epigenetic regulation of cellular memory by the ... - Semantic Scholar

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Annu. Rev. Genet. 2004. 38:413–43 doi: 10.1146/annurev.genet.38.072902.091907 c 2004 by Annual Reviews. All rights reserved Copyright First published online as a Review in Advance on July 8, 2004

Annu. Rev. Genet. 2004.38:413-443. Downloaded from arjournals.annualreviews.org by University of Science & Technology of China on 11/17/08. For personal use only.

EPIGENETIC REGULATION OF CELLULAR MEMORY BY THE POLYCOMB AND TRITHORAX GROUP PROTEINS Leonie Ringrose and Renato Paro ZMBH, University of Heidelberg, 69120 Heidelberg, Germany; email: [email protected]; [email protected]

Key Words PcG, TrxG, chromatin, silencing, activation ■ Abstract During the development of multicellular organisms, cells become different from one another by changing their genetic program in response to transient stimuli. Long after the stimulus is gone, “cellular memory” mechanisms enable cells to remember their chosen fate over many cell divisions. The Polycomb and Trithorax groups of proteins, respectively, work to maintain repressed or active transcription states of developmentally important genes through many rounds of cell division. Here we review current ideas on the protein and DNA components of this transcriptional memory system and how they interact dynamically with each other to orchestrate cellular memory for several hundred genes.

CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PcG AND TrxG PROTEINS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PcG and TrxG Proteins: Evolutionary Similarities and Differences . . . . . . . . . . . . . PcG Protein Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TrxG Protein Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TARGETS AND PRE DESIGN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Target Genes Controlled by the PcG and TrxG Proteins . . . . . . . . . . . . . . . . . . . . . . PRE Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . INTERACTION OF THE PcG AND TrxG PROTEINS WITH THEIR TARGETS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Finding the Target . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Assessing the Activity of the Promoter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Taking the Relay: Installing Silenced or Active States . . . . . . . . . . . . . . . . . . . . . . . Remembering Silence: The Challenge of Cell Division . . . . . . . . . . . . . . . . . . . . . . IMPLICATIONS FOR MAMMALIAN CELLULAR PLASTICITY AND REPROGRAMMING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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INTRODUCTION At the core of development lies the specialization of cells that make up an organism. Cellular proliferation, and thus an increase in size, is the accompanying process, yielding a fully grown organism ready for life. Specialization is most prominently visualized by the differential gene-expression profiles established during the patterning processes. Thus, in order to grow and maintain a specialized state, the particular configurations of gene expression need to be transmitted to daughter cells in the cell lineages. Experiments in the 1960s and 1970s by Hadorn and coworkers demonstrated that cells have an inherent “cellular memory” allowing them to maintain developmental programs determined early in embryogenesis for the rest of development (44). In Drosophila, imaginal discs are cell clusters that are determined during embryogenesis for a particular fate. Upon passage through metamorphosis during the pupal stage, the early determined cellular program is implemented, and the cells differentiate into the external adult structures. Hadorn demonstrated that imaginal discs can be induced to proliferate by prolonged cultivation in the hemolymph of the female abdomen. After retransplantation into larvae undergoing metamorphosis, the imaginal discs form the appropriate structures and appendages for which they were initially programmed. Thus, even in a foreign environment and after more cell divisions than normally encountered during ontogeny, the imaginal disc cells still remember their initial determined state. What is the molecular mechanism that maintains determined states? This question, reduced to the molecular level, inquires how the differential gene-expression patterns defining the specific cell types are maintained during DNA replication and at mitosis. Drosophila melanogaster is an ideal model organism to study such a fundamental question of developmental biology as many of the genes involved in defining developmental states have been characterized. Genetic analyses uncovered, for example, the Hox genes, the highly conserved class of regulators defining the positions of structures and appendages along the anterior-posterior axis. Mutations in Hox genes transform one body segment into the identity of another. However, similar phenotypes were observed in a large class of other mutants that were apparently involved in regulating Hox genes. Detailed genetic and molecular studies demonstrated that these genes, which could be classified into the two antagonizing groups Polycomb (PcG) and trithorax (trxG), were required to maintain gene-expression patterns of important developmental regulators like the Hox genes during cellular proliferation. Thus, the PcG and TrxG proteins appear to form the molecular basis of the cellular memory.

PcG AND TrxG PROTEINS The first Polycomb group (PcG) mutations, extra sex combs (esc) and Polycomb (Pc), were identified in Drosophila in the 1940s. As their names suggest, these mutations cause additional sex combs on the second and third legs of males, instead of only on the first leg, where they usually belong. This phenotype is caused

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by loss of correct Hox gene repression, leading to the transformation of one body segment into the identity of another (63, 64). To date, 18 genes of the Polycomb group have been identified (Supplementary Table 1, follow the Supplemental Material link from the Annual Reviews home page at http://www.annualreviews.org), and many of their products have been characterized (Figure 1). It was not until the 1980s that the genes belonging to the trithorax group (trxG) began to be identified as suppressors of the extra sex comb phenotype. Seventeen genes that fall into this class are listed in Supplementary Table 1 and its legend. Later molecular analysis showed that many of the proteins encoded by the PcG and trxG act in large complexes and modify the local properties of chromatin to maintain transcriptional repression (PcG) or activation (TrxG) of their target genes (Figure 1).

PcG and TrxG Proteins: Evolutionary Similarities and Differences The PcG and TrxG proteins have been fairly well conserved throughout evolution. Figure 1 shows some examples of Drosophila proteins and their mammalian counterparts. Many of these proteins share functional domains but there are also many divergent regions. Perhaps the most stringent test of functional homology is the “rescue” assay, which tests whether a mammalian protein can compensate for a mutation in the corresponding Drosophila gene. Such experiments have shown that cDNAs encoding the mouse Pc homolog (M33) (88), the mouse polyhomeotic (ph) homolog (mph1) (L. Ringrose, unpublished), and the human pleiohomeotic (pho) homolog (YY1) (3) can rescue some aspects of the mutant phenotype for the corresponding Drosophila gene. The fly and mouse ESC/EED proteins are highly conserved (Figure 1), but surprisingly, the mouse EED protein does not rescue the esc mutant phenotype; instead, it acts as a dominant negative (127). Finally, the human MLL gene, which shares some homology with Drosophila trithorax (trx) (Figure 1), gave partial rescue of the Drosophila trx mutant phenotype caused by a truncation of the trx gene that deletes this homologous region (91a). Thus vertebrate proteins can, to some extent, substitute for their fly counterparts; however, none of the mouse proteins has been shown to give full rescue of lethality. This may be due to the fact that the mouse and fly proteins share only limited homology (Figure 1). Alternatively, incomplete rescue may be due to the expression strategies used for the rescue constructs, none of which were expressed from the endogenous Drosophila promoter for the gene in question. A striking difference between Drosophila and vertebrates is the lack of known vertebrate homologs for three sequence-specific DNA-binding proteins that operate with the PcG or TrxG: GAF, Pipsqueak (PSQ), and Zeste (Figure 1). In Drosophila, GAF and PSQ bind to the same DNA sequence (GA)n. PSQ behaves genetically as an enhancer of PcG phenotypes (47, 52), whereas GAF is encoded by the trxG gene Trithorax-like (Trl). In vertebrates, the sequence (GA)n is essential for the function of many promoters, and activities that bind it have been observed

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in mouse embryonic extracts (11), but the molecular identity of the binding factors has proved elusive. Perhaps we should not expect a protein with similarity to GAF or PSQ: In Drosophila, these proteins both recognize the same sequence through unrelated domains (Figure 1). Vertebrates may use yet another domain to recognize this sequence. Whether such a sequence is important for vertebrate PcG/TrxG function remains to be seen. Figure 1 shows that the proteins that are best conserved between Drosophila and vertebrates are those in the group consisting of E(Z), ESC, and SU(Z)12. Stepping evolutionarily further afield, this also holds true in the worm: Caenorhabditis elegans fulfills PcG function using homologs of E(Z) and ESC, and other proteins that appear to have arisen independently (Supplementary Table 1), (100 and references therein). Remarkably, plants also have homologs to E(Z), ESC, and SU(Z)12, but homologs to other PcG proteins have not been identified in the Arabidopsis genome (50).

PcG Protein Complexes A very fertile field of activity in recent years has been the biochemical purification of PcG complexes, from both Drosophila and human sources (Figure 1). We discuss the functions of PcG complexes in more detail below; here we give an overview. The core components of the 1–2-MDa PRC1 complex (Polycomb Repressive Complex 1) are similar in Drosophila and humans, although the Drosophila complex, purified from 0- to 12-hour-old embryos, contains additional accessory proteins, notably Zeste, SCM, and several transcription factors (113). The human complex was purified from HeLa cells and appears to contain far fewer accessory proteins (73). Otte & Kwaks (95) point out that this may be due to the fact that HeLa cells are a poor source of PcG proteins. The Drosophila core PRC1 complex and its mouse counterpart can both be reconstituted by coexpression of four proteins: PC, PH, PSC, and RING in insect cells (39, 70). Importantly, although this core PRC1 complex can block nucleosome remodeling on a chromatin template in vitro, it has no preference for DNA sequences that contain PREs (PcG Response Elements, the sequences that are bound by the PcG in vivo). Inclusion of the Zeste protein by reconstitution enables increased binding to Zeste sites, which are found in PREs (87). The target sites of GAF and PHO are also found in PREs, and both proteins can be coimmunoprecipitated with PcG proteins (107). However, neither GAF nor PHO is found in PcG complexes purified biochemically, suggesting that these interactions may be transient and do not survive purification. A second group has purified the CHRASCH complex (Chromatin associated silencing complex for homeotics) from Drosophila Schneider cells (51, 52). This complex is related to PRC1 (Figure 1) but has an important difference: It contains the PcG protein Pipsqueak (PSQ), which allows the complex to bind specifically to DNA targets from PREs that contain the (GA)n motif. The CHRASCH complex contains a histone deacetylase (HDAC1) in addition to the PC, PH, and PSQ proteins. The differences between CHRASCH and PRC1 may be due to differences

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in PcG composition between cells in culture and embryos, which underlines the importance of the source of material from which complexes are purified. Over the past two years, several groups have independently isolated a second 600-KDa complex, PRC2 (Figure 1). All versions of PRC2, whether from Drosophila embryos (30, 89, 123, 124) or human HeLa cells (19, 67), contain the same three core proteins [ESC, E(Z), and SU(Z)12 (Figure 1)] and a small number of additional proteins, depending on the purification procedure used. In particular, the association of the histone deacetylase, RPD3, is somewhat disputed [for a review of this debate, see (95)]. All PRC2 complexes can methylate lysines 9 and/or 27 on histone H3. This complex may be more evolutionarily ancient than PRC1, as its core members are conserved in plants and in C. elegans. In summary, the purification of PcG complexes identifies minimal subsets of proteins that associate stably with each other and that interact with chromatin components. These stable associations are evolutionarily conserved. However, coimmunoprecipitation studies identify additional interactions (reviewed below), and thus the complexes that survive purification indicate which are the most robust interactions, but they do not tell the whole story. There is strong evidence that the function and composition of these core complexes in vivo is modulated in different tissues (95) and at different target genes (109, 122). Such different properties may be endowed by the different accessory proteins found in different purified preparations and by the many other PcG proteins that interact genetically.

TrxG Protein Complexes Whereas the PcG proteins seem to be dedicated to their target genes, many of the TrxG proteins form complexes that are involved in general transcriptional processes; thus, their function is not limited to epigenetic maintenance (28, 120). Exceptions are the TRX and ASH1 proteins, which are involved more specifically in regulation at PREs (26, 112). Four complexes that contain TrxG proteins have been purified from Drosophila embryos, all with different chromatin-modifying properties. These complexes are reviewed in detail in (119); here we give an overview and an update on recently identified enzymatic activities. The 2-MDa BRM complex (Figure 1) contains the TrxG proteins Brahma (BRM), Moira (MOR), and OSA, and at least four other accessory proteins (27, 96). The BRM complex is highly related to the yeast SWI/SNF nucleosome-remodeling complex, and the BRM protein (SWI2/SNF2 in yeast) functions as the ATPase subunit of this complex, using the energy of ATP hydrolysis to move histones. Multiple related complexes are found in humans (96 and references therein) (examples of homologs are shown in Figure 1). Recently, two distinct versions of the BRM complex have been identified in Drosophila (85). Two other Drosophila complexes of 2 MDa and 500 kDa, respectively, contain the ASH1 and ASH2 TrxG proteins (96). The fourth known Drosophila TrxG complex, TAC1 (99), of 1 MDa, contains the TrxG protein TRX, the histone acetyltransferase CBP (CREB binding protein), and the antiphosphatase Sbf1. The TRX

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protein contains a SET domain, which is highly similar to that of the yeast protein, SET1. The S. cerevisiae SET1 protein was the first histone methyltransferase shown to methylate lysine 4 on histone H3 (H3K4) (111a). This, together with the demonstration that SET1 is in a complex with a yeast homolog of ASH2, gave the first indication that TrxG function in higher eukaryotes may involve H3K4-specific methyltransferases (111a). Indeed, the SET domain containing proteins ASH1 and TRX were both subsequently shown to be histone methyltransferases, primarily targeting H3K4 (7, 120). H3K4 methylation is typically associated with active chromatin (68). Interestingly, an additional Drosophila protein (CG17395) with extensive similarity to SET1 in the SET domain and other regions, was unearthed in the yeast study (111a). This protein may be a novel TrxG methyltransferase and certainly merits further investigation. In summary, the TrxG complexes thus far identified all contain enzymatic activities that help to activate transcription by modifying chromatin properties.

TARGETS AND PRE DESIGN The target genes of the PcG and TrxG proteins carry cis-regulatory elements that enable both the PcG and TrxG to bind and to maintain the status of transcriptional activity of the gene over many cell generations. These elements have dual function as Trithorax and Polycomb response elements, and have been termed PREs [PcG Response Elements (22)], PRE/TREs [Polycomb/trithorax response elements (111, 125)], and CMMs [cellular memory modules (20)]. Here we use the term PRE.

Target Genes Controlled by the PcG and TrxG Proteins The PcG proteins bind to about 100 sites in polytene chromosomes from larval salivary glands (109, 130). The TrxG proteins TRX and ASH-1 bind to many of these sites, and about 80 more in addition (26, 112, 126). Thus in this tissue alone, about 180 loci are bound and may be regulated by the PcG and TrxG proteins. However, the low resolution of polytene mapping (about 100 kb), together with the complex nature of PRE sequences, means that finding PREs and the genes they regulate has relied upon functional assays, which have identified PREs at five loci: the bithorax (BX-C) and Antennapedia (ANT-C) complexes containing the homeotic genes (94 and references therein; 43, 129), the polyhomeotic locus [itself encoding a PcG gene (12)], the engrailed locus (61), and the hedgehog locus (77). In mammals, we know rather less. No PRE elements have so far been identified, but some target genes are known: Similarly to Drosophila, the vertebrate PcG and TrxG proteins act antagonistically on the Hox genes (46). However, they also intervene in the control of cellular proliferation and tumorigenesis (57), a function not yet described in Drosophila. We recently developed an algorithmic approach to identify PREs and their associated genes in the Drosophila genome (111). This analysis predicted 167 PRE sequences, many of which we verified by experimental testing. However, in

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an algorithmic approach such as this, it is important to evaluate both the sensitivity of the algorithm (how many of the real PREs in the genome were found?) and the selectivity (how many of the predictions are real?). We ensured selectivity by setting a stringent cutoff score for the statistical significance of our predictions in the context of the whole genome. This has costs for sensitivity: In the genome search, several experimentally defined PREs that had been detected robustly in smaller data sets escaped detection in the genome, because they scored lower than this minimum cutoff score. Comparison of our predictions with cytological PcG and TrxG binding sites suggests that about half of the PREs in the genome may be missing from the list of 167 candidates. Nevertheless, the identities of the genes associated with those PREs that were found give insights into the wide spectrum of pathways that are regulated by the PcG and TrxG proteins. Here we review target genes for which there is experimental evidence from other sources. In the early Drosophila embryo, a hierarchy of genes divides the body along its length into ever smaller domains. This hierarchy begins with the maternal coordinate genes, whose graded patterns are interpreted into repetitive patterns by three levels of the segmentation genes: the gap, pair rule, and segment polarity genes. The hierarchy ends with the homeotic genes, which give to each parasegment its individual identity (71). The homeotic genes are the best-documented targets of PcG and TrxG regulation. Homeotic gene products are required in each segment throughout development and adult life, and their early domains of activation or repression are maintained in every subsequent cell generation by the PcG and TrxG. In contrast, many of the earlier segmentation gene products that determine homeotic gene expression fade away after a few hours of development and are used in other tissues later on. Interestingly, previous genetic studies have shown that the gap genes hunchback, knirps, and giant and the pair rule gene even skipped are regulated directly or indirectly by the PcG during early embryogenesis (78, 98). Furthermore, PREs have been defined in the segment polarity genes engrailed (61) and hedgehog (77). Our genome-wide analysis (111) predicted PREs in several of these known target genes and in many additional segmentation genes at all levels of the hierarchy. A scan of PRE scores in the segmentation genes revealed three classes: (a) those with a high PRE score, well above the cutoff at 157 (knirps, tailless, even skipped, Tenascin-major, engrailed, and armadillo); (b) those with a score of 85 to 156, comparable to a number of known PREs; these are potential candidates for regulation (hunchback, hairy, wingless, fused, gooseberry, and patched); (c) those with a score below 80; these are less likely to be directly regulated by the PcG and TrxG, or may have PREs with different sequence composition. The finding that so many genes in the segmentation hierarchy may be regulated by the PcG and TrxG is surprising and indicates that the well-characterized transcription factor cascades may be backed up by chromatin mechanisms at every level of the hierarchy, and that epigenetic control exerted by the PcG and TrxG both begins earlier and is more dynamic than previously expected. In addition, the PREs

BEFORE THE HOMEOTICS: THE SEGMENTATION GENES

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at these genes might be used for the control of functions at later developmental stages. Later in development, cell identities are established by patterning of imaginal discs, which are the larval precursors of adult structures. We predict a surprisingly large number of PREs in gene networks that are involved in this patterning (111), suggesting again that decisions made by transcription factors may be backed up by chromatinmediated mechanisms, which may operate for a limited number of cell divisions, before the target gene is reprogrammed. For example, we predicted PREs in ten genes that have a role in eye development, and we have shown that one of these PRE fragments (from eyes absent) behaves as a PRE in transgenic assays (111). However, the mere fact of possessing a PRE does not give any information about the developmental process in which the PRE is deployed. Unraveling which PREs function when and where is far from trivial. In PcG mutants, misregulation of genes that cause early lethality may mask a later role. A recent screen for genes that are required for differentiation within the eye imaginal disc, independent of their requirements for viability, has identified specific roles for the PcG and trxG in eye development (58). The authors show that the effects of PcG and trxG mutants are not simply due to the misregulation of the known PcG/TrxG target Ubx in this tissue, and conclude that the PcG and TrxG proteins have other direct targets in the eye disc. Our predictions suggest that of those reported by the authors, direct targets include the homothorax, eyes absent, and dachshund genes but not eyeless and teashirt, whose sequences contain no detectable PRE characteristics. Another class of genes for which experimental evidence has recently appeared consists of those involved in oogenesis (92). We predicted PREs in 11 genes with a role in the ovary (111), but again, it was not clear if the PcG and TrxG actually regulate development of this tissue. A gain-of-function screen for genes that play a role in ovarian follicle development (92) identified polyhomeotic, and subsequent analysis demonstrated that the PcG genes Scm and Sce are also involved, but no effects of Psc, Su(z)2, Asx, or Pcl were observed. Importantly, the authors demonstrate that PcG function is limited to the somatic cells, where it is required for both proliferation and differentiation, but does not operate in the germline. This work thus underlines the exquisite tissue-specificity of PcG function and suggests that a specific subset of PcG proteins operates in these cells.


FROM HEAD TO TAIL: PREs IN EYE AND OVARY DEVELOPMENT

The above examples suggest that the PcG and TrxG operate on genes at every stage of development, and thus they may be major regulators of cell identity. Two other kinds of targets suggest that the PcG and TrxG may also impinge on the organization of chromosomes and on the cell cycle. Boivin et al. (13) have shown that some telomeres (the ends of chromosomes) contain PRE-like sequences that do not appear to regulate endogenous genes. By showing that PcG proteins associate with these sequences and that the PcG and trxG interact genetically with telomeric silencing, the authors

OTHER TARGETS: TELOMERES AND TUMOR SUPPRESSORS

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identify a role for the PcG and TrxG proteins distinct from their role in regulating developmental genes. A link between the PcG and telomeric function also exists in humans, where the PcG member BMI-1 induces telomerase activity, thereby extending the lifespan of mammary epithelial cells (33). This is not a lone example: Several vertebrate members of the PcG and TrxG play a role in the control of cellular proliferation and tumorigenesis, although very few of the target genes have been identified (reviewed in 57). In Drosophila, we predict PREs in two tumor suppressors [lethal(2), giant larvae, and proliferation disrupter] and two p53-like transcription factors (bifid and H15). Thus the effects of the PcG and TrxG on proliferation may be conserved in flies and vertebrates. Aside from the implications of such control for cancer, the possibility that the PcG and TrxG may directly regulate cell proliferation checkpoints implies that during normal development, the coordinated processes of differentiation and proliferation may be subject to epigenetic control.

PRE Design What makes a PRE? Recent advances bring us closer to the answer to this question, both in terms of the configuration of grouped PREs at endogenous loci and of stripping PREs down to their bare essentials. The well-characterized PREs of the homeotic loci do not work alone. Each homeotic gene is controlled by two or more PRE elements, which are accompanied by tissue-specific enhancers and boundary elements (5, 79, 101, 118). There are also two PREs at the polyhomeotic locus, which appear to differentially regulate the two polyhomeotic transcription units, phP and phD (12). Using PRE prediction, we found that 90% of predicted high-scoring PREs are accompanied by other PRE peaks nearby (111). Without exception, we found a peak at or near the promoter of PRE containing genes. In the rare cases where we found a lone PRE, it was, without exception, at or near (within 800 bp) of the promoter. Almost all of these lone PREs were associated with short genes (