1Stanford University Medical School, Room B211, Beckman Center, 279 Campus Drive, Stanford, California 94305, USA. 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 rep- licated and transcribed in stable, tissue-specific patterns. The basic unit of chromatin assembly is the nucleosome1, which compacts DNA about sevenfold. However, because the overall level of compaction of the ver- tebrate 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 chro- matin: 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 dis- solution. 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 maternal-effect 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 mobility4. Therefore, it is clear that better in vitro assays are needed to tease apart their biological functions. With the advent of genome-wide analysis techniques such as combining chromatin immunoprecipitation with serial analysis of gene expression (ChIP���SAGE) or with massively parallel sequencing (ChIP���Seq), our understanding of chromatin regulation has improved markedly5. These approaches, combined with rapid RNA interference (RNAi) screening and simpler genetic methods, are allowing a new appreciation of the role of the ATP-dependent remodellers in development, particularly in stem cells. Here, we review the key developmental roles of the four classes of ATP- dependent chromatin-remodelling enzyme in Drosophila melanogaster and mice, and we present evidence that these remodellers have an impor- tant role in establishing and maintaining the pluripotency of embryonic stem cells, perhaps as a result of the unique configuration of the chromatin ���landscape��� of a pluripotent cell. These studies show that chromatin remodellers consist of a large number of assembled complexes, some of which are cell-type specific and developmental-stage specific. Many of these assemblies have specialized and largely non-redundant functions during development. ATP-dependent chromatin-remodelling families ATP-dependent chromatin-remodelling complexes seem to have evolved to accommodate the major changes in chromatin regulation that occurred during the evolution of vertebrates from unicellular eukaryotes (Box 1). As an example, complexes of the SWI/SNF family, which is one of the most-studied families of chromatin-remodelling complexes, have lost, gained and shuffled subunits during evolution from yeast to vertebrates. In particular, the transition to vertebrate chromatin-remodelling com- plexes involved the expansion of several of the gene families encoding the subunits and the use of combinatorial assembly, which together are predicted to allow the formation of several hundred complexes. But what is the advantage of combinatorial assembly? One of the surprises of the genomic era is the relatively small number of genes that are present in vertebrates but not in flies (D. melanogaster). Hence, the greater complexity of vertebrates cannot be attributed to an increase in gene number. Instead, the vertebrate genome, which is about 30-fold larger than the fly genome, contains more genetic regulatory information outside protein-coding genes. Perhaps in response to this expansion of the genome, another strategy was used to regulate chromatin: combinatorial diversity. Current evidence indicates that many vertebrate chromatin-regulatory complexes are assembled combinatorially (see ref. 6 for a review), thereby greatly expanding the potential for diverse gene-expression patterns compared with unicellular eukaryotes. Argu- ably, the greatest need for diverse patterns of gene expression occurs in the development and function of the brain, and it may be no accident that an extraordinary diversity of neural phenotypes is emerging from genetic studies of the subunits of chromatin remodellers in the nervous system (see ref. 7 for a review). The evolutionarily conserved SWI-like ATP-dependent chromatin- remodelling complexes can be broadly divided into four main families on the basis of the sequence and structure of the ATPase subunit: SWI/SNF, ISWI, CHD and INO80 complexes. However, many of the predicted SWI/ SNF-like ATPases do not fit any of these classes and await characterization. Chromatin remodelling during development Lena Ho1 & Gerald R. Crabtree1 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. 474 INSIGHT���REVIEW NATURE|Vol 463|28 January 2010|doi10.1038/nature08911 �� 2010 Macmillan Publishers Limited. All rights reserved

Snf6 Snf11 Sn f6 S nf1 1 Arp7 Arp9 Swi2 Snf5 Swp29 Swi1 Swi3 Swi3 Swp82 Swp73 SNR1 BAP111 BRM Actin MOR MOR SAYP BAP55 BAP 60 BAP170, OSA BAF47 BRG1, BRM BAF57 BAF 155 BAF 170 Actin BRD7, 9 BAF200, BAF250A, B Yeast Swi/Snf complex Monomorphic Transcriptional activation Transcriptional activation Transcriptional repression Transcriptional activation Transcriptional repression Tumour suppressors Mouse BAF complexes Polymorphic Drosophila melanogaster BAP complexes Dimorphic Nucleosomes Increased genome size and vertebrate organogenesis Multicellularity, DNA methylation, linking histones DNA-binding domain Bromodomain PHD Chromodomain or chromoshadow domain Polybromo (BAF180) Polybromo (BAF180) BAF53A, B PtdIns(4,5)P2? BAF45A, B, C, D BAF60A, B, C Why does the regulation of a genome require so many functionally non-redundant ATP-dependent chromatin remodellers if they all act to increase nucleosome mobility? Emerging evidence supports at least two possible explanations. First, new roles and molecular functions of chromatin remodellers have been discovered recently. For example, ISWI complexes have been shown to be required for maintaining the higher- order structure of the D. melanogaster male X chromosome8, and INO80 complexes are involved in telomere regulation, chromosome segregation, and checkpoint control and DNA replication during cell division (see ref. 9 for a review). Hence, it is becoming clear that SWI-like remodellers are intricately involved in many aspects of cell biology beyond transcription. Second, even within their traditional role of transcriptional regulation, ATP-dependent chromatin remodellers do not function in a consistent manner. Brahma-associated factor (BAF) complexes, which belong to the SWI/SNF family, can function as both transcriptional activators and repressors and can even switch between these two modes of action at the same gene10. In addition, tissue-specific BAF complexes have been reported to interact with a variety of transcription factors in different cell types (see ref. 11 for a review), allowing the complexes to take on context- dependent functions arising from their different interaction partners. For these reasons, the roles of ATP-dependent chromatin remodelling may be wider, yet more precise and programmatic, than was previously thought. Indeed, modulating the expression of a single target gene can partly suppress the phenotypes of mutations in the BAF complex in the heart12 and in post-mitotic neurons13. This focus on a single target is also seen for polycomb group (PcG) proteins. These proteins mediate tran- scriptional repression and often oppose the function of trithorax group (TrxG) genes such as those encoding BRG1 and MLL (discussed in the During the evolution of multicellularity and complex body plans, the demand for tissue-specific and developmental-stage-specific expression of genes coincides with increased complexity in chromatin organization and in strategies for chromatin regulation. In the figure, the arrows represent the timescale of evolution, and the appearance of specific strategies of chromatin regulation is indicated, together with relevant important developments in eukaryotic evolution. For instance, the chromatin of yeast (Saccharomyces cerevisiae), which is unicellular, is simpler than that of vertebrates and does not contain linker histones or methylated DNA, the latter of which is also rare in Drosophila melanogaster. ATP-dependent chromatin-remodelling enzymes also evolved, and we take SWI/SNF complexes as an example. In the figure, the homologous subunits of these complexes in yeast, D. melanogaster and mice are shown as similar shapes of the same colour, allowing them to occupy specific positions in the illustration of the complex, as in a jigsaw puzzle. The domains that enable the subunits to interact with DNA are depicted at the surface of each protein, as explained in the key. In yeast, these complexes are monomorphic in composition and seem to contribute mainly, if not exclusively, to transcriptional activation and transcriptional elongation. The evolutionary emergence of multicellular organisms was accompanied by the loss of some of the subunits that are present in yeast Swi/Snf complexes (Snf6, Snf11, Swp29 and Swp82) and the gain of others. Unlike in yeast, there are two D. melanogaster SWI/SNF complexes ��� the Brahma (BRM)-associated proteins (BAP) complex and the polybromo-containing BAP (PBAP) complex, and these can mediate transcriptional activation and transcription repression. In the figure, these are depicted collectively as BAP complexes. In the transition to vertebrate complexes, there was a large increase in the number of possible complexes as a result of vertebrates gaining the ability to combinatorially assemble several subunits encoded by gene families. The possible subunits at each position are listed in the figure (for example, BAF60A, B, C indicates that one of these three subunits is present). Some assemblies of the vertebrate SWI/SNF complexes known as brahma-associated factor (BAF) complexes are tissue-specific and have unique developmental roles, for example the npBAF complex (which is specific to neuronal progenitors) and the nBAF complex (which is specific to neurons) (Fig. 1). Other assemblies might coexist in a specific cell type and perhaps target specific genes or function together with specific transcription factors. As in D. melanogaster, PBAF complexes are a subset of mammalian BAF complexes defined by the incorporation of polybromo (also known as BAF180) and BAF200 (also known as ARID2), although these were purified from HeLa extracts and may represent partly assembled complexes. Thus, although the fundamental activity of promoting nucleosome mobility is highly conserved from yeast to humans, additional mechanisms that have not yet been discovered could account for the evolution of functionally different complexes. It is not known whether these ideas can be generalized to other ATP-dependent remodelling enzymes. BRD, bromodomain-containing protein BRG1, brahma-related gene 1 MOR, Moira PHD, plant homeodomain PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate SAYP, supporter of activation of yellow protein SNR1, Snf5-related protein 1. Box 1 | Evolutionary diversification of SWI/SNF complexes 475 NATURE|Vol 463|28 January 2010 REVIEW���INSIGHT �� 2010 Macmillan Publishers Limited. 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