ATP-dependent chromatin-remodeling complexes and pluripotency Perhaps the genetically most well-documented chromatin regulators of the pluripotent state are the ATP-dependent chromatin-remodeling enzymes. In mammalian cells, approximately 30 genes encode ATP-dependent chromatin regulators that can be roughly grouped into families based on the structural features of the ATPase domain. These include Brg, Brahma/Brm, SNF2H, SNF2L, CHD1, and Mi2-beta, all of which play genetically non-redundant roles. These characterized ATPases are assembled into complexes such as BAF (also called mSWI/SNF), NuRD, ISWI, CDH1, and Tip60 and interact with several other subunits, indicating that perhaps several hundred genes are involved in ATP-dependent chromatin regulation.
In mammalian cells, the Brm (Brahma) and Brg ATPases are assembled with 12 other subunits into BAF or mSWI/SNF complexes that share certain homologs with yeast SWI/SNF complexes, but have lost, gained, and shuffled subunits with other classes of ATPases. Highlighting fundamental mechanistic differences in the control of gene expression, mammalian BAF complexes often repress transcription from a distance, whereas the yeast SWI/SNF complex regulates all known targets by activation from promoters. Unlike the homologous complexes in yeast, flies, and worms, most subunits of mammalian BAF complexes are encoded by gene families and the complexes are combinatorially assembled (
Ho et al. 2009b;
Lemon et al. 2001;
Lessard et al. 2007;
Takeuchi & Bruneau 2009;
Wang et al. 1996a,
b;
Wu et al. 2007,
2009). In certain cases (see below), complex composition confers functional specificity to these complexes.
Genetic studies in mice have demonstrated that BAF complexes are essential for early embryonic development and pluripotency. In mice, inactivation of most BAF subunits including the ATPase Brg as well as the BAF47, BAF57, BAF60, BAF155, BAF180, and BAF250a subunits results in early embryonic lethality, and in the case of Brg, BAF47, and BAF155, a failure of formation of pluripotent cells (
Bultman et al. 2006,
Doan et al. 2004,
Gao et al. 2008,
Guidi et al. 2001,
Kim et al. 2001,
Klochendler-Yeivin et al. 2000,
Lickert et al. 2004,
Roberts et al. 2000). Conversely, mice with deletion of the alternative ATPase Brm are viable and approximately 15% larger than controls (
Reyes et al. 1998). Maternally derived Brg is required for zygotic genome activation, a nuclear reprogramming event that establishes totipotency in the cleavage-stage embryo and is required for embryonic development (
Bultman et al. 2000). Consistent with this, nuclear reprogramming of permeabilized somatic human cells using extracts from
Xenopus laevis eggs and early embryos requires Brg, demonstrating the importance of these complexes in the establishment of pluripotency (
Hansis et al. 2004). Brg, BAF155, and other components of the complex were also identified in a large-scale RNAi screen targeted against chromatin regulatory factors as being required for the maintenance of ES cell colony morphology (
Fazzio et al. 2008) and in a screen for genes required for Nanog expression (
Schaniel et al. 2009). Interestingly, in these screens, components not characteristic of esBAF were not detected. Recently, components of esBAF were found to facilitate pluripotency (
Singhal et al. 2010).
BAF or mSWI/SNF complexes have been considered to be general regulators of transcription, suggesting that the essential roles of this complex could simply reflect a general role in transcription. However, several observations argue strongly against a general role, but rather for a specific and programmatic role. First, recent proteomics studies by
Ho et al. (2009b) revealed that pluripotent ES cells express distinctive complexes (termed esBAF) defined by the presence of Brg, BAF155, and BAF60a and the absence of Brm, BAF170, and BAF60c subunits (). These studies indicated that the ATPase Brg is essential for the self-renewal ability of pluripotent ES cells. shRNA-mediated depletion of Brg in ES cells generated small colonies with flattened morphology indicative of spontaneous differentiation. These studies also showed that ES cells require a specific esBAF composition with respect to BAF155 and BAF170 subunits. BAF155 depletion in ES cells diminished ES cell proliferation and increased cell death, whereas enforced expression of BAF170 decreased ES cell competitive self-renewal ability and teratoma formation in immunocompromised mice (
Ho et al. 2009b). Similarly, combinatorial assembly of subunits of the BAF250 family regulates esBAF function. BAF250a and BAF250b subunits are both required to maintain ES cell pluripotency and self-renewal, but they differentially regulate the potential of ES cells to develop into specific lineages (
Gao et al. 2008,
Yan et al. 2008). BAF250a and b are alternative subunits and esBAF complexes contain either one or the other, which imply that these subtypes of complexes are dedicated to different, non-redundant pluripotency programs. Mouse embryos lacking BAF250a (ARID1a) form the ICM but do not gastrulate or form mesoderm. ES cells deficient for BAF250a are capable of differentiating into primitive endoderm- and ectoderm-like cells but cannot generate mesoderm-derived cardiomyocytes (
Gao et al. 2008). Conversely, disruption of BAF250b in ES cells results in downregulation of pluripotency genes, reduced proliferation, and increased expression of lineage-specific genes, including markers of mesodermal differentiation. Interestingly, deletion of components of the related PBAF complex, defined by the signature subunit BAF180 or polybromo, leads not to a reduction in pluripotency, but instead to specific late developmental effects (see below). Confirming the importance of the specific subunit composition of esBAF complexes, only esBAF subunits have been detected in RNAi screens for pluripotency of ES cells (
Fazzio et al. 2008,
Schaniel et al. 2009).
An important question regarding the role of esBAF complexes is whether their function is simply to act in a general way, promoting the transcription of whatever genes are active in a given cell type, or whether they function in a programmatic way as an essential component of the core pluripotency circuit. Genome-wide studies of direct targets also strongly support a programmatic and unexpected function. High-resolution genome-wide analysis of Brg-containing esBAF occupancy in ES cells revealed that these complexes bind approximately 3% of the murine genome with an average footprint of approximately 2.1 kb. Transcriptional start sites show a clear peak; however, most peaks are not at the transcriptional start site and many enhancers and silencers are also sites of Brg binding (
Ho et al. 2009a). Although repression at a distance had been previously demonstrated for the CD4 gene in T cells (), this finding was a surprise because the yeast SWI/SNF complex activates all its genomic targets by binding to promoters. This reinforces the apparent mechanistic difference between SWI/SNF and BAF complexes and suggests caution when generalizing between the two complexes. Biochemical and genetic studies indicated that Brg-containing esBAF complexes directly interact with Oct4 and Sox2 and are required for ES self-renewal and pluripotency (
Ho et al. 2009a,
b). esBAF complexes occupy the enhancers and promoters of nearly all genes of the core pluripotency network, such as Oct4, Sox2, c-myc, KLF4, Sall4, TCF3, and Nanog. In addition, esBAF complexes co-occupy target genes of Oct4, Sox2, and Nanog, suggesting a functional interaction between esBAF complexes and the core pluripotency circuitry (). Microarray analysis of the genes acutely affected by conditional deletion of
Brg in ES cells revealed that Brg-containing esBAF complexes function mainly as transcriptional repressors in pluripotent ES cells. Consistent with a role for these complexes in maintaining the expression of stem-cell-specific genes within the correct range for ES cell function, Brg represses a significant number of differentiation-specific genes as well as many targets of the core pluripotency network in these cells (
Ho et al. 2009a,
b). Altogether, these studies suggest that esBAF functionally interacts with Sox2 and Oct4 to refine the expression of pluripotency genes, while repressing the transcription of differentiation-specific genes. This suggests a revision of the conventional view that Trithorax genes maintain the expression of developmental genes, whereas Polycomb group (PcG) genes repress them, and it implies that in the case of stem cells these regulatory circuitries may be more complex.
Combinatorial assembly of ATP-dependent BAF chromatin-remodeling complexes also orchestrates the development of the nervous system. A switch in subunit composition of neural, SWI/SNF-like BAF chromatin-remodeling complexes underlies the transition from proliferating neural stem/progenitors to postmitotic differentiated neurons (
Lessard et al. 2007). Most compellingly, the self-renewal and proliferative activities of neural stem/progenitor cells require a specialized npBAF complex containing the double–plant-homeodomain (PHD) domain BAF45a/d subunit and the actin-related protein BAF53a assembled on the Brg/Brm ATPases. The dynamic exchange of these progenitor-specific subunits for the homologous BAF45b, BAF45c, and BAF53b subunits in postmitotic neurons orchestrates cell-cycle withdrawal and the acquisition of neuronal properties. The subunits of the npBAF complex are essential for neural-progenitor proliferation, and mice with reduced dosage for the genes encoding its subunits have defects in neural-tube closure similar to those in human spina bifida. BAF45a expression appears sufficient for inducing proliferation of neural progenitors, implying an instructive role of npBAF complexes. In contrast, the BAF45b/BAF53b-containing neuron-specific BAF (nBAF) complex is essential for postmitotic neuronal function, including activity-dependent dendritic outgrowth, via its association with the Ca
2+-responsive dendritic regulator CREST (
Wu et al. 2007). Remarkably, these studies indicated that the highly homologous BAF53a protein, which is a component of neural-progenitor and non-neural BAF complexes, cannot replace BAF53b's role in dendritic development and that this functional specificity of BAF53b is conferred by its actin fold subdomain 2. More recent studies have found that microRNA-mediated regulation of specific subunits of BAF chromatin-remodeling complexes is essential for mitotic exit and the onset of dendritic morphogenesis in the vertebrate nervous system (
Yoo et al. 2009) (). In postmitotic neurons, BAF53a repression is mediated by sequences in the 3′ untranslated region corresponding to the recognition sites for microRNAs miR-9* and miR-124, which are selectively expressed in these cells. Mutation of these sites leads to persistent expression of BAF53a and defective activity-dependent dendritic outgrowth in neurons, whereas overexpression of miR-9* and miR-124 in neural stem/progenitor cells impaired cellular proliferation. Altogether, these studies indicate that functional specificity to ATP-dependent chromatin-remodeling complexes is achieved, at least in part, by miRNA-mediated switching of specific subunits, allowing differential interaction with specific factors that promote cell-lineage commitment and terminal differentiation.
Finally, deletion of Brg, BAF180, and BAF60c subunits in the mouse has been associated with distinct cardiac developmental outcomes. Mice lacking
BAF180 or polybromo have specific defects in formation of the ventricular chambers of the heart that are consistent with a role for this subunit in response to retinoic acid. Interestingly, earlier retinoic acid–dependent processes do not seem to be affected (
Wang et al. 2004). Conditional mutation of
Brg in the heart indicated that Brg maintains cardiomyocytes in an embryonic state (promotes their proliferation and preserves differentiation) by interacting with histone deacetylases (HDACs) and poly (ADP ribose) polymerase (PARP) and controlling developmental gene expression. In adult cardiomyocytes, Brg is turned off but can be reactivated by cardiac stress to induce a pathological program of gene expression by interacting with HDAC and PARP (
Hang et al. 2010). Similarly, RNAi interference of
BAF60c in the early mouse embryo revealed a specific requirement in skeletal and cardiac development (
Lickert et al. 2004). More recent studies have shown that BAF60c is critical to establish the regions of the embryo that give rise to the heart, a function quite different from that of BAF180 in cardiac development. Remarkably, BAF60c appears to have an instructive role in heart development, because its injection into noncardiogenic regions of the embryo can result in the generation of beating cardiomyocytes (
Takeuchi & Bruneau 2009). These studies suggest the existence of a specialized cBAF complex. However, purification of this putative cardiogenic complex has not yet been reported.
NuRD complexes Mammalian nucleosome remodeling deacetylase (NuRD) complexes contain at least six subunits that are encoded by gene families (
Bowen et al. 2004). These complexes possess both ATP-dependent chromatin-remodeling and HDAC activities (
Denslow & Wade 2007). The activity of Hdac1 and Hdac2 within the complexes requires the presence of the chromodomain-containing Mi2a and Mi2b, which are SNF2/SWI2-like ATPase subunits. Other subunits of these complexes include the methyl-CpG-binding proteins Mbd 1/2/3, the metastasis-associated Mta1/2/3 proteins, the WD40-containing RbAP46 and RbAP48 proteins, and two zinc fingers proteins, p66a and p66b. Mi2b-containing NuRD complexes, which possess both transcriptional repressive and activating functions, are required for hematopoietic stem cell self-renewal and multilineage differentiation (
Wade et al. 1999,
Williams et al. 2004,
Yoshida et al. 2008). Several subunits of these complexes are also important for ES cell pluripotency and differentiation. ES cells lacking Mbd3 are viable but fail to form a stable NuRD complex and display a profound defect in differentiation that results in persistent self-renewal. Mbd3-deficient ES cells can be maintained in the absence of leukemia inhibitory factor and can initiate differentiation in embryoid bodies or chimeric embryos, but they fail to commit to developmental lineages, except when induced with retinoic acid (
Kaji et al. 2006). Recent studies indicated that Mbd3 is required for the ICM of blastocysts to develop into mature epiblast after implantation. Expression of the pluripotency factors Oct4, Nanog, or Sox2 and their targets did not seem to be affected in the absence of MBD3 (methyl-binding domain 3), but transcription of genes that are normally expressed at the preimplantation stage and then silenced failed to be repressed. Unlike Mbd3-null ES cells, Mbd3-deficient ICMs grown ex vivo fail to expand Oct4-positive pluripotent cells despite producing robust endoderm outgrowth (
Kaji et al. 2007). Together, these findings define a role for MBD3 in cell-fate commitment of pluripotent ES cells and epiblast formation after implantation.
Interestingly, a subfamily of NuRD complexes (termed NODE for Nanog and Oct4 associated deacetylase) containing Hdac1/2- and Mta1/2- and near absence (or substoichiometric levels) of Mbd3 and Rbbp7 interacts with the pluripotency factors Nanog and Oct4 (
Liang et al. 2008). NODE HDAC activity seems to be comparable to NuRD, and NODE is recruited to Nanog/Oct4 target genes independently of Mbd3 in ES cells. In contrast to Mbd3 loss-of-function, knockdown of NODE subunits in ES cells increased expression of developmentally regulated genes and promoted differentiation. shRNA-mediated depletion of Mta1 also has different effects than MBD3 depletion on target genes. In contrast to Mbd3, which is required to repress preimplantation genes, Mta1 is required to repress lineage-specific factors, such as Gata6 and Foxa2. Thus, a subfamily of NuRD complexes containing Hdac1/2- and Mta1/2 is essential to maintain pluripotency by interacting with components of the core pluripotency circuitry. The question remains whether different NuRD-related complexes possess distinct enzymatic activities and play generic or specialized roles in the regulation of stem cell self-renewal, proliferation, and differentiation.
ISWI complexes The ISWI family of chromatin remodelers contains two to four subunits based on the alternative ATPases SNF2L and SNF2H, the mammalian homologs of the
Drosophila ISWI ATPase (
Eberharter & Becker 2004). ISWI subunits differ in their expression pattern and assemble into at least seven distinct complexes. SNF2L is a component of the NURF complex, together with BPTF and RbpAp46/48. The PHD-domain-containing BPTF subunit appears to mediate the selective recruitment of ISWI complexes to target genes with transcriptionally active histone marks such as H3K4me3 (
Wysocka et al. 2006), but genetic studies on mice lacking the BPTF PHD domain will be essential to confirm this result. BPTF null embryos have growth defects leading to their death by E8.5 (
Goller et al. 2008), and BPTF deletion in ES cells impairs their ability to form the mesodermal, endodermal, and ectodermal lineages (
Landry et al. 2008).
The chromatin-remodeling activity of at least six subfamilies of ISWI complexes, namely hACF, hCHRAC, hWICH, RSF, NoRC, and SNF2H/cohesin, is regulated by the presence of the alternative ATPase subunit SNF2H (
Eberharter & Becker 2004). Snf2h
−/− embryos die during the periimplantation stage, and Snf2h is required for the survival and proliferation of both the trophectoderm and ICM (
Stopka & Skoultchi 2003). As genetic analyses indicate that ISWI complexes play important roles in diverse biological processes (such as transcriptional regulation, heterochromatin replication, chromatin assembly, and the formation of higher-order chromatin structure), it will be interesting to investigate whether combinatorial assembly of ISWI subunits assembled on SNF2H and SNF2L generates a family of heterogeneous complexes with distinct and specialized functions in embryonic and adult stem cells (
Bozhenok et al. 2002,
Eberharter et al. 2001,
Hamiche et al. 1999,
Ito et al. 1999,
Langst et al. 1999,
Poot et al. 2004,
Strohner et al. 2001).
Tip60-p400 complexes The Tip60-p400 family of complexes [whose subunits, on the basis of tagging overexpressed proteins, appear to be composed of Ruvbl1, Ruvbl2, Dmap1, Ep400 (p400), Htatip (tip60), Trrap, Tip49 (TAP54α), Tip48 (TAP54β), BAF53a, β-actin, E(Pc), and MRGBP] possesses both histone acetyltransferase and chromatin-remodeling activities and can act either as positive or negative regulators of transcription (
Ikura et al. 2000,
Cai et al. 2003). Tip60-p400 transcriptional activity seems to be mediated, at least in part, by the incorporation of the histone variant H2AZ into nucleosomes and by the catalysis of histone acetylation at target genes (
Sapountzi et al. 2006,
Squatrito et al. 2006). Embryos lacking Tip60 and Trrap, two components of the Tip60-p400 complexes, also die before implantation (
Gorrini et al. 2007,
Herceg et al. 2001), suggesting a role in early development. Interestingly, Tip60-p400 was recently identified in a large-scale RNAi screen for chromatin-remodeling proteins involved in ES cell function (
Fazzio et al. 2008). Depletion of several subunits of Tip60-p400 complexes inhibited the self-renewal ability of ES cells, impaired their ability to differentiate, and/or generated ES cell colonies with altered morphology without affecting the expression of the pluripotency transcription factors. Chromatin immunoprecipitation experiments indicated that Tip60-p400 colocalizes with the pluripotency factor Nanog and the transcriptionally active histone mark H3K4me3 in ES cells. Interestingly, the authors observed a significant overlap between Tip60-p400 target genes and that of Nanog and further demonstrated that both Nanog and H3K4me3 are required for Tip600-p400 binding at target promoters in ES cells, whereas binding of Tip60-p400 is required to mediate histone H4 acetylation at both activated and repressed target genes in ES cells.
CHD1 complexes Although there is a strong correlation between open chromatin and the undifferentiated state of stem cells, it has long been debated whether open chromatin is necessary for stem cell potential. In support of this idea, RNAi knockdown of the chromatin remodeler Chd1 reduced chromatin decondensation and pluripotency of ES cells (
Gaspar-Maia et al. 2009). Chd1 contains an ATPase SNF2-like helicase domain and belongs to the chromodomain family of proteins (
Woodage et al. 1997). The two chromodomains in Chd1 are essential for recognition of H3K4me2/3 (
Sims et al. 2005) and Chd1 is involved in transcriptional activation in several organisms (
Simic et al. 2003,
Sims et al. 2007,
Stokes et al. 1996). Chromatin immunoprecipitation studies in mouse ES cells indicated that the
Chd1 promoter is bound by several pluripotency-associated factors such as Oct4, Nanog, Sox2, and Zfx (
Chen et al. 2008), highlighting a potential mechanism by which CHD1 complexes function downstream of the pluripotency factors to maintain open chromatin of mouse ES cells and regulate their pluripotency.
Polycomb group genes regulate pluripotency by suppressing developmental as well as metabolic pathways PcG proteins are an evolutionarily conserved family of chromatin regulators known best for their role in establishing and maintaining the silent state of homeotic gene expression during embryonic development (
Ringrose & Paro 2004). Mammalian PcG proteins assemble into at least three biochemically distinct complexes: PRC1, PRC2, and PhoRC. The four core subunits (PHC, CBX, Bmi1, and RING1) of mammalian PRC1 complexes are homologs of
Drosophila Ph, Pc, Psc, and dRing, respectively. Mammalian PRC2 complexes contain EED, SUZ12, and either EZH1 or EZH2. The SET-domain-containing proteins EZH2 and potentially EZH1 of PRC2 are required for the initiation of silencing through the di- and tri-methylation of the K27 residue of histone H3. This modification forms the recruiting mark for the PRC1 complex, which is implicated in the maintenance of gene repression through the formation of higher-order chromatin structures (
Valk-Lingbeek et al. 2004). This process appears to involve Ring1b-mediated monoubiquitination of H2AK119, an activity that is stimulated by the Bmi1 and Mel18 PRC1 subunits (
Elderkin et al. 2007). Although this simple relationship between the two biochemical activities of PRC2 and PRC1 is appealing, genetic evidence in mammals indicates that this sequential action is not used broadly (see below).
A role for PcG proteins in maintaining ES cell identity and pluripotency was first suggested on the basis that most PcG components are required for early embryonic development (mainly PRC2 subunits, see below) (
Pasini et al. 2004,
Shumacher et al. 1996,
Voncken et al. 2003), the self-renewal/maintenance of different types of adult stem cells (
Molofsky et al. 2003,
Park et al. 2003), and the formation of the bivalent chromatin state of stem cells (
Bernstein et al. 2006). EED is required for PRC2 activity and early embryonic development in mice (
Faust et al. 1995,
Shumacher et al. 1996).
Eednull embryos, which lack all detectable H3K27 methylation, display disrupted A/P patterning of the primitive streak during gastrulation and contain excess extraembryonic mesoderm but reduced embryonic mesoderm. Despite the absence of the repressive H3K27me3 mark,
Eednull ES cells can be derived from blastocysts, and chimeric embryo analyses indicated that they are pluripotent, even though they have a tendency to express differentiation-promoting genes (and differentiate spontaneously) in culture (
Boyer et al. 2006,
Chamberlain et al. 2008). Primordial germ cells are specified in
Eednull embryos, suggesting that they can contribute to the germline (
Faust et al. 1995). However, high-contribution
Eednull chimeras have a paucity of mesoderm, suggesting that Eed is required for the specification of embryonic mesoderm (
Faust et al. 1995) and/or for the differentiation or maintenance of multipotent progenitors (
Chamberlain et al. 2008). Similarly, Suz12 is essential for PRC2 activity and its inactivation results in early lethality of mouse embryos (
Pasini et al. 2004). ES cells and the ICM form in the absence of Suz12, and embryos lacking Suz12 produce all three germ layers.
Suz12−/− ES cells are also characterized by global loss of H3K27 tri-methylation (H3K27me3) and higher expression levels of differentiation-specific genes. However, in contrast to Eed, Suz12 is apparently required for differentiation of ES cells in culture, as Suz12
−/− ES cells cannot form neurons after in vitro differentiation, and Suz12
−/− Embryoid bodies fail to form a proper endodermal layer (
Pasini et al. 2007). A molecular explanation for this apparent paradox is not clear, but it may be related to a role of Suz12 in other complexes. Despite the crucial role of EZH2 in the di- and tri-methylation of H3K27 in ES cells, a recent study by Orkin and colleagues showed that EZH2-deficient ES cells can be derived from blastocysts as well as self-renew (
Shen et al. 2008). Surprisingly, known PcG targets (derepressed in EED-deficient ES cells) remained unaffected in EZH2-deficient ES cells and still contained the H3K27me3 repressive mark. This work also revealed that EZH1 exhibits histone methyltransferase activity in vitro and colocalizes with EED at PcG targets. Depletion of EZH1 in EZH2
−/− ES cells was sufficient to remove the repressive H3K27me3 mark from these important developmental targets, demonstrating functional complementation between these two PRC2 subunits. The PRC2-associated PCL2 (Polycomb-like 2) protein was identified in a genome-wide screen for regulators of ES cell self-renewal and pluripotency. Knockdown of
Pcl2 in mouse ES cells resulted in enhanced self-renewal, differentiation defects, and altered patterns of histone methylation (
Walker et al. 2010). Although these studies suggest that PcG proteins may be dispensable for the establishment of pluripotency in ES cells, they suggest that at least some components of PRC2 complexes are required for the maintenance of pluripotency in its strictest meaning (i.e., potential of ES cells to generate all differentiated cell types in a cell-autonomous fashion as well as chimeras with germline potential). At present, it is still not clear why PRC2 mutant embryos die, but it may relate to a failure to assemble mesodermally derived tissues such as blood vessels or withdrawal of essential cytokines and growth factors.
How might PcG genes be involved in regulating aspects of ES cell identity? Genome-wide studies indicated that PcG targets are preferentially activated upon ES cell differentiation, suggesting that they regulate pluripotency by repressing the premature expression of lineagespecific genes (
Bernstein et al. 2006,
Boyer et al. 2006,
Buszczak & Spradling 2006,
Lee et al. 2006) (). Consistently, PRC1 and PRC2 targets in ES cells were enriched in genes involved in developmental patterning, signaling, morphogenesis, and organogenesis (
Boyer et al. 2006,
Lee et al. 2006). A significant subset of PcG target genes was co-occupied by Oct4, Sox2, and Nanog (
Bernstein et al. 2006,
Boyer et al. 2006,
Lee et al. 2006), suggesting functional interaction between PcG proteins and the core pluripotency network (). However, a much larger fraction of combined Oct4/Sox2/Nanog targets are co-occupied by Brg (
Ho et al. 2009a). Finally, recent studies revealed that one of the founding members of the Jumonji C (JmjC) domain protein family, JARID2, forms a stable complex with PRC2 in pluripotent ES cells and promotes its recruitment to target genes while inhibiting its histone methyltransferase activity (
Pasini et al. 2010,
Peng et al. 2009,
Shen et al. 2009). Jarid2-deficient mice form all germ layers and die with defects in the organization of the cardiovascular system at approximately E10.5. In other genetic backgrounds, the mice survive until birth and are fully formed, indicating that pluripotency in the early embryo is not significantly compromised. Surprisingly, Jarid2 is required for the differentiation of mouse ES cells, and activation of genes marked by H3K27me3 and lineage commitments are delayed in JARID2
−/− ES cells. However, one group of investigators found the opposite result, i.e., that Jmjd1a or Jmjd2c depletion leads to enhanced ES cell differentiation (
Loh et al. 2007). One interpretation is that the dynamic regulation of PRC2 activity by JARID2 fine-tunes the relative balance between self-renewal and differentiation decisions in pluripotent ES cells. Why these defects in pluripotency are not seen or are dramatically blunted in the embryo is not clear, but this may become apparent upon a focused analysis of the Jarid2 embryonic phenotype.
One curious feature of the phenotype of PRC2-deficient mice is that the embryos die significantly after gastrulation and slightly before or at the time that an organized vasculature becomes essential for viability (the vascular/oxygenation checkpoint). For example, VEGF-, VEGF receptor-, and calcineurindeficient mice die at about the same time with a similar appearance (
Carmeliet et al. 1996,
Graef et al. 2001,
Fong et al. 1995,
Shalaby et al. 1995). Because cells that simply fail to differentiate properly do not necessarily die, this suggests a fundamental defect in either the metabolism of PRC2-deficient cells or the initiation of a checkpoint-induced cell death. For these reasons, reanalysis of PRC2-deficient embryos may be quite informative and provide a framework for possible mechanisms underlying PRC2 action.
Whereas deletion of any of the PRC2 subunits in mice is embryonic lethal (embryos die with defects in gastrulation 7 to 9 days post-fertilization), mice with deletion of PRC1 subunits, with the exception of Ring1b, are viable, suggesting that the PRC1 complex may be redundant with another mechanism in early development (
Faust et al. 1995,
Pasini et al. 2007). In any case, these genetic observations indicate that it is unlikely that PRC2 functions only to set up later repression by PRC1 (), because this sequential mechanism would lead to similar phenotypes for PRC1 and PRC2 complex family members. However, several PRC1 components are required for the self-renewal/maintenance of different types of multipotent adult stem cells. For example, Bmi1 is required for the maintenance of hematopoietic stem cells (
Lessard & Sauvageau 2003,
Park et al. 2003); leukemic hematopoietic stem cells (
Lessard & Sauvageau 2003); and neural, mammary, lung, and intestinal stem cells (
Dovey et al. 2008,
Liu et al. 2006,
Molofsky et al. 2003,
Pietersen et al. 2008,
Sangiorgi & Capecchi 2008). In addition to Bmi1, several other subunits of PRC1 (Mel18, Phc1/Rae28, Ring1b) and PRC2 (EZH2) complexes are required for hemopoietic stem cell function (
Kajiume et al. 2004,
Kamminga et al. 2006,
Kim et al. 2004,
Ohta et al. 2002). Even though the targets of Polycomb complexes are commonly thought to be developmental genes, a recent study demonstrated that Bmi1 mutant mice show defects in mitochondrial function resulting in the release of reactive oxygen species with subsequent DNA damage. Remarkably, the Bmi defect in many stem cell populations could be repressed with a second mutation in the DNA damage checkpoint gene, CHK2 (
Liu et al. 2009), indicating that a substantial role of Bmi1 in stem cell populations is to control the generation of reactive oxygen species in mitochondria (). If indeed PRC2 functions upstream of PRC1, then there should also be defective mitochondrial function in Suz12, Eed, and Ezh2 mutant mice, possibly explaining early embryonic death. Altogether, these findings support a model in which Polycomb repression could act not only in pluripotent stem cells to ensure proper lineage choice, but also in progenitor cells to guide their further developmental potential by ensuring proper regulation of subtype-specific genes ().
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