Polycomb, Epigenomes, and Control of Cell Identity

Cell, Vol. 112, 599–606, March 7, 2003, Copyright 2003 by Cell Press

Polycomb, Epigenomes, and Control of Cell Identity Valerio Orlando* Dulbecco Telethon Institute Institute of Genetics & Biophysics CNR Via Pietro Castellino 111 80131 Naples Italy

In development, cell identity is maintained by epigenetic functions that prevent changes in cell type-specific transcription programs. Recent insights into gene silencing mechanisms by Polycomb group (PcG) and trithorax group (trxG) proteins reveal that the memory system involves a concerted process of chromatin modification, blocking of RNA polymerase II, and synthesis of noncoding RNA. Remarkably, cell memory is regulated by a balance between repressors and activators that maintains both transcription status and at the same time the possibility of switching to a different state. Establishment and maintenance of cell identity involve pathways that silence specific sets of genes when and where they must be repressed. Lack of silencing results in noisy consequences for the organism, namely altered genetic programs and increased rate of cell transformation. The genes of the Polycomb group (PcG) and trithorax group (trxG) are part of a widely conserved cell memory system that prevents changes in cell identity by maintaining transcription patterns, set in the first stages of embryonic life, throughout development, and in adulthood. PcG and trxG control, respectively, repressed and active transcriptional states of several loci in the genome, including developmentally and cell cycle-regulated genes. Both groups encode components of multiprotein complexes that control chromatin accessibility. Thus, chromatin structure appears to contain the molecular imprint underlying cell memory and epigenetic inheritance. PcG proteins were thought to maintain gene silencing by locking inactive genes in a heterochromatin-like environment that excludes transcriptional activators and that is incompatible with RNA synthesis. In this way, silent chromatin would irreversibly program differentiated cells not to leave their fate. Recent discoveries, however, depict a more dynamic situation in which PcGmediated silencing is the result of an equilibrium between opposing transcriptional forces, which coexist and include activators as well as repressors, that maintain not only terminally determined states but also competence for switching. Unexpectedly, it now appears that general transcription factors (GTFs), the RNA polymerase II complex, and possibly noncoding RNA contribute to this complex cell memory system. PcG and trxG Complexes Imprint Chromatin In Drosophila, approximately fifteen PcG proteins participate in two separate multiprotein complexes: (1) PRC1 *Correspondence: [email protected]

Review

(Polycomb repressive complex 1), which contains PC and most of the characterized PcG polypeptides such as Polyhomeotic (PH), Posterior sex combs (PSC), and components of the basal transcriptional machinery (Saurin et al., 2001); and (2) ESC-E(Z) (extra sex combsEnhancer-of-zeste), which contains ESC, E(Z), the suppressor of position effect variegation (PEV) SU(var)12, and the DNA binding protein Pleiohomeotic (PHO), the homolog of the mammalian YY1 transcription regulator (Cao et al., 2002; Czermin et al., 2002; Kuzmichev et al., 2002; Mu¨ller et al., 2002). The ESC-E(Z) complex acts early in embryogenesis and is thought to set the stage for the long-term memory PRC1 complex. Both PRC1 and ESC-E(Z) complexes are conserved between flies and mammals, although mammalian PRC1 appears to be devoid of GTFs (Levine et al., 2002). Notably, mammalian genomes contain redundant copies of PcG, and the existence of complexes with varied protein composition has been postulated (Satijn and Otte, 1999). The means by which these complexes find their way onto chromatin and convey epigenetic inheritance are unclear. In Drosophila, both PcG and trxG complexes exert their epigenetic function by binding to specialized, switchable modular DNA elements (known as Polycomb response elements [PREs] or cell memory modules [CMMs]) and core promoters (Lyko and Paro, 1999). PREs, in conjuction with promoters, convey heritable silenced transcription patterns in an epigenetic manner. Upon activation, triggered by a transiently expressed activator, the same element is able to maintain the active state indefinitely, including through female germline transmission (Cavalli and Paro, 1998). In mammals, while interaction of some PcG and trxG complexes with promoters has been demonstrated, no PRE-like DNA elements have yet been identified (see below). PcG proteins, with the exception of PHO, do not contain an obvious DNA binding domain. However, PHO binding sites alone are not sufficient to convey epigenetic inheritance of a silenced state (Mohd-Sarip et al., 2002). The same holds for the trxG GAGA factor, which is found together with PcG at repressed sites (Strutt et al., 1997). Remarkably, assembly of a PcG silencing complex on DNA can be triggered by fusing a DNA binding domain to any PcG protein, though silencing in this case cannot be maintained (Poux et al., 2001). Thus, in order to imprint PcG silenced target genes with the proper epigenetic tag, additional components are required. A major advance came from the discovery that mammalian heterochromatin protein 1 (HP1) binds methylated histone H3 tails (Bannister et al., 2001). The key element in this pathway is the conserved protein domain called SET, named after the proteins SU(var)3-9, E(Z), and trithorax (TRX) that contain this domain. The Su(var)3-9 protein regulates heterochromatin formation and PEV. The mammalian SU(var)3-9 SET domain has been shown to be a histone H3-specific methyltransferase (HMT) that trimethylates the ⑀-amino group of lysines; by so doing, HP1 is recruited, leading to heterochromatin formation (Lachner et al., 2001). In particular, methylation of histone H3 at Lys 9 (K9) in the N-terminal

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tail is a primary epigenetic mark that drives assembly of heterochromatic proteins on chromatin. Recent papers have finally provided breakthrough evidence that specific components of the PcG complex and two polypeptides of the trxG, TRX and ASH1, are HMTs, suggesting that the memory system uses a similar pathway to regulate the binding of both PcG and trxG complexes to their target sites (Beisel et al. 2002; Cao et al., 2002; Czermin et al., 2002; Kuzmichev et al., 2002; Milne et al., 2002, Nakamura et al., 2002; Mu¨ller et al., 2002). It has been shown that the E(Z) SET domain methylates H3 at K9 and K27, with a strong preference for the latter (Cao et al., 2002; Czermin et al., 2002; Kuzmichev et al., 2002; Mu¨ller et al., 2002). It is noteworthy that PC has a strong affinity for H3 methylated at K27. The K9/ K27 code is likely to be set in the early embryo by the ESC-E(Z)-PHO complex. The K9 and K27 methylation pattern is strictly ESC-dependent, as RNAi experiments show that if ESC is missing, H3 methylation and PC binding are rapidly lost. After the blastoderm stage, when the long-term memory system sets in, ESC-E(Z)PHO dissociates from the long-term memory Pc complex. Although ESC is not produced in late embryogenesis, E(Z) continues to be needed to maintain silencing and in particular to maintain Pc-complex binding to its target sites. E(Z) temperature-sensitive mutants show loss of histone methylation, dissociation of PcG proteins from polytene chromosomes, and global chromatin decondensation (Rastelli et al., 1993). The latter indicates a role of E(Z) in higher order chromosome structure. However, PcG proteins are not released at all sites; this could reflect a nonhomogenous composition of Pc complexes at all chromosomal sites and perhaps diverse epigenetic codes and/or other PcG HMTs not identified yet that may mediate PcG binding at specific sites. E(Z) mutants also show defects in chromosome condensation (Jones and Gelbart, 1990; Rastelli et al., 1993), similar to defects characteristic of other PcG proteins (Kodjabachian et al., 1998; Lupo et al., 2001). The timing when the epigenetic imprint is set is a crucial step for the memory system. As noted above, ESC-E(Z) acts in the early embryo in combination with the cell fate determination system. Lack of maternal esc and E(z) gene products results in severe homeotic transformation that can be only partially rescued by paternally derived zygotic product (Jones and Gelbart, 1990), although ChIP experiments have shown that PC and TRX are already present on PREs and promoters at this stage (Orlando et al., 1998). PcG loss-of-function experiments in imaginal discs revealed that this initial imprint is stable enough to allow silencing even after transient reactivation of BX-C genes (Beuchle et al., 2001). Thus, transcription is not sufficient to remove the PcG silencing epigenetic tag. Why this is the case remains to be elucidated. It is possible that imposition of a given epigenetic imprint has consequences on multiple aspects of gene organization, such as subnuclear compartimentalization, that cannot be changed. Because of its role in chromosome structure, it will be interesting in the future to investigate the effects of loss of function of E(Z) on the subnuclear organization of silenced genes. On the activation side, the HMTs TRX and ASH1, two SET-domain-containing trxG gene products, were

shown to methylate histone H3 at K4 and K9 and histone H4 at K20. Methylation at histone H3 K4 is generally considered to be a mark of active genes. Remarkably, K4 trivalent methylation is necessary for trxG-dependent transcriptional activation, and it appears to be incompatible with the binding of PcG and HP1 repressor proteins (Beisel et al., 2002). Conversely, these modifications may facilitate binding of the Brahma (BRM) SWI/ SNF-type chromatin remodeling complex and histone acetyl transferases (HATs). The TRX SET domain was also shown to interact with a SNR1, a component of the BRM complex (Rozenblatt-Rosen et al., 1998). The BRM complex appears to be excluded from PcG silenced domains (Armstrong et al., 2002); thus, recruitment, histone methylation, and association of BRM with nucleosomes could occur sequentially via the TRX SET domain and contribute to the maintenance of antirepression. TRX is found together with PcG proteins at repressed PREs and promoters. Though the role of TRX in repressed promoters is not clear (see below), binding to repressed regions may occur since in principle two histone codes could coexist on different tails within the same nucleosome (since each nucleosome contains two copies each of H3 and H4 histones). TRX binding may also depend on other types of protein-protein and nucleic acid-protein interactions. As an example of the former, the SET domains of human ALL-1 and Drosophila TRX and ASH1 proteins associate in vitro and in vivo (Rozovskaia et al., 2000). Interestingly, binding of TRX to its target sites in polytene chromosomes depends on E(Z) (Kuzin et al., 1994), suggesting that SET domains may have a second function as “attractor” centers for other chromatin reprogramming complexes. Is “trimethylated K9 and dimethylated K27” the complete PcG histone code? Trimethyl K9 and dimethyl K27 on histone H3 appear to be a specific mark for PcG chromatin, as this pattern is highly enriched at PcG target sites in polytene chromosomes and poorly represented in regions of constitutive heterochromatin, such as the chromocenter, chromosome 4, and telomeres (Czermin et al., 2002). Yet one should not forget that each lysine residue can receive up to three methyl groups and that the same residues can be/are acetylated. Thus, it is likely that additional levels of complexity (acetylation, ubiquitination, etc.) will have to be considered in order to understand the nucleosome modification pathway regulated by PcG. Appropriate sets of specific antibodies need to be raised to fully discriminate between different combinations and degrees of modification, although in the case of PcG, the absence of K27 methylation could be considered a promising candidate for a PcG epigenetic code. Setting of the Memory and Noncoding RNA As anticipated, PcG and some trxG proteins may bind PREs and promoters both in the repressed and active state. Conversely, Pc repressor can be found at active promoters (Breiling et al., 2001). Thus, since repressive and activating members coexist, maintenance of activation appears to be regulated by a dosage-dependent mechanism that inhibits repression, and TRX appears to be specifically required to prevent PcG from re-silencing (Poux et al., 2002).

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Why do activators bind repressed domains? Recent papers report that the production of noncoding RNA is involved in heterochromatin formation (reviewed by Dernburg and Karpen, 2002). In its original formulation, the model proposed for PcG and epigenetic maintenance of a silenced state of developmentally regulated genes involved heterochromatinization of euchromatic loci scattered in the genome (Paro, 1990). Thus, maintenance of the silenced state of euchromatic genes would be achieved by building up a chromatin structure into one that is typically condensed, inaccessible to various DNA binding proteins, and incompatible with transcription. Recently, however, it has been realized that more “sound” is made in the silent domains than this model would have predicted. Surprisingly, transcription of noncoding RNA appears to be needed to establish gene silencing. This mechanism involves the intersection between two previously unrelated silencing pathways, namely RNAi and histone H3 K9 methylation. In S. pombe, assembly of heterochromatic complexes at centromeres and silencing at the euchromatic MAT locus requires production of short, complementary sense and antisense transcripts. The resulting dsRNA is processed (to give a 22 nt shRNA) by the gene products of RNAi pathway: dicer (dcr), RNA-dependent RNA polymerase (RdRp), and argonaute (ago) (Volpe et al., 2002). Impairment of shRNA production results in the loss of silencing of an ectopic gene in centromeric heterochromatin in conjunction with loss of H3 K9 methylation and recruitment of the HP1-like SWI6 complex (Volpe et al., 2002). The RNAi pathway is required for the establishment of a heterochromatic state but not for its maintenance (Hall et al., 2002). In particular, persistence of the epigenetic tag, H3 K9 methylation, and the characteristic spreading of heterochromatic structure (H3 K9 methylation and bound SWI6 complex) do not depend on the RNAi pathway. Furthermore, silencing of heterochromatic genes derepressed by inhibitors of histone deacetylase cannot be reestablished in RNAi mutants (Hall et al., 2002). Other silencing phenomena such as cosuppression appear to involve the production of aberrant RNA and depend on RNAi (Cogoni and Macino, 1999; Zamore, 2002). In mammals, PcG proteins also accumulate at pericentromeric heterochromatin (Saurin et al., 1998). Interestingly, mouse pericentromeric repeat satellite DNA is heavily transcribed (Rudert et al., 1995). This localization depends on the HMT SUV39H1, which directly interacts with HPC2 and other PcG proteins (Sewalt et al., 2002). Mammalian HP1 was shown to be associated with constitutive heterochromatin in a histone deacetylase (HDAC)- and RNA-dependent manner (Maison et al., 2002). PcG and HP1 systems appear to cooperate in silencing (Ogawa et al., 2002). Intriguingly, HP1 binds RNA and methylated histones by combining a chromodomain and a hinge domain (Muchardt et al., 2002). Thus, at least for HP1, an RNA moiety would be needed to target silencing complexes onto mammalian heterochromatin. This may include long-range interactions (e.g. X chromosome Xist RNA). Notably, association of PcG with murine inactive X has been reported (Mak et al., 2002). Somehow, production of noncoding RNA

could be a general feature of silencing phenomena and epigenetic setting of the genome. In humans, the homolog of the Drosophila PcG PHO protein, the YY1 transcriptional repressor, has been found to be part of a repressive complex bound to the D4Z4 3.3 kb repeat that regulates genes involved in the control of the acioscapulohumeral dystrophy (FSHD; Gabellini et al., 2002). Dystrophic patients carry deletions of these repeats and show complete or alternate derepression of neighboring and more distant FRG1, FRG2, and ANT-1 transcription units. The transcriptional phenotype is dependent on a threshold number (⬍35 kb) of D4Z4 repeats that are deleted. Morpholino interference in HeLa cells against YY1 leads to inappropriate overexpression of the three genes, confirming that a reduction of silencing components leads to the same transcription defect. As no PREs have been identified yet in mammals, yet 50% of the genome is composed by repetitive sequences, it will be interesting to further investigate if and how repeat DNA or repetitive elements could be a major target of PcG proteins in mammalian genome. The copy number threshold effect is reminiscent of dosage-dependent silencing effects observed in cosuppression phenomena (Henikoff, 1998). The role of PcG and RNAi in cosuppression has been investigated. Transcriptional gene silencing (TGS) occurs when promoter homology exists among the transgenes or with the endogenous gene. In contrast, posttranscriptional gene silencing (PTGS) occurs when the homology is among the protein coding sequence. Accordingly, in flies, insertion of multiple copies of fusion genes carrying the white 5⬘ regulatory region and the alcohol dehydrogenase coding region (w-Adh) suppresses the activity of endogenous Adh (Pal-Bhadra et al., 1997, 1999). Silenced copies and the endogenous Adh gene recruit PcG repressing complex, as shown by immunolocalization of polytene chromosomes. This type of TGS is PcG-dependent. Remarkably, the w-Adh fusion can also suppress the reciprocal Adh-w transgene via Adh, also recruited in the silenced state. Adh appears to mediate Adh-w silencing posttranscriptionally, as Adh deletions abolish the cosuppression effect among the reciprocal transgenes (Pal-Bhadra et al., 1999). In this case, PTGS is not PcG-dependent, and no PcG protein is found on the silenced Adh-w transgene. As expected, genes involved in RNAi (piwi/ago) affect Adh PTGS. Surprisingly, piwi mutations also impaired TGS (Pal-Bhadra et al., 2002). The latter effect may indicate possible crosstalk between PcG and RNAi in TGS. The paradox that in heterochromatin active transcription contributes to silencing may explain the finding that no striking differences were observed among repressed and active portions of the BX-C in terms of both restriction enzyme accessibility and DNA superhelical density (Schlossherret al., 1994; Fitzgerald and Bender, 2001). Furthermore, chromatin immunoprecipitation experiments have shown that TBP, GTFs, and other components of the RNA polymerase II complex are bound to PcG-repressed promoters (Breiling et al., 2001). Thus, target genes of PcG in inactive chromatin are in principle permissive to transcription complexes. However, it will be important to know if these complexes move or remain still.

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ON to Be ON Intergenic transcription is a general feature of complex loci like the ␤-globin and BX-C clusters (Lipshitz et al., 1987; Sanchez-Herrero and Akam, 1989; Cumberledge et al., 1990; Ashe et al., 1997; Plant et al., 2001). The role of these transcripts is not well understood. Recent papers report the identification of noncoding transcripts in the BX-C that would impair epigenetic control of gene silencing by PcG proteins (Bender and Fitzgerald, 2002; Hogga and Karch, 2002; Rank et al., 2002). In particular, noncoding RNA is produced at several intergenic regions of the BX-C. The BX-C contains an array of parasegment-specific enhancer and insulator (boundary) elements that reflect the collinear arrangement of the coding transcription units. Remarkably, the expression of each of these noncoding transcripts is parasegmentspecific and precedes the onset of transcription of the corresponding coding unit (Bae et al., 2002). Interestingly, PREs appear to contain intrinsic cryptic promoters (adjacent to the minimal core PRE, e.g., in Fab7) that would produce an RNA with the potential to destabilize the silencing complex (Rank et al., 2002). This transcript is essential for the ability of the PRE to maintain the ON state of a transgene (Rank et al., 2002). However, a transcript in the nontransgenic Fab7 region is also detected in flies carrying a deletion of the Fab7 element, meaning that the wt transcript may start elsewhere. Indeed, several other transcripts identified by in situ hybridization appear to read through BX-C PRE elements (Bae et al., 2002). These data suggest that noncoding RNA is involved in epigenetic transmission of the active state. An intriguing idea, based on the “piggybacking” model, has been proposed for the PRE antisilencing function (Drewell et al., 2002; Rank et al., 2002). Namely, after determination of the active state of particular BX-C gene, a transcription complex would traverse PREs and “write” on chromatin the corresponding epigenetic code that would inhibit assembly of PcG complex (Rank et al., 2002). So far, the effect of mutations that influence the production of some of these transcripts is not clear. It could be that not the nature of the RNA per se but just the transcription process is the resetting event that triggers the epigenetic mark of the ON state. However, the possibility exists that an RNA moiety could locally trigger assembly of an activating complex, as in the case of dosage compensation in flies, to accomplish antisilencing epigenetic function (Akhtar et al., 2000). The effect of mutations that influence the production of some of these transcripts is not clear. However, noncoding RNA may be also used for long-range interactions (e.g., the Xist RNA which coats the inactive X chromosome). Association of PcG with murine inactive X has been reported (Mak et al., 2002). Thus, other noncoding RNA present in the BX-C and the presence of multiple PREs in the BX-C may be enough to compensate for the lack of one specific noncoding RNA species. In addition, the persistence of these RNAs in late embryogenesis may not indicate constitutive transcription. PcG Silencing and the Basal Transcriptional Machinery The general mechanisms by which transcription factors program repression (and activation) at promoters are

not known. An important step involves specific contacts with basal transcription machinery. Segmentation transcription factors like the Kruppel protein may act as either repressors or activators by binding to TFIIB and TFIIE (Sauer et al., 1995). Repressor Even-skipped binds TBP and blocks recruitment of TFIID (Li and Manley, 1998). It is noteworthy that both PC and TRX are bound to PREs and promoters before and while the segmentation genes cascade sets the transcription states of homeotic genes (Orlando et al., 1998). This would allow at least some components of the memory system to take over the job initiated by transcription factors at the level of basal transcription machinery. Indeed, the longterm memory PRC1 complex contains stoichiometric amounts of TAFs and other GTFs (Saurin et al., 2001). Moreover, it was shown that PcG-repressed promoters contain GTFs in vivo (Breiling et al., 2001). TBP coimmunoprecipitates with PC and other members of PRC1. Interestingly, PC appears to contact TFIIB. TFIIB is thought to bridge the binding of TBP to the RNA pol II complex; thus, this could be a potential key for interfering with activation. However, TFIIF, the DNA-TBP-RNA pol II stabilizing factor, is also present at PcG-repressed promoters, suggesting that the repression mechanism acts downstream of RNA pol II complex recruitment. RNAi and genetic analyses have shown that inhibition of transcription by PcG proteins onto a recruited RNA pol II complex appears to be constitutive, as BX-C genes can be reactivated at all times (Beuchle et al., 2001; Breiling et al., 2001). As mentioned, the PRC1 complex contains TAFs. The significance of this interaction is not clear. Interestingly, mammalian HP1␣ and -␥ interact with hTAFII130 (Vassallo and Tanese, 2002). hTAFII130 and the Drosophila homolog dTAFII110 directly contact the SP1 and CREB activation domains. In addition, hTAFII130 increases transcription activation by contacting various hormone receptor activation domains. Thus, it will be interesting to see whether PC or other PcG proteins share the same target in the basal machinery. In vitro, PRC1 and a PcG core complex (PCC, which contains only PcG proteins) blocks RNA pol II but does not inhibit VP16 binding. Interestingly, this repression activity requires a nucleosomal template, suggesting that chromatin organizes this level of regulation (King et al., 2002). The chromatin model for PcG repression has gone as far as the ones for heterochromatin formation. Namely, RNA pol II complex and RNA synthesis machinery are unexpectedly structural components of PcG repressed promoters. In particular, core promoters appear to be the key target of PcG silencing (Figure 1). In this context it is important to note that, with the exception of EZH, none of the other PcG proteins has a function yet. It is likely that many other enzymatic activities are hidden in the PcG complex. These may include activities that target not only nucleosome components but also RNA pol II complex. It will be interesting to investigate how RNA metabolism and elongation are regulated in this scenario. Loss of Memory for a New Life? After embryogenesis, as morphogenesis and organogenesis are being completed, selector genes may un-

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Figure 1. Model for Promoter Reprogramming by PcG and trxG Proteins and Interaction with General Transcription Factors In the early Drosophila embryo, segmentation transcription factors determine homeotic gene promoter status. Either repression or activation involves interactions with GTFs at promoters. The memory system is preset at PREs and promoters and may readily interact with GTFs as transient determination system ceases its action. Repressed promoters are marked by the early ESC-E(Z) complex that sets the nucleosomal stage for the long-term memory PRC1 complex. Meanwhile, PC binding matures by spreading locally around PREs and promoters (blue objects). GTFs and a functional RNA pol II complex remain engaged with the core promoter that retains the possibility to switch. PcGs block RNA pol II constitutively by inhibiting elongation. Conversely, maintenance of the active state is set by TRX-HAT and ASH1 that create an anti-PRC1 epigenetic code. TRX, in combination with the BRM-complex, maintains the active state by an antirepression mechanism.

dergo transient reactivation or repression, depending on an appropriate combination of stimuli. Hox genes are necessary for establishing developmental axes or increasing in number specific cell populations. Therefore, it may happen that Hox genes are transiently reactivated and promptly rerepressed according to their epigenetic mark. Thus, competence either for silencing or activation should be characteristic of Hox and other developmentally regulated promoters. Competence for switching is restricted and relies on the history of that particular gene, perhaps in the light of which factors or epigenomic imprint the gene has encountered (Maurange and Paro, 2002). In this context, the engagement of the RNA pol II complex with PcG reinforces the epigenetic potential of the memory system. In principle, this equilibrium, although potentially risky, makes genes and genomes still amenable to changes, thus more plastic and prone to respond to developmental signals and to possess high potential for cell reprogramming. It has to be emphasized that the long-term memory system not only freezes extreme OFF and ON states but also determines the levels of expression of target genes. In flies, for example, PcG proteins are ubiquitously ex-

pressed and control their own expression (Fauvarque et al., 1995). In mammals, overexpression of EZH2 in tumor cells represses a number of other PcG genes (Varambally et al., 2002). Thus, any perturbation of the programmed levels of silencing components may have a dramatic impact on cell identity. Remarkably, targeted reduction of EZH2 levels by RNAi inhibited cell proliferation, suggesting that EZH2 hyperdosage is directly correlated to tumor malignancy (Varambally et al., 2002). Other PcG genes, like the Myc cooperating gene BMI-1, have been implicated in tumor progression (Bea et al., 2001; van Kemenade et al., 2001; reviewed in Jacobs and van Lohuizen, 2002). Thus, it is likely that PcG participates in a crucial checkpoint that controls cell proliferation. In flies, E(Z) has a role not only in regulating homeotic genes but also in cell proliferation, though the connection with cell cycle genes is not clear (Jones and Gelbart, 1990). Recently, specific murine PcG and trxG gene products have been shown to be involved in cell proliferation control, by interacting with retinoblastoma (Rb), p53, and E2F transcription factors (Dunaief et al., 1994; Jacobs et al., 1999; Dahiya et al., 2001). In particular,

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PcG complexes seem to participate in the Rb repressive pathway that includes histone deacetylase (Luo et al., 1998; Magnaghi-Jaulin et al., 1998). Moreover, PcG proteins have been shown to repress E2F and Myc responsive genes in G0 cells (Ogawa et al., 2002). Concerning just the Rb and PcG pathway, BMI-1 represses p16(Ink4), a negative regulator of cell proliferation (Jacobs et al., 1999). Deregulation of Hox genes is one of the hallmarks of both Drosophila and mammalian PcG and trxG mutant phenotypes. Overexpression of Hox genes has been correlated to cell proliferation and an increase in stem cell population (Antonchuk et al., 2002; Thorsteinsdottir et al., 2002). An attractive hypothesis could be that specific PcG proteins may be epistatic in the control of other PcG members. Overexpression of the former (e.g., EZH2) may downregulate global PcG content and lead to Hox derepression. Hyperexpression of certain Hox genes may in turn impact cell cycle genes. A complete panel of direct PcG (and Hox) target genes in vivo will have to be determined to unravel the genetic network that maintains the delicate compromise between postmitotic and proliferating states. An important link between PcG, Hox cluster regulation, and mammalian development has been recently reported. PLZF, the promyelocytic leukemia zinc finger protein, was shown to directly bind specific cis-elements in the mouse HoxD cluster (Barna et al., 2002). PLZF functions as a transcriptional repressor through its ability to recruit corepressors such as SMRT, N-CoR, Sin3, and HDACs (see Barna et al., 2002, and references therein). PLZF directly binds BMI-1 and recruits PcG proteins in the HoxD cluster. Remarkably, in limb development, PLZF and PcG antagonize specific posterior activating signals such as retinoic acid and Shh, thus determining anterior expression boundaries of HoxD genes (Barna et al., 2002). In APL (acute promyelocytic leukemia), the PLZF gene fuses to the RAR␣ gene (retinoic acid receptor; Rego and Pandolfi, 2002). As PLZFRAR␣ acts in a dominant fashion over PLZF, this may induce aberrant expression of Hox genes that could impact leukemogenesis. Finally, large-scale selective silencing appears to occur as totipotent cells undergo determination and terminal differentiation programs. Global changes in nuclear and chromatin structure are classical parameters that accompany this process. This appears to strongly influence reprogramming of somatic nuclei in trans-determination experiments. Thus, silencing and memory systems may represent primary determinants for cell identity. As the molecules that maintain cell identity appear to shape the genome and in particular chromatin structure, the fascinating possibility exists to reprogram perhaps even terminally differentiated cells by tackling their memory system. References Akhtar, A., Zink, D., and Becker, P.B. (2000). Chromodomains are protein-RNA interaction modules. Nature 407, 405–409. Antonchuk, J., Sauvageau, G., and Humphries, R.K. (2002). HOXB4induced expansion of adult hematopoietic stem cells ex vivo. Cell 109, 39–45. Armstrong, J.A., Papoulas, O., Daubresse, G., Sperling, A.S., Lis,

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