Enhancer of zeste
The CURLEY LEAF gene of Arabidopsis is necessary for stable repression of a floral homeotic gene (AGAMOUS) required to specify stamen and carpel identity in whorls 3 and 4 respectively. AGAMOUS is a MADS box protein (see Drosophila Serum response factor and MEF2). The CURLEY LEAF protein shows extensive homology to Enhancer of zeste, with three regions conserved between the two proteins. First, the C-terminus contains a 115 amino-acid region, the SET domain, previously recognized as a conserved region in the products of E(z), Trithorax and Suppressor of variegation 3-9. Second, residues 655-720 of CLF show 46% identity with the region of E(Z) (residues 538-603) that are rich in cysteine residues, but with an arrangement unlike that of zinc fingers. Third, residues 270-317 of CLF contain seven cysteines with a similar spacing to a region of E(Z) (residues 321-367) containing five cysteines, and there is a small region of sequence similarity at the N termini of these regions (Goodrich, 1997).
The Polycomb-group (PcG) proteins MEDEA (MEA; encodes a Polycomb-group (PcG) protein for which the closest Drosophila homolog is Enhancer of Zeste), FERTILIZATION INDEPENDENT ENDOSPERM, and FERTILIZATION INDEPENDENT SEED2 regulate seed development in Arabidopsis by controlling embryo and endosperm proliferation. All three of these FIS-class proteins are likely subunits of a multiprotein PcG omplex, which epigenetically regulates downstream target genes. The MADS-box gene PHERES1 (PHE1) is commonly deregulated in the fis-class mutants. PHE1 belongs to the evolutionarily ancient type I class of MADS-box proteins that have not yet been assigned any function in plants. Both MEDEA and FIE directly associate with the promoter region of PHE1, suggesting that PHE1 expression is epigenetically regulated by PcG proteins. PHE1 is expressed transiently after fertilization in both the embryo and the endosperm; however, it remains up-regulated in the fis mutants, consistent with the proposed function of the FIS genes as transcriptional repressors. Reduced expression levels of PHE1 in medea mutant seeds can suppress medea seed abortion, indicating a key role of PHE1 repression in seed development. PHE1 expression in a hypomethylated medea mutant background resembles the wild-type expression pattern and is associated with rescue of the medea seed-abortion phenotype. In summary, these results demonstrate that seed abortion in the medea mutant is largely mediated by deregulated expression of the type I MADS-box gene PHE1 (K&oml;hler, 2003).
A feature shared by some Trithorax family members (in Drosophila Trithorax and Enhancer of zeste), and by other proteins that function in chromatin-mediated transcriptional regulation, is the presence of a 130- to
140-amino acid motif dubbed the SET or Tromo domain. Analysis is presented of SET1, a yeast
member of the trithorax gene family that encodes a 1080-amino
acid protein with a C-terminal SET domain. In addition to its SET domain, which is 40-50% identical to
those previously characterized, SET1 also shares dispersed but significant similarity to Drosophila and
human trithorax homologs. To understand SET1 function(s), a null mutant was created. Mutant strains,
although viable, are defective in transcriptional silencing of the silent mating-type loci and telomeres. The
telomeric silencing defect is rescued not only by full-length episomal SET1 but also by the conserved
SET domain of SET1. set1 mutant strains display other phenotypes, including morphological
abnormalities, stationary phase defects, and growth and sporulation defects. Candidate genes that may
interact with SET1 include those with functions in transcription, growth, and cell cycle control. These
data suggest that yeast SET1, like its SET domain counterparts in other organisms, functions in diverse
biological processes including transcription and chromatin structure (Nislow, 1997).
A unique and essential feature of germ cells is their immortality. In Caenorhabditis elegans, germline immortality requires the maternal contribution from four genes, mes-2, mes-3, mes-4 and mes-6. mes-2 encodes a protein similar to the Drosophila Polycomb group protein, Enhancer of zeste. Sequence analysis of the mes-2 cDNA revealed that it encodes a 773 amino acid protein. Most of the similarity between MES-2 and E(z) lies in two domains found in the carboxy-terminal half of each protein. MES-2 and E(z) are 57% identical at the amino acid level in a motif of ~115 amino acids, named the SET domain for three Drosophila proteins: Suppressor of variegation 3-9 [Su(var)3-9]. The fact that SET domains have been found in proteins that are not otherwise similar and in organisms as diverse as plants, yeast and mammals suggests that the SET domain represents an important functional motif. While the function of the SET domain is not known, all characterized SET domain proteins associate with chromatin. Currently there is no evidence that this domain binds to DNA directly; instead it has been suggested that SET-domain-containing proteins associate with their targets through protein-protein interactions. Consistent with the SET domain being an important functional motif, molecular lesions have been identified in highly conserved SET domain residues in two alleles of mes-2 (Holdeman, 1998).
It is thought that Polycomb group proteins form heteromeric complexes and control gene expression by altering chromatin conformation of target genes. As predicted from its similarity to a Polycomb group protein, MES-2 localizes to nuclei. MES-2 is found in germline nuclei in larval and adult worms and in all nuclei in early embryos. By the end of embryogenesis, MES-2 is detected primarily in the two primordial germ cells. The correct distribution of MES-2 requires the wild-type functions of mes-3 and mes-6. It is hypothesized that mes-2 encodes a maternal regulator of gene expression in the early germline; its function is essential for normal early development and the viability of germ cells (Holdeman, 1998).
The current working model for MES protein function is that a maternal supply of wild-type MES proteins is required for proper chromatin structure and gene expression in the nascent germline. Germline development is known to require maternal PIE-1, a transcriptional repressor that keeps somatically expressed genes turned off in the germline of early embryos. Several somatically expressed genes were tested for ectopic expression in the germlines of mes mutants and no evidence is seen for ectopic expression. This argues that the mes gene products act neither in concert with the PIE-1- mediated system, nor as a continuation of it. It is hypothesized that after PIE-mediated repression is lifted, the mes genes participate in establishing or maintaining germline chromatin in a conformation that ensures the proper pattern of gene expression when the germline starts its developmental program in early larvae. The chromosomes in the primordial germ cells of embryos and young larvae display a unique morphology, which is retained in the germ cells of mes mutants. This suggests that the MES proteins do not operate at the level of controlling gross chromosome morphology. Kelly and Fire (1998) find support for an involvement of the MES proteins in controlling some aspect of germline chromatin organization and gene expression: expression of transgenes present in many copies in extrachromosomal arrays is silenced in the germlines of wild-type worms, but desilenced in the germlines of mes mutants. Desilencing can also be achieved in wild-type germlines by placing the transgenes in the context of additional, complex DNA in the array. These findings suggest that the MES+ system participates in keeping at least some genes silenced in the germline, by having an effect on the state of chromatin. It is speculated that defects in the MES system result in aberrant patterns or levels of gene expression, leading to death of the germline (Holdeman, 1998).
Evolutionary conservation of the Extra sex combs-E(z) partner relationship is supported by recent studies of two Caenorhabditis elegans maternal-effect sterile genes, mes-2 and mes-6. Mutant alleles of either mes-2 or mes-6 produce grandchildless phenotypes that result from the limited proliferation and death of germ cells. MES-2 and MES-6 share sequence similarity with E(z) and Esc, respectively, across extensive portions of these proteins. However, unlike E(z) and Esc functions, mes-2 and mes-6 functions appear to be restricted to the germ line; thus far, there is no evidence for their involvement in Hox gene control. Thus, the developmental roles of MES-2 and MES-6 in worms are distinct from those of their E(z) and Esc homologs in flies and mammals. Nevertheless, there is evidence that the basic biochemical partnership between these proteins has been conserved. The spatial and temporal patterns of MES-2 and MES-6 accumulation in nuclei are identical, and mutations in either gene disrupt the spatial distribution or stability of the other protein. One of these mutations, mes-6bn66, substitutes a residue at a position that aligns with the predicted loop region of Esc that is required for binding to E(z) protein. Taken together, these results are consistent with a direct physical interaction between MES-2 and MES-6 in vivo. The conservation of these worm homologs, together with the evolutionary divergence of their developmental functions, may reflect a common biochemical role in chromatin that has been adapted for use in different cell lineages in worms and flies (Jones, 1998 and references therein).
The C. elegans maternal-effect sterile genes, mes-2, mes-3, mes-4, and mes-6, encode nuclear proteins that are essential for germ-line development. They are thought to be involved in a common process because their mutant phenotypes are similar. MES-2 and MES-6 are homologs of Enhancer of zeste and extra sex combs, both members of the Polycomb group of chromatin regulators in insects and vertebrates. MES-3 is a novel protein, and MES-4 is a SET-domain protein. To investigate whether the MES proteins interact and likely function as a complex, biochemical analyses were performed on C. elegans embryo extracts. Results of immunoprecipitation experiments indicate that MES-2, MES-3, and MES-6 are associated in a complex and that MES-4 is not associated with this complex. Based on in vitro binding assays, MES-2 and MES-6 interact directly, via the amino terminal portion of MES-2. Sucrose density gradient fractionation and gel filtration chromatography were performed to determine the Stokes radius and sedimentation coefficient of the MES-2/MES-3/MES-6 complex. Based on those two values, it is estimated that the molecular mass of the complex is ~255 kDa, close to the sum of the three known components. These results suggest that the two C.
elegans Polycomb group homologs (MES-2 and MES-6) associate with a novel partner (MES-3) to regulate germ-line development in C. elegans (Xu, 2001).
Twelve PcG proteins have been cloned so far in Drosophila, but only two of them, E(Z) and ESC, are conserved in worms and also in plants. These data suggest that these two proteins might be distinctive PcG members, which function independently of the other PcG proteins. In Drosophila, E(Z) and ESC are
associated in a complex in vivo and interact with each other directly in vitro. This association is conserved for their mammalian homologs, ENX and EED, and also for their worm homologs, MES-2 and MES-6 (Xu, 2001).
The portion of MES-2 that is important for its interaction with MES-6 in vitro is the N-terminal 194 aa. Similarly, sequences within the N-terminal region of fly E(Z) (amino acids 34-66) and the mouse E(Z) homolog (amino acids 132-160) are responsible for their interactions with ESC homologs. Although there is very
little sequence similarity between the N termini of MES-2 and E(Z) homologs, their tertiary structure and the nature of the interaction between the partners might be conserved. The ESC-binding region of E(Z) is predicted to include a long stretch of helix, and the N-terminal 194 aa of MES-2 is predicted to form multiple long helices (Xu, 2001).
The conservation of the interaction between E(Z) and ESC among worms, flies, and mammals suggests that the molecular mechanism by which these protein partners
function has been maintained throughout evolution. The MES-2/MES-6 complex therefore might regulate gene expression in C. elegans by the same mechanism
used by the E(Z)/ESC complex in Drosophila. Indeed, MES-2 and MES-6 participate in repressing gene expression in C. elegans, as E(Z) and ESC are known to do in Drosophila (Xu, 2001).
The Xenopus homolog of Drosophila Enhancer of Zeste has been identified using a differential display strategy designed to identify
genes involved in early anterior neural differentiation. XEZ codes for a protein of 748 amino acids that is very highly conserved in evolution
and is 96% identical to both human and mouse EZ(H)2. In common with most other Xenopus Pc-G genes and unlike mammalian Pc-G genes,
XEZ is anteriorly restricted. Zygotic expression of XEZ commences during gastrulation, much earlier than other anteriorly localized Pc-G
genes; expression is restricted to the anterior neural plate and is confined later to the forebrain, eyes and branchial arches. XEZ is induced in
animal caps overexpressing noggin; up-regulation of XEZ therefore represents a response to inhibition of BMP signaling in ectodermal cells.
The midbrain/hindbrain junction marker En-2, and hindbrain marker Krox-20, are target genes of XEZ and that XEZ functions to
repress these anteroposterior marker genes. Conversely, XEZ does not repress the forebrain marker Otx-2. XEZ overexpression results in a
greatly thickened floor of the forebrain. These results implicate an important role for XEZ in the patterning of the nervous system (Barnett, 2001).
Polycomb group (PcG) genes are required for stable inheritance of epigenetic states throughout development, a phenomenon termed cellular memory. In Drosophila and mice, the product of the E(z) gene, one of the PcG genes, constitutes the ESC-E(Z) complex and specifically methylates histone H3. It has been argued that this methylation sets the stage for appropriate repression of certain genes. This study reports the isolation of a well-conserved homolog of E(z), olezh2, in medaka. Hypomorphic knock-down of olezh2 resulted in a cyclopia phenotype and markedly perturbed hedgehog signaling, consistent with a previous report on oleed, a medaka esc. Cyclopia was also found in embryos treated with trichostatin A, an inhibitor of histone deacetylase, which is a transient component of the ESC-E(Z) complex. The level of tri-methylation at lysine 27 of histone H3 is substantially decreased in both olezh2 and oleed knock-down embryos, and in embryos with hedgehog signaling perturbed by forskolin. It is concluded that the ESC-E(Z) complex per se participates in hedgehog signaling (Shindo, 2005).
Drosophila Extra sex combs (Esc) and its mammalian homolog, embryonic ectoderm development (eed), are special Pc-G members; they are required early during development when
Pc-G repression is initiated, a process that is still poorly understood. To gain insight into the molecular function of Eed, the yeast two-hybrid method was used to find Eed-interacting proteins. Described is the specific in vivo binding of Eed to Enx1 and Enx2, two mammalian homologs of the essential Drosophila Pc-G gene Enhancer-of-zeste [E(z)]. No direct biochemical interactions are found between Eed/Enx and a previously characterized mouse Pc-G protein complex containing
several mouse Pc-G proteins, including mouse polyhomeotic (Mph1). This suggests that different Pc-G complexes with distinct functions may exist. However, and in support of a bridging role for Enx1, partial colocalization of Enx1 and Mph1 to subnuclear domains may point to more transient
interactions between these complexes (van Lohuizen, 1998).
If the observed interactions between Enx1, Enx2, and Eed are relevant for their in vivo function, one would expect the expression patterns to coincide during development. Since the onset of Eed expression possibly occurs earlier than for most other Pc-G genes, a sensitive, semiquantitative RT-PCR assay was used to monitor the expression patterns of Enx1 and Enx2 during development and in adult mice. Whereas no expression is detected in blastocysts, Enx1 is already highly expressed in day 7.3 mouse embryos and becomes gradually more restricted to specific organs during development, with the highest expression being in the testis. In contrast, Enx2 expression was first detected on day 9 and continues to be expressed at moderate levels in most tissues. These results indicate that there is indeed extensive overlap in onset and tissue distribution between Enx1, Enx2, and Eed: the onset of Enx1 expression parallels that of Eed, whereas in adult mice Enx2 and Eed are expressed in most tissues (van Lohuizen, 1998).
The Polycomb group proteins are involved in maintenance of the silenced state of several
developmentally regulated genes. These proteins form large aggregates with different subunit
compositions. To explore the nature of these complexes and their function(s), the full-length Eed
(embryonic ectoderm development) protein, a mammalian homolog of the Drosophila Polycomb
group protein Esc, was used as a bait in a yeast two-hybrid screen. Several strongly interacting cDNA clones
were isolated. The cloned cDNAs all encode the 150- to 200-amino-acid N-terminal fragment of the
mammalian homolog of the Drosophila Enhancer of zeste [E(z)] protein, Ezh2. The full-length Ezh2
binds strongly to Eed in vitro, and Eed coimmunoprecipitates with Ezh2 from murine 70Z/3 cell
extracts, confirming the interaction between these proteins that was observed in yeast. Mutations T1031A and
T1040C in one of the WD40 repeats of Eed, which account for the hypomorphic and lethal phenotype
of eed in mouse development, block binding of Ezh2 to Eed in a two-hybrid interaction in both yeast and
mammalian cells. These mutations also blocked the interaction between these proteins in vitro. In
mammalian cells, the Gal4-Eed fusion protein represses the activity of a promoter bearing Gal4 DNA
elements. The N-terminal fragment of the Ezh2 protein abolishes the transcriptional repressor activity
of Gal4-Eed protein when these proteins are coexpressed in mammalian cells. Eed and Ezh2 are also found
to bind RNA in vitro, and RNA alters the interaction between these proteins. These findings suggest
that Polycomb group proteins Eed and Ezh2 functionally interact in mammalian cells, an interaction that
is mediated by the WD40-containing domain of Eed protein (Denisenko, 1998).
X inactivation in female mammals is one of the best studied examples of heritable gene silencing and provides an important model for studying maintenance of patterns of gene expression during differentiation and development. The process is initiated by a cis-acting RNA, the X inactive specific transcript (Xist). Xist RNA is thought to recruit silencing complexes to the inactive X, that then serve to establish and maintain the inactive state in all subsequent cell divisions. Most lineages undergo random X inactivation, there being an equal probability of either the maternally (Xm) or paternally (Xp) inherited X chromosome being inactivated in a given cell. In the extraembryonic trophectoderm and primitive endoderm lineages of mouse embryos, however, there is imprinted X inactivation of Xp. This process is also Xist dependent. A recent study has shown that imprinted X inactivation in trophectoderm is not maintained in embryonic ectoderm development (eed) mutant mice. Eed, the mammalian homolog of Drosophila Extra sex combs, and Enx1, the mammalian homolog of enhancer of Zeste, are directly localized to the inactive X chromosome in XX trophoblast stem (TS) cells. The association of Eed/Enx1 complexes is mitotically stable, suggesting a mechanism for the maintenance of imprinted X inactivation in these cells (Mak, 2002).
Drosophila suppressor of zeste 12 (Su(z)12) is a Polycomb group (PcG) transcriptional repressor and is present in E(z)-ESC, a multiprotein complex with methylation activity specific for lysine 9 and 27 of histone H3. Although PcG- and heterochromatin-mediated gene silencing have been considered distinct, mutant flies of Su(z)12 show not only homeotic transformation but also position effect variegation. The mammalian SU(Z)12 directly interacts with heterochromatin protein 1alpha (HP1alpha) and PcG enhancer of zeste 2 (EZH2), the mammalian counterpart of E(z), in vitro and in vivo. Two distinct domains in SU(Z)12 are involved in these interactions, the region between the zinc finger motif and the VEFS (VRN2-EMF2-FIS2-Su(z)12) box for HP1alpha (amino acid residues 479-536) and the VEFS box for EZH2 (amino acid residues 600-639), which are not mutually exclusive. Interestingly this region of the VEFS box has been shown to be critical for the phenotype of the Su(z)12 mutant fly. In addition SU(Z)12 represses transcription activity in the presence of HP1alpha in a reporter assay. These results provide a molecular explanation for the functional link of these epigenetic silencing processes mediated by Su(z)12 (Yamamoto, 2004).
Recent studies have revealed the intrinsic histone methyltransferase (HMTase) activity of the EED-EZH2 complex and its role in Hox gene silencing, X inactivation, and cancer metastasis. This study focuses on the function of individual components. It was found that the HMTase activity requires a minimum of three components -- EZH2, EED, and SUZ12 -- while AEBP2 is required for optimal enzymatic activity. Using a stable SUZ12 knockdown cell line, it has been shown that SUZ12 knockdown results in cell growth defects, which correlate with genome-wide alteration on H3-K27 methylation as well as upregulation of a number of Hox genes. Chromatin immunoprecipitation (ChIP) assay identified a 500 bp region located 4 kb upstream of the HoxA9 transcription initiation site as a SUZ12 binding site, which responds to SUZ12 knockdown and might play an important role in regulating HoxA9 expression. Thus, this study establishes a critical role for SUZ12 in H3-lysine 27 methylation and Hox gene silencing (Cao, 2004).
The importance of histone methylation by the polycomb group proteins was examined in the mouse circadian clock mechanism. Endogenous EZH2, a polycomb group enzyme that methylates lysine 27 on histone H3, co-immunoprecipitates with CLOCK and BMAL1 throughout the circadian cycle in liver nuclear extracts. Chromatin immunoprecipitation revealed EZH2 binding and di- and tri-methylation of H3-K27 on both the Period 1 and Period 2 promoters. A role of EZH2 in cryptochrome-mediated transcriptional repression of the clockwork was supported by overexpression and RNA interference studies. Serum-induced circadian rhythms in NIH 3T3 cells in culture were disrupted by transfection of an RNA interfering sequence targeting EZH2. These results indicate that EZH2 is important for the maintenance of circadian rhythms and extend the activity of the polycomb group proteins to the core clockwork mechanism of mammals (Etchegaray, 2006).
Polycomb-group (Pc-G) genes are required for the stable repression of the homeotic selector genes and other developmentally regulated genes, presumably
through the modulation of chromatin domains. Among the Drosophila Pc-G genes, Enhancer of zeste [E(z)] merits special consideration since it represents one of the Pc-G genes most conserved through evolution. In addition, the E(Z) protein family contains the SET domain, which has recently been linked with histone methyltransferase (HMTase) activity. Although E(Z)-related proteins have not (yet) been directly associated with HMTase activity, mammalian Ezh2 is a member of a histone deacetylase complex. To investigate its in vivo function, mice deficient for Ezh2 were generated. The Ezh2 null mutation results in lethality at early stages of mouse development. Ezh2 mutant mice either cease developing after implantation or initiate but fail to complete gastrulation. Moreover, Ezh2-deficient blastocysts display an impaired potential for outgrowth, preventing the establishment of Ezh2-null embryonic stem cells. Interestingly, Ezh2 is up-regulated upon fertilization and remains highly expressed at the preimplantation stages of mouse development. Together, these data suggest an essential role for Ezh2 during early mouse development and genetically link Ezh2 with eed and YY1, the only other early-acting Pc-G genes (O'Carroll, 2001).
Enhancer of zeste 2 (Ezh2), a SET domain-containing protein, is crucial for development in many model organisms, including early mouse development. In mice, Ezh2 is detected as a maternally inherited protein in the oocyte but its function at the onset of development is unknown. A conditional
allele of Ezh2 was used to deplete the oocyte of this maternal inheritance. The loss of maternal Ezh2 has a long-term effect causing severe
growth retardation of neonates despite 'rescue' through embryonic
transcription from the paternal allele. This phenotypic effect on growth could
be attributed to the asymmetric localization of the Ezh2/Eed complex and the
associated histone methylation pattern to the maternal genome, which is
disrupted in Ezh2 mutant zygotes. During subsequent development, distinct histone methylation patterns were detected in the trophectoderm and the pluripotent epiblast. In the latter, where Oct4 expression continues from the zygote onwards, the Ezh2/Eed complex apparently establishes a unique epigenetic state and plasticity, which probably explains why loss of Ezh2 is
early embryonic lethal and obligatory for the derivation of pluripotent
embryonic stem cells. By contrast, in the differentiating trophectoderm cells
where Oct4 expression is progressively downregulated, Ezh2/Eed complex
is recruited transiently to one X chromosome in female embryos at the onset of X-inactivation. This accumulation and the associated histone methylation are also lost in Ezh2 mutants, suggesting a role in X inactivation. Thus, Ezh2 plays significant and diverse roles during early development, as well as during the establishment of the first differentiated cells, the trophectoderm, and of the pluripotent epiblast cells (Erhardt, 2003).
Polycomb group protein Ezh2 is an essential epigenetic regulator of embryonic development in mice, but its role in the adult organism is unknown. High expression of Ezh2 in developing murine lymphocytes suggests Ezh2 involvement in lymphopoiesis. Using Cre-mediated conditional mutagenesis, a critical role has been demonstrated for Ezh2 in early B cell development and rearrangement of the immunoglobulin heavy chain gene (Igh). Ezh2 is a key regulator of histone H3 methylation in early B cell progenitors. These data suggest Ezh2-dependent histone H3 methylation as a novel regulatory mechanism controlling Igh rearrangement during early murine B cell development (Su, 2003).
E2F is a family of transcription factors that regulate both cellular
proliferation and differentiation. To establish the role of E2F3 in
vivo, an E2f3 mutant mouse strain was generated. E2F3-deficient mice arise at one-quarter of the expected frequency, demonstrating that
E2F3 is important for normal development. To determine the molecular
consequences of E2F3 deficiency, the properties of
embryonic fibroblasts derived from E2f3 mutant mice were analyzed. Mutation of E2f3 dramatically impairs the mitogen-induced,
transcriptional activation of numerous E2F-responsive genes. A number of genes, including B-myb,
cyclin A, cdc2, cdc6, and DHFR, could be identified whose
expression is dependent on the presence of E2F3 but not E2F1. The E2Fs regulate the expression of several proteins that are involved in
early development, including homeobox proteins, transcription factors
involved in cell fate decisions, a number of proteins that determine
homeotic gene transcription, and signaling pathways such as the
TGFbeta and Wnt pathways that are essential for early development. As
an example of the relevance of these findings, it has been reported
that position-effect variegation (PEV) in Drosophila depends
on E2F activity. Loss of E2F activity enhances PEV,
whereas overexpression of E2F activity suppresses PEV in
Drosophila. These data suggested that the
E2Fs themselves have an epigenetic effect by regulating chromatin
structure or, more likely, that the E2Fs control PEV by regulating
genes of the Polycomb group (PcG) family. In this screen, several PcG genes have been identified, like Enhancer of Zeste 2 (EZH2),
Embryonic Ectoderm Development protein (EED) and Homolog of
Polyhomeotic (EDR2/HPH2). The E2F-induced expression of these genes may provide an explanation for the role of
E2F in the regulation of PEV and, more importantly, in development (Muller, 2001).
EZH2, homolog of Drosophila E(z) is highly expressed in metastatic prostate cancer and in lymphomas. EZH2 is a component of the PRC2 histone methyltransferase complex, which also contains EED and SUZ12 and is required for the silencing of HOX gene expression during embryonic development. Both EZH2 and EED are essential for the proliferation of both transformed and non-transformed human cells. In addition, the pRB-E2F pathway tightly regulates their expression and, consistent with this, EZH2 is found to be highly expressed in a large set of human tumors. These results raise the question whether EZH2 is a marker of proliferation or if it is actually contributing to tumor formation. Significantly, it is proposed that EZH2 is a bona fide oncogene, since ectopic expression of EZH2 is found to be capable of providing a proliferative advantage to primary cells and, in addition, its gene locus is specifically amplified in several primary tumors (Bracken, 2003).
Human (EZH2) and mouse (Ezh1) homologs of the Drosophila
Polycomb-group (Pc-G) gene Enhancer of zeste (E[z]) have been isolated, a crucial regulator of homeotic gene
expression implicated in the assembly of repressive protein complexes in chromatin. Mammalian
homologs of E(z) are encoded by two distinct loci in mouse and man, and the two murine Ezh genes
display complementary expression profiles during mouse development. Human EZH1 and murine Ezh1 (and probably also EZH2/Ezh2), are encoded by orthologous loci in mouse and man, and in both comparisons amino acid identities are 98%. EZH2/Ezh2 and E(z) appear to be most closely related, whereas EZH1/Ezh1 is the more divergent of the mammalian gene pairs. Human/mouse EZH2/Ezh2 is 61% and human/mouse EZH1/Ezh1 is 55% identical to E(z). There are four regions of high sequency identity. In addition to the most highly conserved SET domain (86% identity), a 115 amino acid, cysteine-rich region immediately precedes the SET domain (68% identity). Most of the cystein residues originally noted for E(z) are conserved in the mammalian proteins. In the amino-terminal half, two stretches, one of 66 amino acids (domain I; 66% identity) and one of 114 amino acids (domain II: 56% identity), are highly related between mammalian and fly E(z) proteins. Domain II also contains six conserved cysteine residues with unusual spacing and is separated from domain I by a stretch of charged amino acids. The position and sequence of the nuclear localization signal is also conserved (Laible, 1997).
In mice, Ezh1-specific transcripts are detected ubiquitously during embryogenesis, however their relative abundance is generally upregulated during development and in adults reaches 4- to 5-fold higher levels in kidney, brain and skeletal muscle. Ezh1 transcripts remain at a low level in liver, spleen and thymus. Ezh2 transcripts are downregulated as development progresses, to display a more thymus-restricted expression. Human EZH2 and murine Ezh2 are subject to alternative splicing that removes the amino-terminal half of the conserved cysteine-rich region or, in EZH1, part of the SET domain. The E(z) gene family reveals a
striking functional conservation in mediating gene repression in eukaryotic chromatin: extra gene copies
of human EZH2 or Drosophila E(z) in transgenic flies enhance position effect variegation of the
heterochromatin-associated white gene, and expression of either human EZH2 or murine Ezh1 restores
gene repression in Saccharomyces cerevisiae mutants that are impaired in telomeric silencing.
Together, these data provide a functional link between Pc-G-dependent gene repression and inactive
chromatin domains, indicating that silencing mechanism(s) may be broadly conserved in eukaryotes (Laible, 1997).
The finding of mammalian E(z) homologs allows the definition of a SET domain family, and suggests that the SET domain represents an ancient protein module that is intrinsically involved in chromatin-controlled gene regulation. Four subgroups are described. The SET domain of E(z) related proteins (subgroup I) appears to be most similar to that of subgroup II genes, which include the Trithorax homologs and S. cerevisiae SET1. The SET domain of S. cerevisiae Ezl-1 and Drosophila Ash-1 appear to be unique, because in both of these genes the SET domain is not located at the carboxy terminus (subgroup III). Finally, SET domains for subgroup IV genes comprose those present in Drosophila Su(var)3-9 related proteins and human G9a and KG-1 (Laible, 1997).
Polycomb group (PcG) chromatin proteins regulate homeotic genes in both animals and plants. In Drosophila and vertebrates, PcG proteins form complexes and maintain early patterns of Hox gene repression, ensuring fidelity of developmental patterning. PcG proteins in C. elegans form a complex and mediate transcriptional silencing in the germline, but no role for the C. elegans PcG homologs in somatic Hox gene regulation has been demonstrated. Surprisingly, it is found that the PcG homologs MES-2 [E(Z)] and MES-6 (ESC), along with MES-3, a protein without known homologs, do repress Hox expression in C. elegans. mes mutations cause anteroposterior transformations and disrupt Hox-dependent neuroblast migration. Thus, as in Drosophila, vertebrates, and plants, C. elegans PcG proteins regulate key developmental patterning genes to establish positional identity (Ross, 2003).
The three mes genes act upstream of the Hox genes mab-5 and egl-5 during V ray differentiation, and loss of mes activity can restore normal ray development and mating ability to males mutant in the mab-5 activator pal-1. Males lacking mes activity display anterior expansions of tail structures and ectopic expression of the Hox reporter egl-5::gfp and the Hox target lin-32::gfp. This regulation is not restricted to the male tail: mes-2, -3, and -6 also repress lin-39::lacZ expression in the midbody and head and mab-5 activity in a migrating neuroblast. Consistent with a general somatic regulatory function, MES protein expression is widespread in larvae, particularly males. These findings suggest that the regulatory relationship between PcG chromatin proteins and the Hox genes has been conserved in nematodes (Ross, 2003).
Several lines of evidence suggest a functional interaction
between the PcG and trxG proteins. For example, genetic evidence indicates that the Enhancer of zeste [E(z)] gene can be considered both a PcG and a trxG gene. To better understand the molecular interactions in which the E(z) protein is involved, a two-hybrid screen was performed with Enx1/EZH2, a mammalian homolog of E(z), as the target. The human EED protein is shown to interact with Enx1/EZH2. EED is the human homolog of eed, a murine PcG gene that has extensive homology with the Drosophila PcG gene extra sex combs (esc). Enx1/EZH2 and EED coimmunoprecipitate, indicating that they also interact in vivo. However, Enx1/EZH2 and EED do not coimmunoprecipitate with other human PcG proteins, such as HPC2 and BMI1. Furthermore, unlike HPC2 and BMI1, which colocalize in nuclear domains of U-2 OS osteosarcoma cells, Enx1/EZH2 and EED do not colocalize with HPC2 or BMI1. These findings indicate that Enx1/EZH2 and EED are members of a class of PcG proteins that is distinct from previously described human PcG proteins (Sewalt, 1998).
To define the domains that are responsible for the interaction between Enx1 and EED, different parts of Enx1 and EED were subcloned in frame with
the GAL4-DBD and to see whether these proteins could still interact with full-length EED or full-length Enx1. Enx1 comprises two N-terminal domains that show strong homology between Drosophila E(z) and its mammalian homologs. These domains have been designated domains I and II. Furthermore, Enx1 contains a C-terminal cysteine-rich domain and a SET domain. This last domain is found in a number of different proteins such as the Trithorax protein. The region encompassing both the cysteine-rich domain and the SET domain
(aa 498 to 746) does not interact with EED. A region extended toward the N terminus (aa 285 to 746) also
does not interact with EED. In contrast, the region encompassing domain I and a part of domain II (aa 1 to 285) does interact with EED. To analyze this region in more detail, two constructs were made, one containing homology domain I (aa 1 to 195) and the other containing homology domain II (aa 172 to 335). Only the region of Enx1 that contains homology domain I interacts with the EED protein. It is concluded that EED binds to the N-terminal region of Enx1 that encompasses homology domain I (Sewalt, 1998).
EED contains five WD-40 domains which are thought to be involved in protein-protein interactions. The importance of these WD-40 domains for the interaction between Enx1 and EED was tested. Truncated EED
protein constructs were made that contain an increasing number of WD-40 domains. None of the truncated EED proteins, which contain up to four WD-40 domains, interact with Enx1. Only when all five WD-40
domains are present is this truncated EED protein (aa 184 to 535) able to interact with Enx1. The
most N-terminal region of EED, which does not contain WD-40 domains, is not important for
mediating the interaction between Enx1 and EED (Sewalt, 1998).
It has been proposed that Drosophila Esc interacts
with the transcriptional machinery through the WD-40 domains. This model is based on the homology that is
found between Esc and Tup1, a yeast protein that also contains seven WD-40 domains. These WD-40 domains are important for the involvement of the Tup1 protein in the repression of gene activity and in its binding to the DNA-binding homeodomain protein 2. Point mutations in the
WD-40 domains have also been found in several esc mutants. Either one of two point mutations in the second WD-40 domain completely abolishes the interaction in the two-hybrid system between Enx1 and EED. Precisely these two point mutations are responsible for the severe developmental defects in eed mutant mice. It is significant that the ability of the eed535 protein to repress gene activity is also completely abolished by these point mutations. It is therefore tempting to speculate that both the interference with the binding capacity and the repressing abilities of the eed/EED protein through these point mutations contribute to the developmental defects in eed mutant mice. One
immediate consequence of these point mutations can be that the Enx1 protein is no longer able to bind to eed; this leads to a subsequent loss of integrity for the protein complex formed in part by both Enx1/EZH2 and eed (Sewalt, 1998).
Enhancer of Zeste [E(z)] is a Polycomb-group transcriptional repressor and one of the founding members of the family of SET domain-containing proteins. Several SET-domain proteins possess intrinsic histone methyltransferase (HMT) activity. However, recombinant E(z) protein was found to be inactive in a HMT assay. A multiprotein E(z) complex has been isolated from humans that contains extra sex combs, suppressor of zeste-12 [Su(z)12], and the histone binding proteins RbAp46/RbAp48 (see Caf1). This complex, which has been termed Polycomb repressive complex (PRC) 2, possesses HMT activity with specificity for Lys 9 (K9) and Lys 27 (K27) of histone H3. The HMT activity of PRC2 is dependent on an intact SET domain in the E(z) protein. It is hypothesized that transcriptional repression by the E(z) protein involves methylation-dependent recruitment of PRC1. The presence of Su(z)12, a strong suppressor of position effect variegation, in PRC2 suggests that PRC2 may play a widespread role in heterochromatin-mediated silencing (Kuzmichev, 2002).
The polypeptide composition of the PRC2, specifically the presence of Su(z)12, suggests that PRC2 plays a more general role in transcriptional silencing outside of the repression of HOX genes. Su(z)12 is a protein with dual PcG and Su(var) functions, and this, therefore, suggests that PRC2 has functions other than homeotic gene repression and, in fact, may play a more general role in heterochromatin-mediated silencing. The observation that human E(z) can function as an inducer of silencing in yeast and as an enhancer of PEV in Drosophila supports this notion. It is speculated that the requirement for E(z) and ESC during early embryonic development reflects its function in general transcriptional silencing. The multifunctional nature of both the E(z) and Su(z)12 proteins suggests that they may also display biochemical heterogeneity. For example, the heterogeneous elution profile of E(z) on various columns suggests that E(z) exists in several distinct complexes (Kuzmichev, 2002).
Purified PRC2 displayed specificity for K9 and K27 of the histone H3 tail. The complex, under the conditions of the assays used, displayed a strong preference for K27. However, when the H3-tail was used as a GST-fusion protein, PRC2 displayed apparently equal specificity for K9 and K27. Analyses of the amino acid sequence in which these lysines are embedded shows a great deal of conservation. K9 is present within the sequence QTARK9STG, whereas K27 is present within the sequence KAARK27SAP. Therefore, at least two different possibilities can be postulated to account for the specificity observed. In one case, the specificity of PRC2 is relaxed in vitro, under the assay conditions used, and the methylation of K9 is nonspecific because of the sequence similarity of the residues within which K9 resides. An apparently similar situation was observed in studies analyzing the specificity of the histone methyltransferase G9a, which biochemically behaves as a H3-histone methyltransferase that preferentially targets K9 and, to much lower levels, K27. In vivo, however, G9a clearly targets H3-K9: whether or not the extent of H3-K27 methylation is decreased in G9a-null cells is unknown. A second possibility is that E(z) targets both K9 and K27, but that this is a regulated process such that methylation of K9 and/or K27 is modulated by factors that associate with E(z) and/or by other modifications existing in the nucleosome. This second possibility is favored based on the following observations. First, the E(z) protein can be considered to be a PcG as well as a TrxG. Not surprisingly, the analyses demonstrate that E(z) is present in distinct complexes. One of the complexes containing E(z) is PRC2; however; this complex also includes Su(z)12. Su(z)12 is a polypeptide that has been found in genetic analyses to regulate the expression of the HOX genes, but loss of function of Su(z)12 suppresses PEV. Therefore, the presence of Su(z)12 in PRC2 may regulate the methylation sites within the histone H3 tail (Kuzmichev, 2002).
Methylation of histone H3-K9 was shown to be an essential step in the establishment of inactive X chromosome. H3-Lys 9 methylation of the inactive X chromosome is not mediated by Suv39 or by G9a. Studies have demonstrated that the imprinted inactivation of the X chromosome in females is lost in mutant mice lacking eed (the mammalian homolog of ESC). Moreover, studies have also demonstrated that during imprinted X inactivation, the mammalian ESC-E(z) complex is localized to the inactive X chromosome in a mitotically stable manner. It is speculated, in light of the accumulated data, that H3-K9 methylation of the inactive X chromosome might be mediated by E(z) within PRC2 or a PRC2-like complex. Importantly, however, the function of methylation of histone H3 at K27 has not been analyzed in the establishment and/or maintenance of the inactive X chromosome. In light of the results discussed above, it is postulated that methylation of H3-K27 may also be important in the process of X inactivation (Kuzmichev, 2002).
It is proposed that the role of E(z) HMT activity in the repression of homeotic gene expression is to establish a binding site for other PcG proteins. It is suggested that PRC2 is recruited to the HOX gene cluster by a transiently acting repressor, for example, through an EED-YY1/Pho interaction or an RbAp46/48-HDAC/dMi2/Hb interaction. Once recruited, PRC2 methylates K27 on histone H3, and this mark recruits PC1. The PC1 protein can convert this mark into a permanently repressed state through methylation of K9 through the recruitment of the Su(var)3-9 H3-K9-specific histone methyltransferase and/or the recruitment of PRC1. Alternatively and/or additionally, PC1 may stimulate the H3-K9 HMT activity of PRC2. This hypothesis is supported by studies demonstrating that trimethylation of K27 is necessary for binding of PC1 to an H3 tail peptide. These findings are in full agreement with studies demonstrating loss of chromosome binding for several PRC1 components upon inactivation of E(z). Interestingly, immunolocalization experiments using antibodies specific for methylated H3-K9 suggest that almost all of the H3-K9 methylation is concentrated in the chromocenter of Drosophila polytene chromosomes, with almost no staining detectable on the chromosomal arms. In contrast, E(z) and other PcG proteins, with subnuclear localization that is regulated by E(z), bind only to discrete bands along the arms of polytene chromosomes. These observations suggest that methylation at K27, rather than methylation at K9, is more likely to establish a binding site for the PC1 protein. This may explain why methylation of K9 alone was not sufficient to allow PC1 to recognize specifically the H3 tail in vitro. The observed PC1 binding was independent of DNA. However, repression of HOX genes in vivo is dependent on PRE. From these results, it must be concluded that although methylation of the H3 tail is important in creating a recognition site for PC1 binding, stable and specific binding must require additional factors and or modifications. A likely candidate is a nucleosome on the PRE with the histone H3-tail methylated at position 27 (Kuzmichev, 2002).
The presence of the RbAp46 and RbAp48 proteins in the ESC-E(z) complex may be important for several reasons. First, these histone-binding proteins are often found in complexes with enzymes involved in the covalent modification of histones. For example, RbAp46 is essential for substrate recognition by, and enzymatic activity of, the histone acetyltransferase enzyme Hat1. Therefore, it is speculated that the inability to detect HMT activity in preparations of recombinant E(z) protein is owing, in part, to the lack of the RbAp46/RbAp48. Another implication of the presence of RbAp proteins in the E(z) complex is that they might facilitate interaction with HDACs. During development, GAP proteins facilitate repression of the HOX genes. GAP proteins, such as Hunchback, are short-lived. Hunchback represses HOX genes by recruiting the Drosophila homolog of human Mi-2 protein, a constituent of the NuRD complex which also contains HDACs 1 and 2 and RbAp46/RpAp48. An interesting possibility is that HDACs or RbAp proteins initially recruited by Hunchback can later recruit PRC2 containing HMT activity via interaction with E(z). This may constitute a switch from short-term to long-term repression (Kuzmichev, 2002).
In support of this hypothesis, PRC2 contains E(z) and RbAp proteins. In addition, there is strong experimental evidence for an interaction between HDACs and E(z). One function of the E(z)-HDAC interaction is to deacetylate histones so that the E(z)-containing complex can methylate them. A similar mechanism was found to operate in yeast, in which methylation of H3-K9 by Clr4 requires deacetylation of H3-K9 and Lys 14 (K14) by Clr6 and Clr3, respectively. A similar mechanism is likely to operate in higher eukaryotes because acetylation and methylation are mutually exclusive marks, and methylation of H3-K9 by Suv39h1 requires deacetylation of this residue. The findings demonstrating two distinct ESC-E(z) complexes, one of which coelutes with HDAC1, raises the possibility that the PRC2 can transiently associate with an HDAC complex. This observation raises the possibility that PRC2 HDAC1 may be a highly-specialized complex dedicated to the methylation of H3-K27, which apparently is not acetylated in vivo in higher eukaryotes. Therefore, it is possible that these two different ESC-E(z) multiprotein complexes establish different marks on the histone H3 tail (Kuzmichev, 2002).
The Eed-Enx1 Polycomb group complex has been implicated in the maintenance of imprinted X inactivation in the trophectoderm lineage in mouse. Recruitment of Eed-Enx1 to the inactive X chromosome (Xi) also occurs in random X inactivation in the embryo proper. Localization of Eed-Enx1 complexes to Xi occurs very early, at the onset of Xist expression, but then disappears as differentiation and development progress. This transient localization correlates with the presence of high levels of the complex in totipotent cells and during early differentiation stages. Functional analysis demonstrates that Eed-Enx1 is required to establish methylation of histone H3 at lysine 9 and/or lysine 27 on Xi and that this, in turn, is required to stabilize the Xi chromatin structure (Silva, 2003).
In eed-/- XX embryos Enx1 protein does not localize to Xi. This result is consistent with in vitro analysis demonstrating that the eed3354SB mutation disrupts a WD40 domain required for the interaction of Eed with Enx1. Thus, in the absence of functional Eed protein, the Enx1 HMTase cannot be directed toward specific targets. Eed/Enx1 complexes methylate H3-K9 and K27 in vitro, with a strong preference for K27. Failure to localize Enx1 clearly accounts for the absence of H3-K9/K27 methylation on Xi in eed-/- embryos (Silva, 2003).
It should be noted that, while previous studies have identified H3-K9 methylation as an early mark of silent chromatin on the inactive X chromosome, more recent data indicates that this could be attributable to cross-reactivity of di/tri-meH3-K9 antisera toward di/tri-meH3-K27. New antisera highly specific for di/tri-meH3-K9 detect pericentromeric heterochromatin, but not Xi, while the tri-meH3-K27 antibody used in this study detects Xi, but not pericentromeric heterochromatin. It is also possible that Xi is di/trimethylated both at K9 and K27 and that this configuration is not recognized by the novel di/trimethylH3-K9 antisera (Silva, 2003).
H3-K9/K27 methylation is shown to serve to stabilize the Xi chromatin structure. Thus, in eed-/- embryos, H3-K9 hypoacetylation and loss of H3-K4 methylation on Xi are not seen in a significant number of cells. Moreover, both the Xa and Xi alleles of two X-linked genes were seen to be expressed in a similar proportion of cells. These observations provide a basis for explaining reactivation of the X-linked GFP transgene in trophectoderm cells of eed-/- embryos. However, other studies have not observe reactivation of the GFP transgene in cells of the embryo proper, leading to the conclusion that Eed is required for the maintenance of imprinted, but not random, X inactivation. This difference can be accounted for by two factors. First, it is probable that reactivation of any given locus on Xi is sporadic and progressive. Since embryos exhibit mosaic expression of the XGFP transgene because of random X inactivation, a relatively small increase in the proportion of cells expressing the transgene, as observed for endogenous X-linked genes in this study, would be difficult to quantify. A second factor, is that additional levels of epigenetic silencing, for example, DNA methylation, play a more significant role in maintenance of X inactivation in cells of the embryo proper compared with the trophectoderm, potentially masking the effects of failure to establish H3-K9/K27 methylation (Silva, 2003 and references therein).
Initiation and propagation of X inactivation occurs coincident with cellular differentiation and involves a large nonprotein-coding RNA, the X inactive-specific transcript (Xist). Xist RNA spreads over the X chromosome and is thought to induce chromosome inactivation through recruitment of as yet unidentified silencing factors. The silencing function of Xist RNA has been shown to map to a short tandemly repeated element at the 5' end of the transcript (Silva, 2003 and references therein).
The banded localization of Eed-Enx1 complexes on Xi parallels that observed for Xist RNA. There could be an interaction, either direct or indirect, between Xist and the Eed-Enx1 complex. This view is further supported by observations reported in this study. (1) Recruitment of Eed-Enx1 complexes occurs extremely rapidly after the onset of stable Xist RNA accumulation in differentiating XX ES cells. (2) Eed-Enx1 recruitment occurs in response to expression of ectopic Xist RNA transgenes, both in undifferentiated XY ES cells and also in XYTg15 blastocysts. (3) Eed-Enx1 complexes are not recruited in response to expression of Xistinv mutant RNA, which fails to elicit X inactivation (Silva, 2003).
While the data support the idea that Eed-Enx1 complexes are recruited by Xist RNA, they do not prove that this interaction is direct. In fact, localization of Eed-Enx1 to Xi is first seen to occur at the morula stage, when imprinted X inactivation is initiated, whereas Xist RNA expression is detected earlier, in cleavage stage embryos. High levels of Eed-Enx1 complexes are available during the cleavage stages, suggesting that a factor(s) required for the interaction of the complex with Xist is absent (Silva, 2003).
Also relevant to the question of whether or not the Eed-Enx1 complex interacts directly with Xist RNA is the observation that eed-/- embryos are able to initiate X inactivation. This is, presumably, at least in part, attributable to recruitment of an HDAC complex that can deacetylate H3-K9. It is possible that components of the PcG complex other than Eed and Enx1 are still assembled in eed-/- embryos and that these confer H3-K9 HDAC activity. In the Drosophila ESC (Eed) protein, an equivalent mutation disrupts EZ (Enx1) recruitment but does not appear to ablate complex formation. An alternative scenario is that the complex responsible for H3-K9 deacetylation is a direct target of Xist RNA and that the Eed-Enx1 HMTase is then recruited by this complex. In support of this idea, heritable silencing at Drosophila homeotic genes is initiated by recruitment of the dMi2 protein, a component of an HDAC and chromatin remodeling complex, by Hunchback. Moreover, Eed protein can interact with type 1 HDACs, both in mammalian cells and in Drosophila. Thus, HDAC complexes may recruit Eed-Enx1 to target sites, including Xi, rather than vice versa (Silva, 2003).
It is interesting to note that the time during which high levels of Eed-Enx1 complex are available corresponds closely to the window of opportunity during which cells are responsive to Xist RNA. On the basis of this consideration, a model is proposed that speculates that Xist RNA recruits HDAC and Eed-Enx1 complexes, which lead to establishment of a primary level of chromatin silencing. Only during early differentiation stages would levels of these complexes be sufficient to establish chromosome-wide primary silencing. This would explain why expression of Xist in more differentiated cell types cannot induce X inactivation. It is further suggested that maintaining localization of the HDAC/Eed-Enx1 complexes is Xist RNA dependent. This would account for reversibility and Xist dependence of silencing in undifferentiated ES cells or during early differentiation stages. Extinguishing Xist expression would result in delocalization of HDAC/Eed-Enx1 complexes, loss of H3-K9/K27 methylation, increased H3-K9 acetylation, and, hence, reactivation of Xi (Silva, 2003).
To account for the fact that X inactivation does subsequently become stabilized and Xist independent, it is suggested that the chromatin modifications induced by the HDAC and Eed-Enx1 complex provide a template for recruitment of other silencing components. These could be responsible for further histone N-terminal modifications, for example, global H4 deacetylation, and also for DNA methylation at CpG islands and recruitment of macroH2A1.2. It should be noted that proteins of the PRC1 PcG group complex are not localized to Xi at any stage and are therefore unlikely to be involved in maintaining X inactivation in late development (Silva, 2003).
A key finding from these experiments is that recruitment of Eed-Enx1 to Xi is temporally regulated, rather than lineage specific, and that this, in turn, appears to relate to temporal regulation of overall levels of Eed and, to a lesser extent, Enx1 proteins. A similar expression profile has been reported for ESC (Eed) protein in Drosophila embryogenesis. It is striking that these factors are expressed at highest levels in totipotent or multipotent precursors and during early stages of differentiation. One interpretation of this observation is that Eed-Enx1 complexes are components of the machinery required to confer genome plasticity. Thus, like X inactivation during the window of opportunity, silent chromatin at other Eed-Enx1 targets may be reversible if the primary signal (for example Hunchback at homeotic loci in Drosophila) is removed. This would provide cells of the early embryo with the capacity to activate regions of the genome in response to specific differentiation signals, contrasting with the situation in differentiated cells, where heritable silencing is highly stable and is normally irreversible (Silva, 2003).
The Polycomb group (PcG) protein Eed is implicated in regulation of imprinted X-chromosome inactivation in extraembryonic cells but not of random X inactivation in embryonic cells. The Drosophila homolog of the Eed-Ezh2 PcG protein complex achieves gene silencing through methylation of histone H3 on lysine 27 (H3-K27), which suggests a role for H3-K27 methylation in imprinted X inactivation. This study demonstrates that transient recruitment of the Eed-Ezh2 complex to the inactive X chromosome (Xi) occurs during initiation of X inactivation in both extraembryonic and embryonic cells and is accompanied by H3-K27 methylation. Recruitment of the complex and methylation on the Xi depend on Xist RNA but are independent of its silencing function. Together, these results suggest a role for Eed-Ezh2-mediated H3-K27 methylation during initiation of both imprinted and random X inactivation and demonstrate that H3-K27 methylation is not sufficient for silencing of the Xi (Plath, 2003).
The Ezh2 protein endows the Polycomb PRC2 and PRC3 complexes with histone lysine methyltransferase (HKMT) activity that is associated with transcriptional repression. Ezh2 expression is developmentally regulated in the myotome compartment of mouse somites and its down-regulation coincides with activation of muscle gene expression and differentiation of satellite-cell-derived myoblasts. Increased Ezh2 expression inhibits muscle differentiation, and this property is conferred by its SET domain, required for the HKMT activity. In undifferentiated myoblasts, endogenous Ezh2 is associated with the transcriptional regulator YY1. Both Ezh2 and YY1 are detected, with the deacetylase HDAC1, at genomic regions of silent muscle-specific genes: their presence correlates with methylation of K27 of histone H3. YY1 is required for Ezh2 binding because RNA interference of YY1 abrogates chromatin recruitment of Ezh2 and prevents H3-K27 methylation. Upon gene activation, Ezh2, HDAC1, and YY1 dissociate from muscle loci, H3-K27 becomes hypomethylated and MyoD and SRF are recruited to the chromatin. These findings suggest the existence of a two-step activation mechanism whereby removal of H3-K27 methylation, conferred by an active Ezh2-containing protein complex, followed by recruitment of positive transcriptional regulators at discrete genomic loci are required to promote muscle gene expression and cell differentiation (Cartetti, 2004).
These results indicate that Ezh2 is recruited at the chromatin of selected muscle regulatory regions by the transcriptional regulator YY1. Both can be coimmunoprecipitated from myoblast and not myotube cell extracts, and the proteins colocalize at the same muscle chromatin regions in a developmentally regulated manner. The interaction of endogenous YY1 and Ezh2 is likely to be mediated by the PcG EED protein because recombinant YY1 and Ezh2 do not directly associate. Previous reports have demonstrated a negative role for YY1 in regulating muscle gene expression through interaction with distinct nucleotides within the CarG-box [CC(A+T-rich)6GG], one of the DNA elements required for muscle-specific gene transcription. Transcriptional activation coincides with replacement of YY1 by the serum response factor (SRF), whose interaction with the CarG-box is required for muscle-specific transcription to proceed. These data suggest a two-step activation model of muscle gene expression. In the repressed state, YY1 recruits a complex containing both Ezh2 and HDAC1 that silences transcription through histone methylation (H3-K27) and deacetylation. Transcriptional activation entails the initial removal of the YY1-Ezh2-HDAC1 repressive complex and subsequent recruitment of the activators SRF (which replaces YY1) and the MyoD family of transcription factors and associated acetyltransferases. Since YY1 binding tolerates a substantial nucleotide heterogeneity in its DNA recognition sites, muscle and non-muscle-specific CarG-less regulatory regions may be also occupied and regulated in a similar manner. In contrast, Ezh2 does not appear to promiscuously regulate expression of all muscle-specific genes as indicated by the transient coexpression of Ezh2 and myogenin in the myotome of developing embryos and lack of Ezh2 recruitment and H3-K27 methylation at the myogenin promoter. Distinct histone methyltransferases and deacetylases have been shown to modify histones at the myogenin promoter (Cartetti, 2004 and references therein).
Polycomb group complexes 2 and 3 are involved in transcriptional silencing. These complexes contain a histone lysine methyltransferase (HKMT) activity that targets different lysine residues on histones H1 or H3 in vitro. However, it is not known if these histones are methylation targets in vivo because the human PRC2/3 complexes have not been studied in the context of a natural promoter because of the lack of known target genes. RNA expression arrays and CpG-island DNA arrays were used to identify and characterize human PRC2/3 target genes. Using oligonucleotide arrays, a cohort of genes were identified whose expression changes upon siRNA-mediated removal of Suz12 [Drosophila homolog Su(z)12], a core component of PRC2/3, from colon cancer cells. To determine which of the putative target genes are directly bound by Suz12 and to precisely map the binding of Suz12 to those promoters, a high-resolution chromatin immunoprecipitation (ChIP) analysis was combined with custom oligonucleotide promoter arrays. Additional putative Suz12 target genes were identified by using ChIP coupled to CpG-island microarrays. HKMT-Ezh2 and Eed, two other components of the PRC2/3 complexes, colocalize to the target promoters with Suz12. Importantly, recruitment of Suz12, Ezh2 and Eed to target promoters coincides with methylation of histone H3 on Lys 27 (Kirmizis, 2004).
Identification of mammalian PcG target genes has remained elusive for two main reasons. First, the majority of the previous PcG studies focused mostly on the biochemical purification and in vitro characterization of the activities of the PcG complexes and second, the lack of DNA-binding domains within PcG proteins makes the search for their target loci difficult. In this present study, the first known direct target genes of mammalian PcG complexes has been identified. To do so, RNAi was first used to identify genes deregulated by the loss of Suz12 protein in colon cancer cells. Next, Suz12 was shown to bind directly to the promoter of one of these genes (MYT1). Other members of the PRC2/3 complexes were shown to colocalize with Suz12 at the MYT1 promoter. Most importantly, recruitment of Suz12, Ezh2, and Eed to the MYT1 promoter was shown to correlate with methylation of H3-K27. To demonstrate that this silencing mechanism is not unique to MYT1, other Suz12 target genes were identified using a ChIP assay coupled to a CpG island microarray. Similarly to MYT1, the other target promoters of Suz12 are bound by the PRC2/3 components and are characterized by H3-K27 methylation. Thus, the first identified human PcG target genes all appear to be regulated by the histone methylase activity of the PRC complexes (Kirmizis, 2004).
The Suz12 target gene MYT1 was originally cloned from a human brain cDNA library on the basis of its ability to bind cis-regulatory elements of the glia-specific myelin proteolipid protein (PLP) gene and is suggested to be the prototype of the C2HC-type zinc finger protein family. More recently, the Xenopus ortholog of MYT1 (X-MYT1) was identified as a transcriptional activator because it could induce expression of an N-tubulin promoter reporter construct in transient transfection assays. Dominant-negative forms of X-MYT1 inhibited normal neurogenesis, suggesting that X-MYT1 is essential for inducing neuronal differentiation. Intriguingly, a recent report shows that the Xenopus ortholog of Ezh2 (XEZ) is expressed exclusively in the anterior neural plate during early Xenopus embryogenesis, and it was postulated that XEZ might be involved in delaying anterior neuronal differentiation (Barnett, 2001). Based on the current findings, it is possible that Ezh2 delays neuronal differentiation, via the PRC2/3 complexes, by repressing the activity of the MYT1 gene. In addition to MYT1, four additional promoters were identified as being robustly bound by components of the PRC2/3 complexes; each of these promoters is also characterized by high levels of H3-K27. Although a link between components of the PRC2/3 complexes and Wnt1, the cannabinoid receptor (CNR1), or the potassium channel KCNA1 have not been previously reported, it is intriguing to note that these mRNAs are expressed at very low levels in most human tissues, suggesting that they may be generally silenced by the PRC complexes. In support of this hypothesis, some of these target genes were shown to be bound by PRC2/3 components in other cell lines, such as the human MCF7 and mouse F9 (Kirmizis, 2004).
Noncoding RNAs (ncRNA) participate in epigenetic regulation but are poorly understood. This study characterized the transcriptional landscape of the four human HOX loci at five base pair resolution in eleven anatomic sites, and identified 231 HOX ncRNAs that extend known transcribed regions by more than 30 kilobases. HOX ncRNAs are spatially expressed along developmental axes, possess unique sequence motifs, and their expression demarcate broad chromosomal domains of differential histone methylation and RNA polymerase accessibility. A 2.2 kilobase ncRNA was identified residing in the HOXC locus, termed HOTAIR, which represses transcription in trans across 40 kilobases of the HOXD locus. HOTAIR interacts with Polycomb Repressive Complex 2 (PRC2) and is required for PRC2 occupancy and histone H3 lysine-27 trimethylation of HOXD locus. Thus, transcription of ncRNA may demarcate chromosomal domains of gene silencing at a distance; these results have broad implications for gene regulation in development and disease states (Rinn, 2007).
By analyzing the transcriptional and epigenetic landscape of the HOX loci at high resolution in cells with many distinct positional identities, a panoramic view was obtained of multiple layers of regulation involved in maintenance of site-specific gene expression. The HOX loci are demarcated by broad chromosomal domains of transcriptional accessibility, marked by extensive occupancy of RNA polymerase II and H3K4 dimethylation and, in a mutually exclusive fashion, by occupancy of PRC2 and H3K27me3. The active, PolII-occupied chromosomal domains are further punctuated by discrete regions of transcription of protein-coding HOX genes and a large number of long ncRNAs. These results confirm the existence of broad chromosomal domains of histone modifications and occupancy of HMTases over the Hox loci, and extend on those observation in several important ways (Rinn, 2007).
First, by comparing the epigenetic landscape of cells with distinct positional identities, it was showm that the broad chromatin domains can be programmed with precisely the same boundary but with diametrically opposite histone modifications and consequences on gene expression. The data thus functionally pinpoint the locations of chromatin boundary elements in the HOX loci, the existence of some of which have been predicted by genetic experiments. One such boundary element appears to reside between HOXA7 and HOXA9. This genomic location is also the switching point in the expression of HOXA genes between anatomically proximal versus distal patterns and is the boundary of different ancestral origins of HOX genes, raising the possibility that boundary elements are features demarcating the ends of ancient transcribed regions. Second, the ability to monitor 11 different HOX transcriptomes in the context of the same cell type conferred the unique ability to characterize changes in ncRNA regulation that reflect their position in the human body. This unbiased analysis identified more than 30 kb of new transcriptional activity, revealed ncRNAs conserved in evolution, mapped their anatomic patterns of expression, and uncovered enriched ncRNA sequence motifs correlated with their expression pattern -- insights which could not be gleamed from examination of EST sequences alone. The finding of a long ncRNA that acts in trans to repress HOX genes in a distant locus is mainly due to the ability afforded by the tiling array to comprehensively examine the consequence of any perturbation over all HOX loci. The expansion of a handful of Hox-encoded ncRNAs in Drosophila to hundreds of ncRNAs in human HOX loci suggests increasingly important and diverse roles for these regulatory RNAs (Rinn, 2007).
An important limitation of the tiling array approach is that while improved identification of transcribed regions is obtained, the data does not address the connectivity of these regions. The precise start, end, patterns of splicing, and regions of double-stranded overlap between ncRNAs will need to be addressed by detailed molecular studies in the future (Rinn, 2007).
The results uncovered a new mechanism whereby transcription of ncRNA dictates transcriptional silencing of a distant chromosomal domain. The four HOX loci demonstrate complex cross regulation and compensation during development. For instance, deletion of the entire HOXC locus exhibits a milder phenotype than deletion of individual HOXC genes, suggesting that there is negative feedback within the locus. Multiple 5' HOX genes, including HOXC genes, are expressed in developing limbs, and deletion of multiple HOXA and HOXD genes are required to unveil limb patterning defects. The results suggest that deletion of the 5' HOXC locus, which encompass HOTAIR, may lead to transcriptional induction of the homologous 5' HOXD genes, thereby restoring the total dosage of HOX transcription factors. How HOX ncRNAs may contribute to cross-regulation among HOX genes should be addressed in future studies (Rinn, 2007).
HOTAIR ncRNA is involved in Polycomb Repressive Complex 2-mediated silencing of chromatin. Because many HMTase complexes lack DNA binding domains but possess RNA binding motifs, it has been postulated that ncRNAs may guide specific histone modification activities to discrete chromatin loci. This study has shown that HOTAIR ncRNA binds PRC2 and is required for robust H3K27 trimethylation and transcriptional silencing of the HOXD locus. HOTAIR may therefore be one of the long sought after RNAs that interface the Polycomb complex with target chromatin. A potentially attractive model of epigenetic control is the programming of active or silencing histone modifications by specific noncoding RNAs. Just as transcription of certain ncRNA can facilitate H3K4 methylation and activate transcription of the downstream Hox genes (Sanchez-Elsner, 2006; Schmitt, 2005), distant transcription of other ncRNAs may target the H3K27 HMTase PRC2 to specific genomic sites, leading to silencing of transcription and establishment of facultative heterochromatin. In this view, extensive transcription of ncRNAs is both functionally involved in the demarcation of active and silent domains of chromatin as well as being a consequence of such chromatin domains (Rinn, 2007).
Several lines of evidence suggest that HOTAIR functions as a bona fide long ncRNA to mediate transcriptional silencing. First, full length HOTAIR is detected in vivo and in primary cells, but not small RNAs derived from HOTAIR indicative of miRNA or siRNA production. Second, depletion of full length HOTAIR led to loss of HOXD silencing and H3K27 trimethyation by PRC2, and third, endogenous or in vitro transcribed full length HOTAIR ncRNA physically associated with PRC2. While these results do not rule out the possibility that RNA interference pathways may be subsequently involved in PcG function, they support the notion that the long ncRNA form of HOTAIR is functional. The role of HOTAIR is reminiscent of XIST, another long ncRNA shown to be involved in transcriptional silencing of the inactive X chromosome. An important difference between HOTAIR and XIST is the strictly cis-acting nature of XIST. HOTAIR is the first example of a long ncRNA that can act in trans to regulate a chromatin domain. While a trans repressive role for HOTAIR was observed, the data do not permit ruling out a cis-repressive role in the HOXC locus. siRNA-mediated depletion of HOTAIR was substantial but incomplete; further, the proximity between the site of HOTAIR transcription and the neighboring HOXC locus may ensure significant exposure to HOTAIR even if the total pool of HOTAIR in the cell were depleted. The precise location of HOTAIR at the boundary of a silent chromatin domain in the HOXC locus makes a cis-repressive role a tantalizing possibility. Judicious gene targeting of HOTAIR may be required to address its role in cis-regulation of chromatin (Rinn, 2007).
The discovery of a long ncRNA that can mediate epigenetic silencing of a chromosomal domain in trans has several important implications. First, ncRNA guidance of PRC2-mediated epigenetic silencing may operate more globally than just in the HOX loci, and it is possible that other ncRNAs may interact with chromatin modification enzymes to regulate gene expression in trans. Second, PcG proteins are important for stem cell pluripotency and cancer development; these PcG activities may also be guided by stem cell or cancer-specific ncRNAs. Third, Suz12 contains a zinc finger domain, a structural motif that can bind RNA, and EZH2 and EED both have in vitro RNA binding activity. The interaction between HOTAIR and PRC2 may also be indirect and mediated by additional factors. Detailed studies of HOTAIR and PRC2 subunits are required to elucidate the structural features that establish the PRC2 interaction with HOTAIR. As is illustrated in this study, high throughput approaches for the discovery and characterization of ncRNAs may aid in dissecting the functional roles of ncRNAs in these diverse and important biological processes (Rinn, 2007).
Genetic studies have demonstrated that Bmi1 promotes cell proliferation and stem cell self-renewal with a correlative decrease of p16INK4a expression. Polycomb genes EZH2 and BMI1 repress p16 expression in human and mouse primary cells, but not in cells deficient for pRB protein function. The p16 locus is H3K27-methylated and bound by BMI1, RING2, and SUZ12. Inactivation of pRB family proteins abolishes H3K27 methylation and disrupts BMI1, RING2, and SUZ12 binding to the p16 locus. These results suggest a model in which pRB proteins recruit PRC2 to trimethylate p16, priming the BMI1-containing PRC1L ubiquitin ligase complex to silence p16 (Kotake, 2007).
The mammalian pRB family proteins, pRB, p107, and p130 (also known as pocket proteins), play a key role in controlling the G1-to-S transition of the cell cycle and maintaining differentiated cells in a reversible quiescent or permanent senescent arrest state. The pocket proteins are hypophosphorylated in cells exiting mitosis as well as in quiescent cells, where they bind to and negatively regulate the function of the E2F family transcription factors. In cells entering the cell cycle, extracellular mitogens first induce the expression of D-type cyclins, which bind to and activate CDK4 and CDK6, leading to the phosphorylation of pRB family proteins, causing functional inactivation by E2F dissociation, thereby promoting a G1-to-S transition. Inhibition of CDK4 and CDK6 by the INK4 family of CDK inhibitors (p16INK4a, p15INK4b, p18INK4c, and p19INK4d) retains pRB family proteins in their hypophosphorylated, growth-suppressive states and prevents G1-to-S progression. Disruption of the INK4-RB pathway, consisting of INK4-cyclinDs-CDK4/6-RB-E2Fs, deregulates G1-to-S control and represents a common event in the development of most, if not all, types of cancer (Kotake, 2007).
Among the major challenges toward a better understanding of G1 control by the INK4-pRB pathway is how different INK4 genes are regulated, thereby linking G1 control to different cellular pathways. INK4 proteins are relatively stable, and the primary regulation of INK4 is through transcriptional control. The expression of each of the INK4 genes is distinctly different during development, in different adult tissues, and in response to different cellular conditions. There have been only a few reports wherein a transcriptional regulator has been demonstrated to bind to an INK4 promoter by either gel shift or chromatin immunoprecipitation (ChIP) assay. Identification of factors that directly bind to INK4 promoters holds the key to linking different cellular pathways to G1 control by the INK4-pRB pathway, but these links remain disproportionately poorly understood in comparison with knowledge of the function of the INK4-pRB pathway (Kotake, 2007).
To elucidate the molecular mechanisms regulating p16 expression, whether p16 gene expression is directly regulated by BMI1, an oncogene that encodes a transcriptional repressor of the Polycomb group (PcG) of proteins, was directly tested. Deletion of Bmi1 retards cell proliferation, causes premature senescence in mouse embryonic fibroblasts (MEFs), and reduces the number of hematopoietic stem cells, with an associated up-regulation of p16 (and to a lesser extent of p19Arf). Codeletion of p16 (or p16-Arf) partially rescues the proliferative defects of Bmi1-null cells, providing genetic evidence supporting a functional interaction between the Bmi1 and p16 genes. However, whether BMI1 directly binds to and regulates the transcription of the p16 gene has not been demonstrated. A notable feature of p16 is its high level of expression in virally transformed cells and its inverse correlation with pRB function, suggesting a negative regulation of p16 gene expression by pRB. Therefore whether pRB and BMI1 collaboratively repress p16 expression was also examined (Kotake, 2007).
These results provide the first biochemical evidence supporting a direct regulation of p16 transcription by the PRC2 histone methyltransferase complex and the BMI1-RING2-containing PRC1 histone ubiquitin ligase complex. Both H3K27 methylation at and BMI1/RING2 binding to the p16 locus require the function of the pRB family proteins, linking for the first time H3K27 methylation and the function of BMI1 with the pRB proteins. The detailed biochemical mechanism by which pRB family proteins collaborate with BMI1 to repress p16 transcription is yet to be determined. In repeated attempts, binding of pRB to the p16 locus could not be detected. The simplest model suggested by these results is that the pRB family proteins are either involved in regulating the enzymatic activity or the recruitment of PRC2 to the p16 locus. H3K27 methylation by PRC2 would then facilitate recruitment of the BMI1-containing PRC1L complex to ubiquitinate H2A, leading to p16 silencing (Kotake, 2007).
The results also suggest a regulatory loop between p16 and the pocket proteins, with p16 acting as an upstream activator of the pocket proteins and the pocket proteins repressing p16 transcription as negative feedback. INK4 proteins are intrinsically stable and, once synthesized, stably bind to and inhibit the activity of CDK4/6 by both interfering with ATP binding and by reducing the cyclin-CDK4/6 surface. Without a mechanism for repressing INK4 expression, mitogen-induced cyclin D synthesis would not be able to compete off INK4 from CDK4/6, and displaced, monomeric cyclin D proteins would be rapidly degraded, leaving a constitutive activation of RB function and locking cells in a permanent G1-arrested state. Repression of p16 expression by pRB family proteins thus also constitutes a feedback loop to set up a balance between INK4-mediated inhibition and cyclin D-mediated activation of G1 progression. This function of p16, however, must be repressed in stem cells, which undergo continuous proliferation and self-renewal in vivo. It is speculated that one mechanism to achieve this is through expression of BMI1 in the stem cell compartment (Kotake, 2007).
The Polycomb Group Protein EZH2 is a transcriptional repressor involved in controlling cellular memory and has been linked to aggressive prostate cancer. The functional role of EZH2 in cancer cell invasion and breast cancer progression has been investigated. EZH2 transcript and protein are consistently elevated in invasive breast carcinoma compared with normal breast epithelia. Tissue microarray analysis, which included 917 samples from 280 patients, demonstrate that EZH2 protein levels are strongly associated with breast cancer aggressiveness. Overexpression of EZH2 in immortalized human mammary epithelial cell lines promotes anchorage-independent growth and cell invasion. EZH2-mediated cell invasion requires an intact SET domain and histone deacetylase activity. This study provides compelling evidence for a functional link between dysregulated cellular memory, transcriptional repression, and neoplastic transformation (Kleer, 2003).
Prostate cancer is a leading cause of cancer-related death in males and is second only to lung cancer. Although effective surgical and radiation treatments exist for clinically localized prostate cancer, metastatic prostate cancer remains essentially incurable. Through gene expression profiling, the polycomb group protein enhancer of zeste homolog 2 (EZH2) is found to be overexpressed in hormone-refractory, metastatic prostate cancer. Small interfering RNA (siRNA) duplexes targeted against EZH2 reduce the amounts of EZH2 protein present in prostate cells and also inhibit cell proliferation in vitro. Ectopic expression of EZH2 in prostate cells induces transcriptional repression of a specific cohort of genes. Gene silencing mediated by EZH2 requires the SET domain and is attenuated by inhibiting histone deacetylase activity. Amounts of both EZH2 messenger RNA and EZH2 protein are increased in metastatic prostate cancer; in addition, clinically localized prostate cancers that express higher concentrations of EZH2 show a poorer prognosis. Thus, dysregulated expression of EZH2 may be involved in the progression of prostate cancer, as well as being a marker that distinguishes indolent prostate cancer from those at risk of lethal progression (Varambally, 2002).
Polycomb-repressive complex 2 (PRC2)-mediated histone methylation plays an important role in aberrant cancer gene silencing and is a potential target for cancer therapy. S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin A (DZNep) induces efficient apoptotic cell death in cancer cells but not in normal cells. DZNep effectively depletes cellular levels of PRC2 components EZH2, SUZ12, and EED and inhibits associated histone H3 Lys 27 methylation (but not H3 Lys 9 methylation). By integrating RNA interference (RNAi), genome-wide expression analysis, and chromatin immunoprecipitation (ChIP) studies, a prominent set of genes were identifed, selectively repressed by PRC2 in breast cancer, that can be reactivated by DZNep. The preferential reactivation of a set of these genes by DZNep, including a novel apoptosis affector, FBXO32, contributes to DZNep-induced apoptosis in breast cancer cells. These results demonstrate the unique feature of DZNep as a novel chromatin remodeling compound and suggest that pharmacologic reversal of PRC2-mediated gene repression by DZNep may constitute a novel approach for cancer therapy (Tan, 2007).
Polycomb group proteins are essential for early development in metazoans, but their contributions to human development are not well understood. The Polycomb Repressive Complex 2 (PRC2) subunit SUZ12 has been mapped across the entire nonrepeat portion of the genome in human embryonic stem (ES) cells. SUZ12 is distributed across large portions of over two hundred genes encoding key developmental regulators. These genes are occupied by nucleosomes trimethylated at histone H3K27, are transcriptionally repressed, and contain some of the most highly conserved noncoding elements in the genome. PRC2 target genes are preferentially activated during ES cell differentiation and the ES cell regulators OCT4, SOX2, and NANOG cooccupy a significant subset of these genes. These results indicate that PRC2 occupies a special set of developmental genes in ES cells that must be repressed to maintain pluripotency and that are poised for activation during ES cell differentiation (Lee, 2006).
It was striking that SUZ12 occupied many families of genes that control development and transcription. These included 39 of 40 of the homeotic genes found in the HOX clusters and the majority of homeodomain genes. SUZ12 bound homeodomain genes included almost all members of the DLX, IRX, LHX, and PAX gene families, which regulate early developmental steps in neurogenesis, hematopoiesis, axial patterning, tissue patterning, organogenesis, and cell-fate specification. SUZ12 also occupied promoters for large subsets of the FOX, SOX, and TBX gene families. The forkhead family of FOX genes is involved in axial patterning and tissue development from all three germ layers. Mutations in members of the SOX gene family alter cell-fate specification and differentiation and are linked to several developmental diseases. The TBX family of genes regulates a wide variety of developmental processes such as gastrulation, early pattern formation, organogenesis, and limb formation. Thus, the genes preferentially bound by SUZ12 have functions that, when expressed, promote differentiation. This is likely to explain, at least in part, why PRC2 is essential for early development and ES cell pluripotency (Lee, 2006).
A remarkable feature of PRC2 binding at most genes encoding developmental regulators was the extensive span over which the regulator occupied the locus. For the majority (72%) of bound sites across the genome, SUZ12 occupied a small region of the promoter similar in size to regions bound by RNA polymerase II. For the remaining bound regions, SUZ12 occupancy encompassed large domains spanning 2–35 kb and extending from the promoter into the gene. A large portion of genes encoding developmental regulators (72%) exhibited these extended regions of SUZ12 binding. In some cases, binding encompassed multiple contiguous genes. For instance, SUZ12 binding extended ~100 kb across the entire HOXA, HOXB, HOXC, and HOXD clusters but did not bind to adjacent genomic sequences, yielding a highly defined spatial pattern. In contrast, clusters of unrelated genes, such as the interleukin 1-β cluster, were not similarly bound by SUZ12. Thus, genes encoding developmental regulators showed an unusual tendency to be occupied by PRC2 over much or all of their transcribed regions (Lee, 2006).
Previous studies have noted that many highly conserved noncoding elements of vertebrate genomes are associated with genes encoding developmental regulators. Given SUZ12's strong association with this class of genes, the possibility that SUZ12 bound regions are associated with these highly conserved elements was investigated. Inspection of individual genes suggested that SUZ12 occupancy was associated with regions of sequence conservation. Eight percent of the approximately 1,400 highly conserved noncoding DNA elements were found to be associated with the SUZ12 bound developmental regulators. Using entries from the PhastCons database of conserved elements (Siepel, 2005), it was found that SUZ12 occupancy of highly conserved elements was highly significant. Since PRC2 has not been shown to directly bind DNA sequences, it is expected that specific DNA binding proteins occupy the highly conserved DNA sequences and may associate with PRC2, which spreads and occupies adjacent chromatin. Thus, the peaks of SUZ12 occupancy might not be expected to precisely colocate with the highly conserved elements, even if these elements are associated with PRC2 recruitment (Lee, 2006).
The observation that OCT4, SOX2, and NANOG are bound to a significant subset of developmental genes occupied by PRC2 supports a link between repression of developmental regulators and stem cell pluripotency. Like PRC2, OCT4 and NANOG have been shown to be important for early development and ES cell identity. It is possible, therefore, that inappropriate regulation of developmental regulators that are common targets of OCT4, NANOG, and PRC2 contributes to the inability to establish ES cell lines in OCT4, NANOG, and EZH2 mutants (Lee, 2006).
Polycomb group protein Ezh2, one of the key regulators of development in organisms from flies to mice, exerts its epigenetic function through regulation of histone methylation. This study reports the existence of the cytosolic Ezh2-containing methyltransferase complex, and the function of this complex has been tied to regulation of actin polymerization in various cell types. Genetic evidence supports the essential role of cytosolic Ezh2 in actin polymerization-dependent processes such as antigen receptor signaling in T cells and PDGF-induced dorsal circular ruffle formation in fibroblasts. Revealed function of Ezh2 points to a broader usage of lysine methylation in regulation of both nuclear and extra-nuclear signaling processes (Su, 2005; full text of article).
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