcG repressor, SU(Z)12. A
recombinant complex reconstituted from these four subunits methylates lysine-27 of histone H3. Mutations in the E(z) SET domain disrupt methyltransferase activity in vitro and HOX gene repression in vivo. These results identify E(Z) as a PcG protein with enzymatic activity and implicate histone methylation in PcG-mediated silencing (Müller, 2002).
Histone H3 is an attractive candidate to be the primary functional target for the Esc-E(z) complex in vivo. Previous work on heterochromatin proteins has established a molecular model that links H3 methylation and gene repression. In this case, SUV39H HMTase methylates H3-K9 and this modification
creates a binding site for the chromodomain of HP1. Similarly, H3 methylation by Esc-E(z) might provide a binding site for another chromodomain protein, such as Polycomb (PC), a component of the PRC1 complex, whose chromodomain is required for chromatin localization in vivo. Targeting of PRC1 via histone methylation performed by Esc-E(z) is consistent with several in vivo results that imply synergy between these complexes. Rather than supplying binding sites for specific proteins, Esc-E(z) mediated methylation
could influence chromatin more generally by altering adjacent nucleosome interactions needed to package the chromatin fiber (Müller, 2002).
Besides histone methylation, genetic and biochemical tests link histone deacetylation to PcG-mediated repression and hyperacetylation to trxG-dependent active states. It will be important to test for regulatory interactions, or crosstalk, among the various tail modifications. For example, H3-K27 acetylation would be anticipated to antagonize H3-K27 methylation, similar to the mutual exclusion of these two modifications on H3-K9. Likewise, interplay between phospho-H3-S28 and methylation of H3-K27 could resemble the inhibitory crosstalk between H3-S10 and H3-K9. This possibility could enable kinases and phosphatases to make regulatory inputs to the PcG/trxG system. It will be important to determine whether additional PcG/trxG proteins possess or
interact with histone-modifying activities, and also to define the histone modification states on PcG-repressed and trxG-activated genes in vivo (Müller, 2002).
The HOX genes of Drosophila provide the best example of heritable silencing by PcG proteins. Recent work has shown that derepression of HOX genes after removal of Pc or Psc in proliferating cells can be reversed if the
depleted protein is resupplied within a few cell generations. These results led to the
proposal that silenced HOX genes bear a heritable molecular mark that targets them for PcG repression and that this mark can be maintained for a few cell generations, even after HOX genes are derepressed. Could the
proposed mark reflect, at least in part, H3-K27 methylation by the Esc-E(z) complex? Histone lysine methylation is a very stable modification, so is well-suited for a long-term molecular mark. E(z) mutants show an unusually long delay before HOX misexpression is detected in imaginal disc clones (72 hr for robust misexpression of UBX) and a long delay is observed in temperature-shift
experiments with an E(z) allele that behaves as a null at restrictive temperature. In addition, of all the PcG mutants tested, only Su(z)12 mutants show a comparably long delay in release from silencing in imaginal disc clones. In contrast, removal of the PRC1 components Psc or Ph triggers rapid loss of repression (Müller, 2002).
Remarkably, Esc, E(z), and SU(z)12 are conserved in many organisms, where they appear to function together as repressors in a wide array of developmental processes. Mammalian complexes that resemble the
Esc-E(z) complex are implicated in multiple processes including early embryonic patterning, HOX gene regulation, and hematopoiesis. Intriguingly, mouse homologs of Esc and E(z) associate with the inactive X chromosome in trophoblast stem cells, suggesting a direct role in X-inactivation. In C. elegans, the Esc- and E(z)-related proteins MES-6 and MES-2 form a stable complex and are required for germline development and gene silencing. The conserved partnership extends to plants, where proteins related to Esc, E(z),
and SU(z)12 are cohort regulators in several Arabidopsis developmental pathways. A striking example is VRN2, a SU(z)12 relative, which is
required for long-term gene silencing in response to vernalization. Further work in these systems should address if histone methylation by Esc-E(z) complexes represent an evolutionary ancient mechanism to mark chromatin for heritable repression during development (Müller, 2002).
Polycomb group (PcG) proteins play important roles in
maintaining the silent state of HOX genes. Recent studies have
implicated histone methylation in long-term gene silencing. However, a
connection between PcG-mediated gene silencing and histone methylation
has not been established. This study reports the purification and
characterization of an EED-EZH2 complex, the human counterpart of the Drosophila ESC-E(Z) complex. The
complex specifically methylates nucleosomal histone H3 at lysine 27 (H3-K27). Using chromatin immunoprecipitation assays, it is shown that
H3-K27 methylation colocalizes with, and is dependent on, E(z)
binding at an Ultrabithorax (Ubx) Polycomb
response element (PRE), and that this methylation correlates with
Ubx repression. Methylation on H3-K27 facilitates binding of
Polycomb (Pc), a component of the PRC1 complex, to histone H3
amino-terminal tail. Thus, these studies establish a link between
histone methylation and PcG-mediated gene silencing (Cao, 2002).
To understand the function of histone methylation, attempts were made to
identify histone methyltransferase (HMTase) using a systematic biochemical approach. Certain fractions derived from HeLa cell nuclear
pellet contained high levels of HMTase activity toward nucleosomal
histone H3. To identify the enzyme(s) present in these fractions, the
proteins were further fractionated in a DEAE5PW column, which separated the HMTase activities into two peaks. The present study focuses on the second peak.
After fractionation on phenyl sepharose and hydroxyapatite columns, the active fractions were further purified
through a gel filtration Superose 6 column. Analysis of the fractions
derived from this column indicates that the HMTase activity elutes
between fraction 47 and 50 with an estimated mass of about 500 kD. Silver staining of an SDS-polyacrylamide gel containing these fractions revealed that six major polypeptides copurify with the enzymatic activity. Because the largest protein band neither
cofractionates with the HMTase activity in the hydroxyapatite column, nor coimmunoprecipitates with the other components, it is concluded that this largest band is not a part of the HMTase protein complex (Cao, 2002).
To identify the proteins that copurify with the HMTase activity, the protein bands were excised and analyzed by a combination of peptide
mass fingerprinting and mass spectrometric sequencing.
In addition to RbAp48, a polypeptide present in many protein complexes
involved in histone metabolism, several human PcG proteins, including
EZH2, SUZ12, and EED, were identified in the HMTase complex. A zinc finger transcriptional repressor named AEBP2 was also
identified. Whether this protein is involved in
targeting the complex remains to be determined. EZH2 contains a SET
domain, a signature motif for all known histone lysine
methyltransferases, except the H3-K79 methyltransferase DOT1, and is therefore likely to be the
catalytic subunit. However, recombinant EZH2 made in Escherichia coli or baculovirus-infected SF9 cells has no detectable HMTase activity, indicating that either a posttranslational modification or
other components in the complex are required for the HMTase activity.
This is consistent with previous results in which a partial EZH2
protein containing the SET domain was used (Cao, 2002).
Although mammalian EZH2 and EED, and their respective homologs in
Drosophila and Caenorhabditis elegans, are known to interact directly, the presence of SUZ12 in such a complex has not been previously reported. To verify that these proteins are components of the same protein complex, antibodies against each of these proteins were generated. Western blot analysis of the column fractions derived from the last two columns indicates that these proteins copurify with the HMTase activity. To further confirm that the copurified proteins exist as a single protein complex, the last column fractions were immunoprecipitated with an antibody to SUZ12. All five proteins coimmunoprecipitate. Because a protein complex containing Drosophila Esc and E(z), respective homologs of EED and EZH2, has been previously named the ESC-E(Z) complex, the human counterpart is referred to as the EED-EZH2 complex. Although both EED-EZH2 and Esc-E(z) complexes physically associate with HDACs, the purified complex neither contains any HDAC polypeptide nor possesses detectable HDAC activity. It is possible that a different protein complex containing EED, EZH2, and HDAC may exist. Alternatively, HDACs may be recruited to target sites through direct interaction with EED, yet may not exist as a stable subunit of EED-EZH2 complexes. Further work is needed to differentiate these possibilities (Cao, 2002).
To characterize the substrate specificity of the EED-EZH2
complex, equivalent amounts of histone H3 that exist alone, in complex with other core histones, and in mono- or
oligo-nucleosome forms were subjected to methylation by equal amounts of the enzyme. The EED-EZH2 complex
is capable of methylating all forms of histone H3, but shows a strong
preference for H3 in oligonucleosome forms (Cao, 2002).
Attempts were made to identify the residue methylated by the EED-EZH2
complex. Because oligonucleosomes are preferred substrates, they were
subjected to methylation by the EED-EZH2 complex in the presence of
S-adenosyl-L-[methyl-3H]methionine
(3H-SAM). After purification, the labeled H3 was subjected
to microsequencing followed by liquid scintillation counting. Neither
K4 nor K9 released numbers of counts clearly greater than background.
However, a small radioactive peak was detected in cycle 27.
Given that the recovery efficiency decreases with each microsequencing cycle, the detection of a small peak on cycle 27 indicates that K27 is
likely to be the site targeted by the EED-EZH2 complex. To confirm this
possibility, each of the five potential methylation sites on
H3 were mutated and the effect of the mutation on the ability of H3 to
serve as a substrate for the enzyme was compared. As a control, the ability of these H3 mutants to be methylated by SUV39H1 was also analyzed. Mutation of
K27 completely abolishes the ability of H3 to serve as a substrate,
whereas mutations of other sites have little effect. As expected, only mutation of K9 affects the SUV39H1-mediated H3 methylation. These data, led to the conclusion that K27 is the predominant site, if not the only site, that is targeted for methylation by the EED-EZH2 complex (Cao, 2002).
To gain insight into the function of H3-K27 methylation in vivo, a polyclonal antibody was generated against a dimethyl-K27 H3 peptide. This
antibody is highly specific for mK27 when evaluated by peptide
competition and enzyme-linked immunosorbent assay. Western
blot analysis with the H3-mK27-specific antibody demonstrates
that H3-K27 methylation occurs in a variety of multicellular organisms,
including human, chicken, and Drosophila . However, it does
not appear to occur in the budding yeast Saccharomyces cerevisiae (Cao, 2002).
Given that both H3-K27 methylation as well as the EED-EZH2 counterpart
exist in Drosophila , whether the ESC-E(Z) complex is responsible for H3-K27 methylation was examined in this organism. Several E(z)
temperature-sensitive mutant alleles have been characterized, one of which,
E(z)61, contains a
Cys-to-Tyr substitution (C603Y) in the cysteine-rich region immediately
preceding the SET domain. When reared continuously at
18°C (permissive temperature), E(z)61 homozygotes exhibit no detectable mutant phenotype and maintain wild-type expression patterns of HOX genes, such as Ubx. However, at 29°C (restrictive temperature), E(z)61 produces multiple homeotic phenotypes due to derepression of HOX genes,
which correlates with loss of polytene chromosome binding by the
E(Z)61 protein and disruption of chromosome
binding by Polycomb (PC) and other PRC1 components. Given that chromosome binding by E(Z)61 protein is abolished at 29°C, H3-K27 methylation should be correspondingly reduced in the mutants at 29°C, if E(Z) is responsible for H3-K27 methylation. Western blot analysis of
the histones from wild-type and E(z)61 fly embryos at 18° and 29°C demonstrate that the H3-K27 methylation is abolished in the E(z)61 embryos at
29°C. However, these conditions do not have a
detectable effect on H3-K9 methylation. It is therefore concluded that functional E(Z) protein is required for H3-K27 methylation in vivo (Cao, 2002).
To understand the functional relation between E(z)-mediated H3-K27
methylation and HOX gene silencing, a study was carried out of E(z) binding, H3-K27 methylation, and recruitment of PC, a core component of the PRC1
complex, to the major PRE of the Ubx gene in
S2 tissue culture cells by chromatin immunoprecipitation (ChIP). Consistent with the involvement of E(z) in H3-K27 methylation, ChIP
analysis of a 4.4-kb region that includes this PRE showed
precise colocalization of E(z) binding and H3-K27 methylation. In contrast, similar colocalization was not observed for
mK9, indicating that H3-K9 methylation, or at least K9-dimethylation,
is independent of E(z) binding. To further verify the importance of E(z)
binding for H3-K27 methylation, attempts were made to disrupt Esc-E(z)
complex activity using RNA interference (RNAi). It was reasoned that
depletion of the Esc protein, a direct binding partner of E(z) and a
component of the Esc-E(z) complex, would result in disruption of PRE
binding by E(z). Depletion of Esc with RNAi results in greatly reduced PRE binding by E(z), loss of H3-K27 methylation, and concomitant loss
of PC binding. Depletion of PC in S2 cells has
been shown to result in derepression of Ubx.
Therefore, these data collectively suggest that the Esc-E(z) complex is
critical not only for H3-K27 methylation, but also for PC binding to
the PRE region, and that H3-K27 methylation is associated with
Ubx repression (Cao, 2002).
To examine the relation between E(z) binding, H3-K27 methylation, and
Ubx gene repression in vivo, wing imaginal discs were dissected from homozygous E(z)61 larvae that had
been either reared continuously at 18°C or shifted from 18° to
29°C ~48 hours before dissection, and analyzed E(z) binding and
H3-K27 methylation in the same Ubx PRE region by ChIP. Consistent with previous studies demonstrating disruption of polytene chromosome binding by both E(z)61 and PC proteins at 29°C, ChIP analysis showed loss of E(z)61 and PC binding to this PRE at restrictive temperature.
In addition, H3-K27 methylation colocalizes with E(z) binding at
permissive temperature, but is lost along with E(z) binding at 29°C.
In contrast, similar changes in H3-K9 methylation were not observed
under the same conditions. Under normal conditions,
Ubx is not expressed in wing discs due to PcG-mediated
silencing. Similar inactivation of an
E(z) temperature-sensitive allele during larval
development has been shown to result in derepression of Ubx
in wing discs. Thus, Ubx PRE-associated
nucleosomes appear to be targeted by E(z)-mediated H3-K27 methylation,
which correlates with PC binding and repression of Ubx.
Collectively, these data suggest that H3-K27 methylation plays an
important role in the maintenance of Ubx gene silencing (Cao, 2002).
The chromodomain of the heterochromatin protein HP1 specifically
binds to H3 tails that are methylated at K9 by the HMTase SUV39H1. Given that PC contains a chromodomain and that loss of E(z) function abolishes H3-K27
methylation as well as Pc binding to the Ubx PRE, it is possible that methylation of H3-K27 by Esc-E(z)
facilitates PRE binding by PC, analogous to the effect of H3-K9
methylation on nucleosome binding by HP1. To test this possibility,
Drosophila PC was generated using the rabbit reticulocyte
transcription/translation-coupled system and it was incubated with
biotinylated H3 peptides with or without K27 methylation in the
presence of streptavidin-conjugated Sepharose beads. Analysis by
peptide pull-down assay indicated that methylation on K27 facilitates binding of Pc to the H3 peptide. Binding of Pc to the peptides is
specific because the chromodomain-containing protein HP1 fails to bind to the same peptides under the same conditions (Cao, 2002).
Previous studies strongly suggest that the chromodomain of PC is
necessary and sufficient for targeting PC to specific chromosomal locations in vivo because mutations in the PC chromodomain abolish the
ability of PC to bind to chromatin in vivo. In
addition, a chimeric PC/HP1 protein, in which the HP1 chromodomain is
replaced by the PC chromodomain, binds to both heterochromatin and PcG
target sites in euchromatin. To evaluate the
contribution of the chromodomain in the preferential binding of PC to
K27 methylated peptide, a PC mutant was generated in which two of the
highly conserved amino acids Trp-47 and Trp-50 were changed to Ala.
These two amino acids were chosen because the corresponding amino acids
in the HP1 chromodomain have been shown to directly contact the methyl
group of an H3-mK9 peptide. The mutant
PC does not preferentially bind to the K27 methylated peptide, suggesting that the chromodomain of PC is responsible
for the preferential binding to the H3-mK27 (Cao, 2002).
Collectively, these studies support a model in which
Esc-E(z)-mediated H3-K27 methylation serves as a signal for the
recruitment of the PRC1 complex by facilitating PC binding. Recruitment of PRC1 in turn prevents the access of nucleosome remodeling factors, such as SWI/SNF, leading to the formation of a repressive chromatin state. Although this model is attractive, it does not exclude the possibility that protein-protein interaction also contributes to the recruitment of
PRC1 to PREs. Indeed, a recent study indicates that PC transiently associates with the Esc-E(z) complex during early embryogenesis. These studies established a correlation between H3-K27 methylation and PcG silencing. Further work is needed to establish the
exact role of H3-K27 methylation in PcG silencing (Cao, 2002).
The Polycomb Group proteins are required for stable long-term maintenance of transcriptionally repressed states. Two distinct Polycomb Group complexes have been identified, a 2-MDa PRC1 complex and a 600-kDa complex containing the ESC and E(Z) proteins together with the histone deacetylase RPD3 and the histone-binding protein p55. There are at least two embryonic ESC/E(Z) complexes that undergo dynamic changes during development and a third larval E(Z) complex that forms after disappearance of ESC. A larger embryonic ESC complex has been identified containing RPD3 and p55, along with E(Z), that is present only until mid-embryogenesis, while the previously identified 600-kDa ESC/E(Z) complex persists until the end of embryogenesis. Constitutive overexpression of ESC does not promote abnormal persistence of the larger or smaller embryonic complexes and does not delay a dissociation of E(Z) from the smaller ESC complex or delay appearance of the larval E(Z) complex, indicating that these changes are developmentally programmed and not regulated by the temporal profile of ESC itself. Genetic removal of ESC prevents appearance of E(Z) in the smaller embryonic complex, but does not appear to affect formation of the large embryonic ESC complex or the PRC1 complex. The ESC complex is already bound to chromosomes in preblastoderm embryos and genetic evidence is presented that ESC is required during this very early period (Furuyama, 2003).
Polycomb group (PcG) proteins are required to maintain stable repression of the homeotic genes and others throughout development. The PcG proteins ESC and E(Z) are present in a prominent 600-kDa complex as well as in a number of higher-molecular-mass complexes. A 1-MDa ESC/E(Z) complex has been identified and characterized that is distinguished from the 600-kDa complex by the presence of the PcG protein Polycomblike (PCL) and the histone deacetylase RPD3. In addition, the 1-MDa complex shares with the 600-kDa complex the histone binding protein p55 and the PcG protein SU(Z)12. Coimmunoprecipitation assays performed on embryo extracts and gel filtration column fractions indicate that, during embryogenesis E(Z), SU(Z)12, and p55 are present in all ESC complexes, while PCL and RPD3 are associated with ESC, E(Z), SU(Z)12, and p55 only in the 1-MDa complex. Glutathione transferase pulldown assays demonstrate that RPD3 binds directly to PCL via the conserved PHD fingers of PCL and the N terminus of RPD3. PCL and E(Z) colocalize virtually completely on polytene chromosomes and are associated with a subset of RPD3 sites. As shown for E(Z) and RPD3, PCL and SU(Z)12 are also recruited to the insertion site of a minimal Ubx Polycomb response element transgene in vivo. Consistent with these biochemical and cytological results, Rpd3 mutations enhance the phenotypes of Pcl mutants, further indicating that RPD3 is required for PcG silencing and possibly for PCL function. These results suggest that there may be multiple ESC/E(Z) complexes with distinct functions in vivo (Tie, 2003).
Mammalian Polycomb group (PcG) protein YY1 can bind to Polycomb response elements in Drosophila embryos and can recruit other PcG proteins to DNA. PcG recruitment results in deacetylation and methylation of histone H3. In a CtBP mutant background, recruitment of PcG proteins and concomitant histone modifications do not occur. Surprisingly, YY1 DNA binding in vivo is also ablated. CtBP mutation does not result in YY1 degradation or transport from the nucleus, suggesting a mechanism whereby YY1 DNA binding ability is masked. These results reveal a new role for CtBP in controlling YY1 DNA binding and recruitment of PcG proteins to DNA (Srinivasan, 2004).
To determine whether YY1 can recruit PcG proteins to DNA, chromatin immunoprecipitation (ChIP) assays were performed in a transgenic Drosophila embryo system consisting of hsp70-driven GALYY1 and a reporter construct containing the LacZ gene under control of the Ultrabithorax (Ubx) BXD enhancer and the Ubx promoter adjacent to GAL4-binding sites (BGUZ). The BGUZ reporter is expressed ubiquitously during embryogenesis but is selectively repressed in a PcG-dependent manner by GALYY1 and GALPc. Embryos were either left untreated or heat shocked to induce GALYY1 expression. After immunoprecipitation with various antibodies, the region surrounding the GAL4-binding sites in the BGUZ reporter was detected by PCR. Prior to heat shock, no GALYY1 could be observed at the reporter gene. After heat shock, GALYY1 binding to the reporter gene was easily detected. Interestingly, concomitant with GALYY1 binding, there was an increase in binding of the Polycomb (Pc) and Polyhomeiotic (Ph) proteins. Thus, YY1 DNA binding results in PcG recruitment to DNA (Srinivasan, 2004).
Binding of PcG proteins to PRE sequences is known to cause deacetylation of histone H3 and methylation on Lys 9 and Lys 27. Interestingly, induction of GALYY1 binding to the reporter gene resulted in loss of histone H3 acetylation on K9 and K14. Simultaneously, there was a gain of methylation on histone H3 Lys 9 and Lys 27. Therefore, YY1 binding to the BGUZ reporter results in the recruitment of PcG proteins to DNA and subsequent post-translational modifications of histones characteristic of PcG complexes (Srinivasan, 2004).
The presence of PcG proteins and the status of histone H3 modifications at the Ubx promoter region, which is 4 kb downstream of the GALYY1-binding site, were determined. To avoid amplification of the endogenous Ubx promoter, immunoprecipitated samples were amplified with primers spanning the Ubx-LacZ boundary. Interestingly, the presence of Pc and Ph was detected at the promoter after GALYY1 induction. The presence of GALYY1 at this site was also detected. The GAL4 protein alone does not bind to the Ubx promoter region, indicating specificity for YY1 sequences. The induced GAL4 protein was functional, however, because it efficiently bound to the GAL4-binding site in the BGUZ reporter. Binding by GALYY1 could, therefore, be due to either cryptic YY1-binding sites present at the promoter, physical association of GALYY1 with other proteins bound at the promoter, or interactions via looping of DNA between the GAL4-binding sites and the Ubx promoter. Again, induction of GALYY1 resulted in loss of acetylation of H3K9 and H3K14 and simultaneous gain of methylation on H3K9 and H3K27. These results are consistent with studies that have reported spreading of PcG proteins and histone modifications to flanking DNA (Srinivasan, 2004).
PHO and YY1 bind to the same DNA sequence, and PHO-binding sites have been identified in multiple PREs. Therefore, it was reasoned that YY1 would bind to endogenous PREs and perhaps increase recruitment of PcG proteins. For this, the major Ubx PRE (PRED), that contains multiple PHO-binding sites located in the bxd region, was examined. As expected, upon GALYY1 induction, GALYY1 was detected at this endogenous PRE site. In addition, YY1 binding was accompanied by an increase in Pc and Ph signals when compared with no heat shock controls and a loss of H3 K9 and H3 K14 acetylation and gain of H3 K9 and H3 K27 methylation. Thus, YY1 can bind to an endogenous PRE and can augment PcG recruitment (Srinivasan, 2004).
These results clearly indicated that YY1 DNA binding results in recruitment of PcG proteins, histone deacetylases (HDACs), and histone methyltransferases (HMTases) to DNA. To determine whether the Drosophila E(z) protein (which possesses HMTase activity) was involved, whether YY1 transcriptional repression was lost in an E(z) mutant background was examined. The results are consistent with the observation that E(z) specifically methylates histone H3 on Lys 27, which creates a binding site for the chromodomain of Pc. Thus, the repression observed with GALYY1 requires function of the E(z) PcG protein (Srinivasan, 2004).
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).
The Extra sex combs (ESC) protein is a Polycomb group (PcG) repressor that is a key noncatalytic subunit in the ESC-Enhancer of zeste [E(Z)] histone methyltransferase complex. Survival of esc homozygotes to adulthood based solely on maternal product and peak ESC expression during embryonic stages indicate that ESC is most critical during early development. In contrast, two other PcG repressors in the same complex, E(Z) and Suppressor of zeste-12 [SU(Z)12], are required throughout development for viability and Hox gene repression. A novel fly PcG repressor, called ESC-Like (ESCL), is described whose biochemical, molecular, and genetic properties can explain the long-standing paradox of ESC dispensability during postembryonic times. Developmental Western blots show that ESCL, which is 60% identical to ESC, is expressed with peak abundance during postembryonic stages. Recombinant complexes containing ESCL in place of ESC can methylate histone H3 with activity levels, and lysine specificity for K27, similar to that of the ESC-containing complex. Coimmunoprecipitations show that ESCL associates with E(Z) in postembryonic cells and chromatin immunoprecipitations show that ESCL tracks closely with E(Z) on Ubx regulatory DNA in wing discs. Furthermore, reduced escl+ dosage enhances esc loss-of-function phenotypes and double RNA interference knockdown of ESC/ESCL in wing disc-derived cells causes Ubx derepression. These results suggest that ESCL and ESC have similar functions in E(Z) methyltransferase complexes but are differentially deployed as development proceeds (Wang, 2006).
ESC and E(Z), and their homologs, are functional partners in the chromatin of plants, invertebrates, and mammals. Working together, they control a diverse array of developmental processes, including flower and seed differentiation in Arabidopsis thaliana, germ line development in Caenorhabditis elegans, X chromosome inactivation in mice, and Hox gene repression in flies and mammals. Recent studies show that this partnership reflects a requirement for ESC in potentiating the histone methyltransferase activity of E(Z) (Wang, 2006).
In light of this functional interdependence, a paradox is presented by developmental studies in Drosophila melanogaster, which show that ESC is primarily needed during early embryogenesis, whereas E(Z) is required throughout embryonic, larval, and pupal development. Analysis of ESCL, which can replace ESC in E(Z) HMTase complexes in vitro, provides a plausible solution to this puzzle. ESCL expression is largely complementary to that of ESC, peaking during later developmental stages, and functional studies show that ESCL is partially redundant with ESC in imaginal tissues. These results, together with prior genetic data that address esc time of action, indicate that ESC predominates in embryos, whereas both ESCL and ESC make functional contributions during postembryonic development (Wang, 2006).
Phenotypic analyses of esc loss-of-function mutants provided the original evidence that the primary time of ESC action is during embryogenesis. Although complete loss of esc+ product is embryonic lethal and yields wholesale misexpression of Hox genes, it was shown that maternally provided esc+ product provides sufficient function during embryogenesis to enable zygotically null esc– animals to survive to adulthood. These esc– adults are fertile, healthy, and phenotypically normal except for minor homeotic transformations such as extra sex combs on the meso- and meta-thoracic legs. In contrast, animals that are zygotically null for any other PcG subunit of the ESC-E(Z) complex or PRC1 fail to survive beyond early pupal stages, with most dying by the embryonic/L1 stage (Wang, 2006).
Additional experiments with a conditional esc allele further delimited the main time of ESC function to a period of mid-embryogenesis extending from about the onset of gastrulation (about 3 h at 25°C) until germ band shortening (approximately 9 to 12 h). An independent study that measured phenotypic rescue by a heat-inducible esc+ transgene confirmed that the time of ESC action begins at about 3 h of embryogenesis. These genetically determined times of esc+ function coincide with the accumulation of ESC protein, which peaks during mid-embryogenesis and declines by the end of embryogenesis (Wang, 2006).
However, full consideration of the genetic evidence also indicates that ESC does contribute to postembryonic PcG repression, particularly in imaginal tissues. Analysis of esc– larvae showed modest defects in Hox gene repression in imaginal discs as well as in the central nervous system. In particular, this study attributed the extra sex combs phenotype of esc– larvae to misexpression of the Scr Hox gene in the T2 and T3 leg discs. In addition, production of extra sex combs from patches of esc– tissue generated by somatic recombination during larval development indicates that the time of ESC action extends into the larval period, at least in leg discs (Wang, 2006).
A postembryonic role is consistent with the detection of ESC on the Ubx gene in wing discs and with the overlapping roles of ESC and ESCL in Ubx repression in disc-derived MCW12 cells. This result might explain why esc– wing discs did not produce homeotic phenotypes even after sufficient passage to ensure depletion of maternal esc+ product; presumably, both ESC and ESCL would need to be disrupted in this tissue to yield robust Hox misexpression. Finally, although it is much less abundant at late developmental times than in embryos, ESC is detected by Western blotting in larval and pupal extracts. Thus, the genetic and molecular data together indicate that ESC does function during postembryonic stages, albeit with a more modest overall contribution than its critical role in embryos (Wang, 2006).
These considerations imply that the developmental division of labor between ESC and ESCL is not simply that ESC functions only in embryos and ESCL takes over for subsequent stages. Rather, although ESC does predominate early, as evidenced by the global loss of H3 K27 methylation in esc– embryos, postembryonic development appears to involve both ESC and ESCL. It was originally hypothesized that late developmental functions of the esc locus might be executed by an esc+ isoform distinct from the embryonic version. The current data confirm that multiple ESC-related proteins do operate during fly development, with a late-acting version supplied by a second copy of the esc gene (Wang, 2006).
The functional context for ESCL during postembryonic development is presumably as a subunit in E(Z)-containing complexes with histone methyltransferase activity. The fact that ESCL can assemble in place of ESC and restore HMTase activity to a reconstituted E(Z) complex indicates that the biochemical roles of ESCL and ESC are similar. ESCL/ESC functional overlap could reflect a mixture of postembryonic E(Z) complexes, with some containing ESCL and others containing ESC. The simplest version of this scenario would entail four-subunit postembryonic HMTase complexes similar to the embryonic core complex of E(Z), SU(Z)12, NURF-55, and ESCL or ESC. However, postembryonic E(Z) complexes have yet to be purified, so their molecular compositions are not yet known. In fact, there is evidence that larval E(Z) complexes may differ from embryonic E(Z) complexes in features besides the ESCL/ESC subunit. For example, the SIR2 histone deacetylase has been reported to associate with larval but not embryonic E(Z) complexes. Much remains to be determined about postembryonic E(Z) complexes, including subunit compositions and characterization of presumed HMTase activity (Wang, 2006).
Although the catalytic subunit E(Z) contains the conserved SET domain, studies on fly, worm, and mammalian homologs reveal that the ESC subunit is also critical for HMTase function. The single loss of ESC from the fly complex or loss of its homolog, MES-6, from the C. elegans complex yields subcomplexes with little or no HMTase activity in vitro. In agreement with this, genetic removal of ESC eliminates most or all methyl-H3 K27 in fly embryos, loss of MES-6 eliminates most or all methyl-H3 K27 in worm germ lines and early embryos, and loss of EED removes most or all methyl-H3 K27 from embryonic mouse cells. The mechanism by which ESC and its relatives potentiate the activity of HMTase complexes is not known. An in vitro study argues against a role for fly ESC in mediating stable contacts with nucleosome substrate. In contrast, loss of ESC by RNA interference in fly S2 cells leads to dissociation of E(Z) from chromatin targets (Wang, 2006).
A biochemical analysis of the human EED-EZH2 complex (also called PRC2) has revealed an intriguing difference in the HMTase depending upon the subtype of EED subunit present in the complex. Multiple isoforms of EED are expressed in HeLa cells that differ in the extents of their N-terminal tails through use of alternative translation start sites. 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. Taken together with other studies, this suggests that EED is a regulatory subunit that can influence both substrate specificity and catalytic efficiency of the HMTase (Wang, 2006).
In light of this finding, it seems possible that ESCL-E(Z) complexes might also have HMTase activity with altered lysine specificity. However, both ESCL-E(Z) and ESC-E(Z) recombinant complexes showed similar specificities for H3 K27 in H3/H4 tetramers and no methylation of mammalian histone H1 by either of these recombinant complexes was detected in vitro. It is noted that the human H1 K26 methylation site is embedded in an ARKS sequence, which is also present surrounding H3 K27. This sequence is not conserved in Drosophila histone H1, suggesting that the ability of certain EZH2 complexes to methylate H1 may not be conserved in the fly system. However, there may well be other relevant methylation substrates besides histone H3 and it remains possible that alternative ESC isoforms could alter lysine specificities for these other substrates (Wang, 2006).
Based upon their temporal expression profiles, it seems clear that esc and escl have distinct functions in a developmental context. Their temporal division of labor is most clearly demonstrated by esc– escl+ embryos, which show extreme homeotic transformations accompanied by dramatically reduced levels of methylated H3 K27. This division could be entirely a consequence of differential transcriptional controls built into their divergent promoters. That is, ESC and ESCL could be functionally identical proteins that are just expressed at peak levels at different times. Alternatively, the two proteins may possess intrinsic differences that are also important during development but are not revealed by the assays applied so far. One possibility is that ESC and/or ESCL may play a role in methylation of nonhistone proteins. The only nonhistone proteins yet identified that fly E(Z) complexes can methylate are two subunits of the core complex itself, E(Z) and SU(Z)12. It is not clear if this self-methylation is functionally relevant and, in any case, it occurs at comparable levels with the ESC- and ESCL-containing recombinant complexes (Wang, 2006).
It is also possible that ESC and ESCL could differ in contributions to E(Z) complexes besides HMTase activity. These other functions could include interacting with and recruiting histone deacetylases, mediating physical interactions with PRC1 components, recruiting E(Z) complexes to target loci, and influencing the way E(Z) complexes interact with other (non-K27) histone tail modifications. Indeed, there is evidence for differential association of histone deacetylases with E(Z) complexes at embryonic versus larval stages, which parallels temporal changes in ESC and ESCL abundance. At the same time, ESC and ESCL functions must overlap enough to account for the sufficiency of either one to maintain Ubx repression in at least some postembryonic cells (Wang, 2006).
Definitive answers will require promoter swap experiments in which ESCL is placed under control of the ESC promoter and vice versa, to determine which combinations provide genetic rescue of esc and escl mutations in vivo. Along with this approach, a complete understanding of the developmental role of ESCL will require generation of escl mutant alleles and systematic analysis of the phenotypic consequences of escl loss of function (Wang, 2006).
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).
Polycomb-group (PcG) proteins are highly conserved epigenetic transcriptional repressors that play central roles in numerous examples of developmental gene regulation. Four PcG repressor complexes have been purified from Drosophila embryos: PRC1, PRC2, Pcl-PRC2 and PhoRC. Previous studies described a hierarchical recruitment pathway of PcG proteins at the bxd Polycomb Response Element (PRE) of the Ultrabithorax (Ubx) gene in larval wing imaginal discs. The DNA-binding proteins Pho and/or Phol are required for target site binding by PRC2, which in turn is required for chromosome binding by PRC1. This study identified a novel larval complex that contains the PcG protein Polycomblike (Pcl) that is distinct from PRC1 and PRC2 and which is also dependent on Pho and/or Phol for binding to the bxd PRE in wing imaginal discs. RNAi-mediated depletion of Pcl in larvae disrupts chromosome binding by E(z), a core component of PRC2, but Pcl does not require E(z) for chromosome binding. These results place the Pcl complex (PCLC) downstream of Pho and/or Phol and upstream of PRC2 and PRC1 in the recruitment hierarchy (Savla, 2008).
Drosophila Polycomb-group (PcG) genes were originally identified as negative regulators of Hox genes. PcG-mediated silencing in Drosophila occurs in essentially two broadly defined stages: assumption of transcriptional repression responsibilities from gene-specific transcription factors in early embryos, followed by maintenance of the silenced state through many cycles of cell division beginning in mid-late-stage embryos and continuing throughout the remainder of development (Savla, 2008).
Although much of the genetic analysis of PcG functions and studies of the mechanisms by which PcG proteins are targeted to specific genomic sites have focused on their activities in larval tissues, in vitro biochemical analyses have focused on PcG complexes isolated from embryos: PRC1, PRC2 and PhoRC. PRC1 possesses multiple chromatin modifying activities in vitro suggesting that it, among PcG complexes, might be most directly responsible for preventing transcription. The primary functions of PhoRC and PRC2 appear to be to recruit and/or stabilize target site binding by PRC1, and potentially other PcG proteins. PhoRC includes the DNA-binding PcG protein Pleiohomeotic (Pho), which binds to sites within Polycomb Response Elements (PREs) that serve as docking platforms for PcG proteins. Pho directly interacts with components of both PRC1 and PRC2, and is required for recruitment of both complexes. The E(z) subunit of PRC2 trimethylates histone H3 at lysine 27 (H3K27me3), facilitating recruitment of PRC1 (Savla, 2008).
A variant of PRC2 has recently been described that includes the PcG protein Polycomblike (Pcl). On the basis of gel filtration analysis of native complexes in embryo nuclear extracts and the stoichiometry of the purified Pcl-PRC2 complex, it appears that the majority of embryonic Pcl is present in Pcl-PRC2, but that the other PRC2 core subunits, E(z), Su(z)12, Esc and NURF55 (also known as Caf1 - FlyBase), predominantly are in a complex(es) lacking Pcl. It has been proposed that inclusion of Pcl in PRC2 is required for high levels of H3K27me3 in vivo, although the in vitro histone methyltransferase activity of Pcl-PRC2 is indistinguishable from that of PRC2 lacking Pcl. In this study, a larval Pcl-containing complex is identified that is distinct from PRC2 and PRC1 and shown to be required for chromosome binding by these PcG complexes (Savla, 2008).
In order to examine potential differences between embryonic and larval
stage PcG complexes, larval nuclear extracts were fractionated over a Superose 6
gel filtration column, and western blots of the fractions were probed with anti-E(z)
and anti-Pcl antibodies. Larval E(z)-containing complexes have a relative mass
of ~500 to 600 kDa, similar to that of embryonic PRC2 complexes that lack
Pcl. However, Pcl was undetectable in E(z)-containing fractions and appeared to be in a complex with a relative mass of ~1500 kDa. This is different
from the fractionation profile of Pcl from embryo extracts, in which it
co-fractionates with E(z) in native complexes with relative mass estimates in
the range of ~650 kDa to 1000 kDa, suggesting that, unlike its association with a subset of PRC2 complexes in embryos, Pcl functions as a component of a distinct complex in larvae, which will be referred to as the Pcl-Complex (PCLC) (Savla, 2008).
In order to further investigate the relationship of Pcl with other PcG
proteins and its role in PcG-mediated silencing in larvae, chromatin
immunoprecipitation (ChIP) assays were performed on wing imaginal discs. The
PcG maintains the transcriptional silence of the Hox gene
Ultrabithorax (Ubx) in the epithelial cells of wing discs. Other
PcG proteins, including the DNA-binding proteins Pho and Phol and components
of the PRC1 and PRC2 complexes, have previously been shown to be present at
the major PRE in the Ubx cis-regulatory bxd region in this tissue.
Consistent with a previous report, Pcl also was detected at the bxd PRE, and appears to largely colocalize with E(z) and Phol (Savla, 2008).
A hierarchical relationship among PcG proteins at
the bxd PRE in which Pho and/or Phol are required, but are not
necessarily sufficient, for recruitment of PRC2, which in turn facilitates
recruitment of PRC1. In order to determine how Pcl might fit into this recruitment pathway, ChIP assays were performed on E(z) mutant wing
imaginal discs. E(z)61 is a temperature-sensitive allele
that displays nearly wild-type activity at 18°C, but strongly reduced
activity at 29°C. Following shift from 18°C to 29°C, bxd PRE
binding by E(z)61 protein is rapidly lost and along with it the
detection of H3K27me3 and Pc in this region. ChIP assays of wing discs dissected from E(z)61 larvae 24 hours following shift from 18° to 29°C confirmed loss of E(z) from the PRE, but revealed no effect on Pcl and Phol binding to PRE fragments 3 and 4, but a slight decrease of both proteins at the PRE 2 fragment. It is speculated that Pcl and Phol signals at this proximal edge of the PRE are partly due to protein-protein cross-links, which might be reduced in the absence of PRC2. Retention of Pcl at the PRE in the absence of E(z) and by extension absence of PRC1, which requires PRC2 for binding to this region, confirms that Pcl is not a stable subunit of larval versions of either PRC1 or PRC2 and is consistent with its inclusion in a distinct complex (Savla, 2008).
Flies that are homozygous for null Pcl alleles die as embryos and
no conditional Pcl alleles exist, precluding reciprocal experiments
on Pcl mutant larvae. Therefore, transgenic fly lines were generated
that contain inserts of a pWIZ-Pcl construct, which expresses Pcl shRNA under
the control of Gal4, permitting inducible RNAi-mediated knockdown of Pcl in
combination with Gal4 drivers. Individuals that contain both pWIZ-Pcl
and P{GAL4-da.G32}, which constitutively expresses Gal4, died as early pupae and exhibited dramatically reduced levels of Pcl in wing
imaginal discs. E(z) levels were not affected. ChIP assays of these Pcl-depleted wing discs confirmed reduced Pcl levels at the bxd PRE and revealed commensurate loss of E(z). Thus, although Pcl does not require PRC2 for PRE binding, Pcl, presumably functioning as a subunit of PCLC, is needed for stable binding of PRC2 to the bxd PRE. Phol remains at the PRE in the absence of Pcl (Savla, 2008).
In order to determine whether Pcl (like components of PRC1 and PRC2)
requires Pho and/or Phol for PRE binding, ChIP assays were performed using
wing imaginal discs from phol81A; pho1 larvae.
Consistent with their role in recruiting other PcG proteins, Pcl
was lost from the bxd PRE in the absence of Pho and Phol. These
observations at the bxd PRE also appear to generally apply to PcG-binding sites
throughout the genome (Savla, 2008).
These results demonstrate the existence of a distinct Pcl protein complex
in larvae that is required for recruitment of PRC2 to chromosomal target sites
and/or to stabilize its binding. As previously described, E(z), as a core
subunit of PRC2, is required for target site binding by PRC1. Therefore, Pcl is indirectly required for chromosome binding by PRC1 as well, although direct interaction with PRC1 cannot be ruled out, similar to the way in which Pho may contribute to target site binding by PRC1 by interacting both
with PRC2 subunits and with Pc, a core subunit of PRC1
(Savla, 2008 and references therein).
In vitro histone methyltransferase assays of Pcl-PRC2 show that its
activity and specificity for methylation of H3K27 are essentially
indistinguishable from that of PRC2 complexes lacking Pcl. ChIP analysis of
Pcl mutant embryos has shown that Pcl does not seem to be required
for target site binding by other PRC2 subunits, but that it may be needed for
high levels of trimethylation of H3K27. One
explanation for these observations is that the contribution of Pcl to Pcl-PRC2
in embryos might be to mediate interaction with other proteins that are yet to
be identified. In larvae, Pcl exists as a subunit of a distinct complex. Given
the ability of Pcl to directly interact with several PRC2 subunits, colocalization of Pcl and E(z) at the PRE, and dependence of E(z) on Pcl for binding to the bxd PRE and other genomic sites, it is likely that
PCLC is closely associated with PRC2 at target sites in larvae. In both
embryos and larvae, some of the activities attributed to Pcl might, upon
further inspection, be due to the activities of other Pcl-associated proteins,
the close apposition of which with PRC2 and other PcG complexes may be
mediated by Pcl. The differential deployment of Pcl as a subunit of PRC2 and
as a subunit of PCLC at distinct developmental stages is intriguing and might
reflect the different molecular activities needed for establishment of
silencing in embryos and maintenance of the silenced state in larval tissues. A more detailed understanding of the mechanisms by which Pcl contributes to PcG silencing will require identification of the other proteins contained within the larval PCLC complex and the potential biochemical activities of the complex (Savla, 2008).
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