Subunits of mammalian SWI/SNF (mSWI/SNF, also called BAF) complexes have recently been implicated as tumor suppressors in a number of human malignancies. To understand the full extent of their involvement, we conducted a proteomic analysis of purified endogenous mSWI/SNF complexes. Our studies revealed several new dedicated, stable subunits not found in the yeast SWI/SNF complex including Bcl7a, b and c, Bcl11a and b, Brd9 and SS18. Incorporating these novel members, we determined the frequency of mSWI/SNF subunit mutations in recent exome- and whole-genome sequencing studies of primary human tumors. Surprisingly, mSWI/SNF subunits are mutated in 19.6% of all human tumors reported in 44 exome sequencing studies. Our analysis suggests that specific subunits protect against cancer in specific tissues. In addition, we find that mutations to more than one subunit, which we define as a type of compound heterozygosity, are prevalent in certain cancers. Our studies demonstrate that mSWI/SNF is the most frequently mutated chromatin-regulatory complex (CRC) in human cancer and that in contrast to other known tumor suppressors and oncogenes surveyed, mSWI/SNF is broadly mutated, similar to TP53. Thus, proper functioning of these polymorphic chromatin regulatory complexes may constitute a major mechanism of human tumor suppression.

New methods for the genome-wide analysis of chromatin are providing insight into its roles in development and their underlying mechanisms. Current studies indicate that chromatin is dynamic, with its structure and its histone modifications undergoing global changes during transitions in development and in response to extracellular cues. In addition to DNA methylation and histone modification, ATP-dependent enzymes that remodel chromatin are important controllers of chromatin structure and assembly, and are major contributors to the dynamic nature of chromatin. Evidence is emerging that these chromatin-remodelling enzymes have instructive and programmatic roles during development. Particularly intriguing are the findings that specialized assemblies of ATP-dependent remodellers are essential for establishing and maintaining pluripotent and multipotent states in cells.An essential aspect of building a mammalian cell is packing 1.7 metres of DNA into a 5-micrometre nucleus in a form that allows it to be replicated and transcribed in stable, tissuespecific patterns. The basic unit of chromatin assembly is the nucleosome 1 , which compacts DNA about sevenfold. However, because the overall level of compaction of the vertebrate genome is several thousand fold, relatively little of the DNA in vertebrates is present on simple nucleosomal templates in vivo. Instead, most chromatin is present in undefined, highly compacted structures that remain available for the induction of developmental programs that specify cell fate and morphogenesis.At least three processes control the assembly and regulation of chromatin: DNA methylation (see ref. 2 for a review); histone modifications (see ref. 3 for a review); and ATP-dependent chromatin remodelling, which is the focus of this Review. ATP-dependent remodelling seems to be crucial for both the assembly of chromatin structures and their dissolution. About 30 genes encode the ATPase subunits of these complexes in mammals. With few exceptions, these ATPases seem to be genetically non-redundant, with mutation of the encoding genes often having severe effects on the early embryo or giving rise to maternaleffect phenotypes (in which the phenotype of the embryo reflects the genotype of the mother). Indeed, in many cases, the genes encoding the ATPases or their subunits are haploinsufficient (that is, one copy is insufficient for development), indicating that their role in specific processes is rate limiting. Despite their genetic non-redundancy, the various ATPases seem to have similar activities when studied in vitro: they all increase nucleosome mobility 4 . Therefore, it is clear that better in vitro assays are needed to tease apart their biological functions.Correspondence should be addressed to G.R.C. (crabtree@stanford.edu). Author Information Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2011 March 18. NIH-PA Au...

Mammalian SWI/SNF [also called BAF (Brg/Brahma-associated factors)] ATP-dependent chromatin remodeling complexes are essential for formation of the totipotent and pluripotent cells of the early embryo. In addition, subunits of this complex have been recovered in screens for genes required for nuclear reprogramming in Xenopus and mouse embryonic stem cell (ES) morphology. However, the mechanism underlying the roles of these complexes is unclear. Here, we show that BAF complexes are required for the self-renewal and pluripotency of mouse ES cells but not for the proliferation of fibroblasts or other cells. Proteomic studies reveal that ES cells express distinctive complexes (esBAF) defined by the presence of Brg (Brahma-related gene), BAF155, and BAF60A, and the absence of Brm (Brahma), BAF170, and BAF60C. We show that this specialized subunit composition is required for ES cell maintenance and pluripotency. Our proteomic analysis also reveals that esBAF complexes interact directly with key regulators of pluripotency, suggesting that esBAF complexes are specialized to interact with ES cell-specific regulators, providing a potential explanation for the requirement of BAF complexes in pluripotency.BAF complexes ͉ BAF155 ͉ Brg E S cells are pluripotent cells capable of both limitless selfrenewal and differentiation into all embryonic lineages. These abilities are conferred by various mechanisms, including transcription factors (1-3), possibly Polycomb complexes (4, 5), microRNAs (6), and histone modification enzymes (7) that work in coordination to maintain the expression of pluripotency genes while repressing lineage-determinant genes. The involvement of such mechanisms in pluripotency has been investigated extensively in recent years (reviewed in ref. 8), but the role of chromatin remodeling enzymes remains unclear.The mammalian genome encodes about 30 SWI2/SNF2-like ATPases, which are assembled into SWI/SNF-like complexes with ATP-dependent chromatin remodeling activity. Of these, Brg and Brm are alternative ATPases of a family of 2-MDa multisubunit SWI/SNF or BAF complexes and make up the prototypic mammalian SWI/SNF-like chromatin remodeling complexes (9, 10). BAF complexes have been shown to be essential for many aspects of mammalian development (11-13). A role of BAF complexes in pluripotency is suggested by observations that deletion of Brg, BAF155 (or Srg3), and BAF47 (or hSNF5) all lead to peri-implantation lethality and failure of the totipotent cells that give rise to both the inner cell mass and trophoblast to survive and grow (14-16). The catalytic ATPase subunit, Brg, also was recovered in screens for factors essential for nuclear reprogramming (17) and to ES cell morphology (18). In addition, ES cells lacking BAF250 have defects in ES cell maintenance and differentiation (19,20). However, the mechanism by which BAF complexes help to establish and maintain pluripotency is not understood.In vitro, BAF complexes use energy generated from ATP hydrolysis to alter DNA-nucleosome contacts (21) and can also e...

We report here the discovery and characterization of a gene, ELABELA (ELA), encoding a conserved hormone of 32 amino acids. Present in human embryonic stem cells, ELA is expressed at the onset of zebrafish zygotic transcription and is ubiquitous in the naive ectodermal cells of the embryo. Using zinc-finger-nuclease-mediated gene inactivation in zebrafish, we created an allelic series of ela mutants. ela null embryos have impaired endoderm differentiation potential marked by reduced gata5 and sox17 expression. Loss of Ela causes embryos to develop with a rudimentary heart or no heart at all, surprisingly phenocopying the loss of the apelin receptor (aplnr), which we show serves as Ela's cognate G protein-coupled receptor. Our results reveal the existence of a peptide hormone, ELA, which, together with APLNR, forms an essential signaling axis for early cardiovascular development.

Epigenetic modifications must underlie lineage-specific differentiation as terminally differentiated cells express tissue-specific genes, but their DNA sequence is unchanged. Hematopoiesis provides a well-defined model to study epigenetic modifications during cell-fate decisions, as multipotent progenitors (MPPs) differentiate into progressively restricted myeloid or lymphoid progenitors. While DNA methylation is critical for myeloid versus lymphoid differentiation, as demonstrated by the myeloerythroid bias in Dnmt1 hypomorphs1, a comprehensive DNA methylation map of hematopoietic progenitors, or of any multipotent/oligopotent lineage, does not exist. Here we examined 4.6 million CpG sites throughout the genome for MPPs, common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and thymocyte progenitors (DN1, DN2, DN3). Dramatic epigenetic plasticity accompanied both lymphoid and myeloid restriction. Myeloid commitment involved less global DNA methylation than lymphoid commitment, supported functionally by myeloid skewing of progenitors following treatment with a DNA methyltransferase inhibitor. Differential DNA methylation correlated with gene expression more strongly at CpG island shores than CpG islands. Many examples of genes and pathways not previously known to be involved in choice between lymphoid/myeloid differentiation have been identified, such as Arl4c and Jdp2. Several transcription factors, including Meis1, were methylated and silenced during differentiation, suggesting a role in maintaining an undifferentiated state. Additionally, epigenetic modification of modifiers of the epigenome appears to be important in hematopoietic differentiation. Our results directly demonstrate that modulation of DNA methylation occurs during lineage-specific differentiation and defines a comprehensive map of the methylation and transcriptional changes that accompany myeloid versus lymphoid fate decisions.

To understand the mechanism underlying the roles of these complexes in ES cells, we performed high-resolution genome-wide mapping of the core ATPase subunit, Brg, using ChIP-Seq technology. We find that esBAF, as represented by Brg, binds to genes encoding components of the core ES transcriptional circuitry, including Polycomb group proteins. esBAF colocalizes extensively with transcription factors Oct4, Sox2 and Nanog genome-wide, and shows distinct functional interactions with Oct4 and Sox2 at its target genes. Surprisingly, no significant colocalization of esBAF with PRC2 complexes, represented by Suz12, is observed. Lastly, esBAF colocalizes with Stat3 and Smad1 genome-wide, consistent with a direct and critical role in LIF and BMP signaling for maintaining self-renewal. Taken together, our studies indicate that esBAF is an essential component of the core pluripotency transcriptional network, and might also be a critical component of the LIF and BMP signaling pathways essential for maintenance of self-renewal and pluripotency.BAF complexes ͉ Brg ͉ SWI/SNF ATP-dependent chromatin remodeling E mbryonic stem cells (ES) maintain an epigenetic state that enables both self-renewal and differentiation into all embryonic lineages (1). Recent studies reveal that in ES cells Oct4, Sox2, Nanog, and Klf4 elaborate a core transcriptional circuitry (2-4), working in coordination with Polycomb complexes (5, 6), microRNAs (7), and histone modification enzymes (8) to stably maintain the expression of pluripotency genes, and to repress lineage determinant genes. This transcriptional circuitry is kept in exquisite balance, because it can be perturbed both by reducing or increasing the levels of core regulators such as Oct4, Sox2, and Nanog, causing ES cells to lose self-renewal ability and/or pluripotency (9-14). At the same time, ATP-dependent chromatin remodeling enzymes in the Tip60/p400 and SWI/SNF families have been recently shown to be crucial for the maintenance and function of ES cells (15). Recent findings by us and others have shown that components of mammalian SWI/SNF (or BAF, Brg/Brahma Associated Factors) complex, Brg, BAF155, and BAF250A are crucial for the proliferation, self-renewal and pluripotency of ES cells (16,17,18). ES cells deficient in Brg maintain the expression of Oct4, Sox2, and Nanog for several cell divisions but rapidly lose colony morphology and proliferative capacity characteristic of ES cells (16). Upon prolonged absence of Brg, remaining ES cells down-regulate pluripotency markers such as Oct4 and Sox2 (16,17), reflecting the complete loss of ES cell identity secondary to the effects of Brg depletion or suggesting that Brg is required to maintain stable expression of these markers over many cell divisions. In addition, the composition of BAF complexes in ES cells (esBAF) is biochemically and functionally specialized. esBAF complexes are defined by the incorporation of Brg but not Brm, BAF155 but not BAF170, and BAF60A but not BAF60C (16). When esBAF complexes are altered by enforced incorpora...

Signaling by the cytokine LIF and its downstream transcription factor, STAT3, prevents differentiation of pluripotent embryonic stem cells (ESCs) by opposing MAP kinase signaling. This contrasts with most cell types where STAT3signaling induces differentiation. We find that STAT3binding across the pluripotent genome is dependent upon Brg, the ATPase subunit of a specialized chromatin remodeling complex (esBAF) found in ESCs. Brg is required to establish chromatin accessibility at STAT3 binding targets, in essence preparing these sites to respond to LIF signaling. Moreover, Brg deletion leads to rapid Polycomb (PcG) binding and H3K27me3-mediated silencing of many Brg-activated targets genome-wide, including the target genes of the LIF signaling pathway. Hence, one crucial role of Brg in ESCs involves its ability to potentiate LIF signaling by opposing PcG. Contrary to expectations, Brg also facilitates PcG function at classical PcG target including all four Hox loci, reinforcing their repression in ESCs. These findings reveal that esBAF does not simply antagonize PcG, but rather, the two chromatin regulators act both antagonistically and synergistically with the common goal of supporting pluripotency.

Preeclampsia (PE) is a gestational hypertensive syndrome affecting between 5 and 8% of all pregnancies. Although PE is the leading cause of fetal and maternal morbidity and mortality, its molecular etiology is still unclear. Here, we show that ELABELA (ELA), an endogenous ligand of the apelin receptor (APLNR, or APJ), is a circulating hormone secreted by the placenta. but not knockout pregnant mice exhibit PE-like symptoms, including proteinuria and elevated blood pressure due to defective placental angiogenesis. In mice, infusion of exogenous ELA normalizes hypertension, proteinuria, and birth weight. ELA, which is abundant in human placentas, increases the invasiveness of trophoblast-like cells, suggesting that it enhances placental development to prevent PE. The ELA-APLNR signaling axis may offer a new paradigm for the treatment of common pregnancy-related complications, including PE.