SWI/SNF-Brg1 Regulates Self-Renewal and ... - Wiley Online Library

EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS SWI/SNF-Brg1 Regulates Self-Renewal and Occupies Core Pluripotency-Related Genes in Embryonic Stem Cells BENJAMIN L. KIDDER,a STEPHEN PALMER,a JASON G. KNOTTa,b a

EMD Serono Research Institute, Inc., Rockland, Massachusetts, USA; bDevelopmental Epigenetics Laboratory, Department of Animal Science, Michigan State University, East Lansing, Michigan, USA

Key Words. Epigenetics • Genomics • Microarray • Embryonic stem cells • Self-renewal • Pluripotent

ABSTRACT The SWI/SNF-Brg1 chromatin remodeling protein plays critical roles in cell-cycle control and differentiation through regulation of gene expression. Loss of Brg1 in mice results in early embryonic lethality, and recent studies have implicated a role for Brg1 in somatic stem cell self-renewal and differentiation. However, little is known about Brg1 function in preimplantation embryos and embryonic stem (ES) cells. Here we report that Brg1 is required for ES cell self-renewal and pluripotency. RNA interference-mediated knockdown of Brg1 in blastocysts caused aberrant expression of Oct4 and Nanog. In ES cells, knockdown of Brg1 resulted in

phenotypic changes indicative of differentiation, downregulation of self-renewal and pluripotency genes (e.g., Oct4, Sox2, Sall4, Rest), and upregulation of differentiation genes. Using genome-wide promoter analysis (chromatin immunoprecipitation) we found that Brg1 occupied the promoters of key pluripotency-related genes, including Oct4, Sox2, Nanog, Sall4, Rest, and Polycomb group (PcG) proteins. Moreover, Brg1 co-occupied a subset of Oct4, Sox2, Nanog, and PcG protein target genes. These results demonstrate an important role for Brg1 in regulating self-renewal and pluripotency in ES cells. STEM CELLS 2009;27:317–328

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION Mouse embryonic stem (ES) cells, derived from the inner cell mass (ICM) of day 3.5 blastocysts, are capable of differentiating into all cells of the embryo proper. ES cell pluripotency is conferred in part through the expression of core transcription factors Oct4, Sox2, and Nanog [1, 2]. In mouse and human ES cells, Oct4, Sox2, and Nanog form a regulatory loop to maintain pluripotency by activating self-renewal genes and repressing lineage-specific genes [1, 2]. Reduced expression of Oct4, Sox2, or Nanog in ES cells results in a loss of pluripotency and differentiation [3– 6]. Furthermore, forced expression of Oct4, Sox2, Klf4, and c-Myc induces pluripotency in human and mouse somatic cells [7–10]. Although these studies lend insight into how transcription factor networks regulate ES cell pluripotency, further work is necessary to fully understand the molecular mechanisms involved in regulating ES cell pluripotency and inducing reprogramming in somatic cells. Recently, a number of chromatin immunoprecipitation and DNA microarrays (ChIP-chip) studies have provided insight into how epigenetic modifications of chromatin structure contribute to the control of pluripotency in ES cells. ChIP-chip experiments have identified global binding of transcription factors in human and mouse ES cells, including Oct4, Sox2, and Nanog [1], polycomb complex proteins [11, 12], and Klf proteins [13]. In addition, histone modifications H3K4me3 and

H3K27me3, termed “bivalent domains,” which correlate with lineage-specific gene repression in ES cells, have furthered our understanding of the ES cell epigenetic landscape. These results and others imply that pluripotency is maintained through the net action of transcription factors and epigenetic modifiers at genespecific loci and regulatory regions. Given the complexity of the ES cell epigenome it is plausible that other epigenetic modifiers, such as ATP-dependent chromatin remodeling enzymes, are also important for regulating pluripotency in ES cells. Brahma-related gene 1 (Brg1)-dependent chromatin remodeling complexes represent a subclass of SWItch/Sucrose NonFermentable (SWI/SNF) ATP-dependent remodelers that have been shown to play key roles in proliferation and differentiation in a number of different tissues and cell types [14]. Brg1 acts as the core catalytic subunit of these multisubunit complexes and facilitates gene activation and repression by displacing nucleosomes proximal to gene promoters. At the chromosomal level, regulation of Brg1 is achieved via subunit composition and interactions with tissue-specific transcription factors [15–18]. Disruption of Brg1 function perturbs cell-cycle regulation and differentiation in many cellular lineages, including muscle, neuronal, lymphoid, and myeloid [19 –23]. Furthermore, complete loss of Brg1 during early embryonic development triggers arrest during the blastocyst stage [24], around the time ES cells are derived from the ICM of normal blastocysts. Moreover, disruption of other SWI/SNF subunits, such as BAF250a [25], BAF250b [26], and INI1 [27], results in loss of ES cell self-

Author contributions: B.L.K.: conception and design, collection of data, data analysis and interpretation, manuscript writing; S.P.: conception and design, financial support, final approval of manuscript; J.G.K.: conception and design, collection of data, data analysis and interpretation, financial support, manuscript writing, final approval of manuscript. Correspondence: Jason G. Knott, Ph.D., Developmental Epigenetics Laboratory, Department of Animal Science, Michigan State University, East Lansing, Michigan 48824, USA. Telephone: 517-432-5446; Fax: 517-353-1699; e-mail: [email protected] Received July 28, 2008; accepted for publication October 26, 2008; first published online in STEM CELLS EXPRESS December 4, 2008. ©AlphaMed Press 1066-5099/2009/$30.00/0 doi: 10.1634/stemcells.2008-0710

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renewal and/or developmental arrest at the blastocyst stage. Altogether, these experimental findings support a role for SWI/ SNF chromatin remodeling proteins in early embryonic development and ES self-renewal. However, to date there is very little known about the biological role of SWI/SNF-Brg1 in preimplantation embryos and ES cells. Here we report that Brg1 is required for ES cell self-renewal and pluripotency. RNA interference (RNAi)-mediated knockdown of Brg1 in early embryos and ES cells resulted in a loss of pluripotency, downregulation of pluripotency-related genes, and upregulation of lineage-specific genes. Using genome-wide ChIP-chip analysis, we identified global Brg1 binding targets in ES cells. Brg1 binds to a significant number of genes important for pluripotency and differentiation. Many of these target genes encode transcription factors and epigenetic modifiers. These results support a role for Brg1 in maintenance of pluripotency and extend our current knowledge on how SWI/SNF chromatin remodeling contributes to regulation of gene expression in mammalian cells.

MATERIALS

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METHODS

Superovulation, Embryo Collection, Microinjection, Embryo Culture, and Outgrowth Analysis B6D2/F1 female mice aged 6 – 8 weeks (Jackson Laboratory, Bar Harbor, ME, http://www.jax.org) were superovulated as previously described [28] and mated with B6D2/F1 males. Fertilized one-cell embryos were collected in M2 medium (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), washed, and cultured in potassium simplex optimized medium (KSOM) with amino acids (Specialty Media, Phillipsburg, NJ, http://www.specialtymedia.com). Microinjections were carried out as described previously [28]. In brief, 5–10 pl of 100 ␮M Brg1, Ini1, or control short interfering RNA (siRNA; Dharmacon, Inc., Lafayette, CO, http://www. dharmacon.com) was injected into the cytoplasm of one-cell embryos. Following injection, embryos were cultured in KSOM for 3– 4 days. Outgrowth analysis was carried out on day 4 blastocysts by removing zona pellucidae with acid Tyrode (Sigma-Aldrich), washing in M2 medium, and culturing in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% fetal bovine serum and 1,000 U/ml leukemia inhibitory factor (LIF). After 96 hours the percentage of blastocysts that attached and underwent outgrowth was calculated. All animals were treated in accordance with Institution Animal Care and Use Committee guidelines under current approved protocols at EMD Serono and Michigan State University.

Microarray and Quantitative Real-Time Polymerase Chain Reaction Analysis of Brg1 siRNA Blastocysts Total RNA from pools of 15 Brg1 siRNA and control blastocysts (a total of four biological replicates) were used for linear, two-round amplification by in vitro transcription and target cRNA preparation according to the Affymetrix Small Sample Prep Technical Bulletin (Affymetrix, Santa Clara, CA, http://www.affymetrix.com). For each replicate 15 ␮g of fragmented cRNA was hybridized to Affymetrix 430 v2.0 GeneChips and then processed according to the manufacturer’s instructions. Analysis was as follows: CEL files were normalized using the RMA algorithm (ArrayAssist; Stratagene, La Jolla, CA, http://www.stratagene.com). Analysis of variance was performed on all groups using a Benjamini and Hochberg False Discovery Rate correction. Genes whose expression differed by at least 1.4-fold (between control and Brg1 siRNA samples) with a p value ⬍5% were considered differentially expressed. For validation of microarrays, real-time quantitative real-time polymerase chain reaction (Q-RT-PCR) analysis was carried out as described previously [28] using TaqMan probes and an ABI Prism 7900HT thermocycler (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.

com). Green fluorescent protein and upstream binding factor were used for normalization.

Immunofluorescence Blastocyst immunofluorescence was performed as previously described, with slight modifications [29]. Briefly, day 4 blastocysts were fixed, permeabilized, washed, blocked, and incubated with a 1/1,000 dilution of anti-Brg1 (H-88) and anti-Oct4 (C-10) antibodies overnight at 4°C (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), and washed three times. For secondary detection, samples were incubated in a 1/2,000 dilution of Alexa Fluor 488 and 594 (Molecular Probes, Eugene, OR, http://probes. invitrogen.com), washed, mounted in Vectashield (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) containing 4,6-diamidino-2-phenylindole, and imaged using a spinning disc confocal module (CARV; Atto Bioscience, Rockville, MD, http:// www.atto.com) [30]. ES cell immunofluorescence was as follows: ES cells were fixed for 15 minutes with 4% paraformaldehyde, washed three times with phosphate-buffered saline (PBS), permeabilized with PBST (PBS-0.05% Tween-20), blocked with 1% bovine serum albumin in PBS, and incubated with 1/200 dilution of anti-Brg1 (H-88), anti-Oct4 (C-20), anti-Gata4 (H-112), and antiGata6 (C-20) antibodies overnight at 4°C (Santa Cruz Biotechnology). The following day, samples were washed three times with PBST and incubated with fluorescein isothiocyanate- or Texas Red-conjugated secondary antibodies and Hoechst 33258 diluted 1/2,000 for 30 minutes at room temperature. Samples were rinsed with 1⫻ PBS and 3⫻ PBST and mounted in ProLong Gold.

ES Cell Culture ES cells were cultured as previously described [31]. Briefly, R1 ES cells, obtained from American Type Culture Collection (Manassas, VA, http://www.atcc.org), were cultured on irradiated mouse embryonic fibroblasts (iMEFs) in medium containing high-glucose DMEM supplemented with fetal calf serum (FCS), LIF, L-glutamine, nonessential amino acids, and ␤-mercaptoethanol. Feederfree R1 ES cells were cultured on gelatin-coated dishes and gradually transitioned into serum-free medium (ESGRO complete clonal grade medium; Chemicon, Temecula, CA, http://www.chemicon. com). Detailed culture conditions are given in supporting Methods available online.

ES Cell Transfection ES cells cultured on iMEFs were harvested using 0.25% trypsin (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) to dissociate cells, centrifuged in 10% FCS DMEM to inactivate trypsin, and resuspended in 100 ␮l of ES cell nucleofector solution (Amaxa Inc., Gaithersburg, MD, http://www.amaxa.com). Five micrograms of plasmid DNA was added, and program A-23 (Nucleofector; Amaxa) was used to transfect ES cells. Post-transfection, ES cell medium was added, and ES cells were incubated at 37°C with 5% CO2 on iMEFs. ES cells were transfected every 48 hours for up to 240 hours or selected in Zeocin (Invitrogen) for up to 18 days. Alkaline phosphatase staining was performed using a kit from Millipore (Billerica, MA, http://www.millipore.com) according to the manufacturer’s guidelines.

RNAi Vector Design BLOCK-iT RNAi Designer software (Invitrogen) was used to design Brg1-short hairpin RNA (shRNA) oligonucleotides. Three sets of Brg1-shRNA oligonucleotides were chosen from the software design and cloned into pENTR/H1 according to Invitrogen’s protocol.

RNA Isolation, Reverse Transcription, and Q-RTPCR of ES Cells Reverse transcription and Q-RT-PCR was performed as previously described, with some modifications [32]. Briefly, total RNA was extracted from Brg1-shRNA ES cells using an RNeasy Mini Kit (Qiagen, Valencia, CA, http://www1.qiagen.com) and DNasetreated using Turbo DNA-free (Ambion, Austin, TX, http://www.

Kidder, Palmer, Knott et al. ambion.com) for 30 minutes at 37°C. Reverse transcription was performed using Superscript III with random hexamer primers (Invitrogen). Q-RT-PCR was performed using TaqMan assays and TaqMan stem cell pluripotency low-density arrays with TaqMan Universal PCR Master Mix reagents or SYBR Green PCR Master Mix reagents (Applied Biosystems).

Chromatin Immunoprecipitation and DNA Microarray Analysis Detailed ChIP-chip protocol is given in supporting Methods available online. The Brg1 antibody was purchased from Santa Cruz Biotechnology (H-88, SC-10768), and the RNA polymerase II antibody was purchased from Upstate (clone CTD4H8, 05-623; Upstate, Charlottesville, VA, http://www.upstate.com). As negative controls, mouse IgG (12–371) and rabbit IgG (PP64B) antibodies were purchased from Upstate. Mouse R1 ES cells (feeder-free; 1 ⫻ 108) were harvested and chemically crosslinked with 1% formaldehyde (Sigma-Aldrich) for 20 hours at 4°C. Cells were pelleted, washed with 1⫻ PBS, washed twice with lysis buffer, and flashfrozen in liquid nitrogen. Pellets were resuspended in pre-immunoprecipitation dilution buffer. Cells were sonicated using a Branson Sonifier 450D (Branson, Danbury, CT, http://www.sonifer.com) at 50% amplitude, with 12 1-minute pulses in ice water. Postsonication, samples were centrifuged and flash frozen in liquid nitrogen. Sonicated cell extracts equivalent to 2 ⫻ 106 cells were used in subsequent immunoprecipitations. Samples were precleared with protein G Dynabeads (Dynal Biotech, Carlsbad, CA, http://www. invitrogen.com/dynal) in 1,000 ␮l of dilution buffer. Cell extracts were incubated with 1 ␮g of antibody overnight at 4°C. Chromatinantibody complexes were isolated with 100 ␮l of protein G Dynabeads and washed one time with low-salt buffer, one time with high-salt buffer, one time with LiCl wash buffer, and twice with TE buffer. Protein/DNA complexes were eluted from the beads at 65°C with occasional vortexing. Crosslinking was reversed by addition of NaCl and incubation overnight at 65°C. Extracts were then treated with RNase A and proteinase K, and DNA was purified using an Upstate EZ ChIP kit. DNA was amplified using a GenomePlex Whole Genome Amplification Kit (Sigma-Aldrich), DNase-treated, and labeled with a GeneChip WT Double-Stranded DNA Terminal Labeling Kit. Labeled DNA was hybridized to Affymetrix mouse promoter 1.0R tiling arrays, washed, and scanned. Immunoprecipitated and control sample biological duplicates were used for ChIPchip analysis.

Identification of Brg1 Binding Regions and Gene Annotation Detailed ChIP-chip data analysis can be found in supporting information. Quantile normalization, including probe intensity computation and log2 adjustment was applied to Affymetrix tiling array data using CisGenome (http://www.biostat.jhsph.edu/⬃hji/cisgenome/). Peak detection was done using the TileMap [33] (http://biogibbs.stanford.edu/ ⬃jihk/TileMap/index.htm) application in CisGenome. MA statistics was applied to analyze the tiling array data [33, 34]. Enrichment peaks were annotated with the closest gene and defined by the distance upstream to the transcription start site (TSS) and the distance downstream of the TSS.

Confirmation of Brg1 Binding Regions Using Q-RT-PCR To design primers for Q-RT-PCR, Brg1 ES cell ChIP-enriched genomic DNA regions were imported into Primer3 (http://frodo.wi. mit.edu/). Real-time Q-RT-PCR was performed on nonamplified Brg1 ES cell ChIP DNA, Input DNA, and IgG control DNA using SYBR Green Master Mix reagents with an ABI Prism 7900HT sequence detection system. Primers are listed in supporting information Table 3.

Microarray Analysis of Published Data Annotated Brg1 ChIP-chip regions were compared with publicly available microarray datasets to evaluate developmental expression patterns of Brg1-bound genes. Affymetrix ES cell and embryoid

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body (EB) microarray expression data (GSE2972) [35] was downloaded from Gene Expression Omni. Analysis is given in supporting information. Global preimplantation-stage microarray expression data (Agilent Technologies, Palo Alto, CA, http://www.agilent. com) from unfertilized eggs; one-cell, two-cell, four-cell, and eightcell embryos; morula; and blastocysts [36] were analyzed using the NIA Array Analysis Tool software (National Institute of Aging, Baltimore, MD, http://lgsun.grc.nia.nih.gov/ANOVA) [37]. Gene expression data of 61 tissues (Genomics Institute of the Novartis Research Foundation, San Diego, CA, http://symatlas.gnf.org) were also analyzed using the NIA Array Analysis Tool software [37]. Detailed analysis is given in the supporting information.

RESULTS SWI/SNF-Brg1 Is Required for Establishment of Pluripotency in Mouse Blastocysts Earlier studies demonstrated that Brg1 is essential for preand peri-implantation development in mice [24]. Furthermore, a recent study showed that maternal Brg1 is essential for zygotic genome activation in mouse two-cell embryos [38]. To examine the role of embryonic Brg1 in blastocyst development we used an RNAi approach. In preliminary experiments, we injected Brg1 siRNA or control siRNA into fertilized one-cell embryos and cultured them in vitro for 3 days. Brg1 siRNA did not affect development past the twocell stage. Immunofluorescence analysis revealed that Brg1 protein was downregulated after the two-cell stage, around the four-cell stage (48 hours postinjection), and remained low through the blastocyst stage (data not shown). Embryos injected with control siRNA showed normal blastocyst development, hatching, and outgrowth formation (Fig. 1A–1E). In contrast, embryos injected with Brg1 siRNA developed normally to the blastocyst stage but failed to hatch and undergo outgrowth (Fig. 1A–1E). In addition, Brg1 knockdown embryos were morphologically similar to control embryos and had a comparable number of cells. Furthermore, knockdown of Ini1 mRNA (a subunit of the SWI/SNF-Brg1 complex) mimicked the phenotype of Brg1 siRNA-injected embryos (supporting information Fig. 1A). Fertilized one-cell embryos injected with Ini1 siRNA developed normally to the blastocyst stage, but they also failed to hatch and undergo outgrowth (supporting information Fig. 1B, 1C). Altogether, these results are consistent with the Brg1-null phenotype and support a role for Brg1 in blastocyst development. To evaluate the effect of Brg1 silencing on global gene expression changes, we performed transcriptome analysis on day 4 Brg1 knockdown and control blastocysts representing a total of four biological replicates. Genes whose expression differed by at least 1.4-fold with a p value less than 5% were considered differentially expressed (Materials and Methods). We found 1,055 genes upregulated and 956 genes downregulated in Brg1 knockdown blastocysts compared with control blastocysts (Fig. 2A; supporting information Table 1A). Hierarchical clustering performed on differentially expressed genes revealed two distinct groups (Fig. 2A). Genes important for maintaining pluripotency such as Oct4 and Nanog were upregulated, whereas Bmp4 and Wnt6/9 were downregulated, in Brg1 knockdown blastocysts compared with control blastocysts (supporting information Table 1A). QRT-PCR was used to confirm the expression of some of these genes (Brg1, Oct4, Nanog) in day 4 Brg1 knockdown blastocysts (Fig. 2B). Furthermore, Ingenuity Pathway Analysis (IPA) (Fig. 2C) and the DAVID Functional Annotation Tool were used to functionally annotate differentially expressed

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Figure 1. Brg1 is required for normal development of mouse blastocysts. (A): Micrographs of day 4 con siRNA blastocysts (AI) and Brg1 siRNA blastocysts (AII). Arrows indicate hatching or hatched embryos. (B): Summary of preimplantation development. Results represent the average ⫾ SEM from five experiments. For Brg1 siRNA-injected embryos, 110 embryos were examined, and for con siRNA-injected embryos, 86 embryos were examined. Filled bars, one-cell embryos injected with con siRNA; open bars, one-cell embryos injected with Brg1 siRNA. Asterisk denotes statistical significance (p ⬍ .05) between Brg1 siRNA and con siRNA hatching embryos. (C): Average total cell number of day 4 blastocysts. Filled bars, con siRNA blastocysts; open bars, Brg1 siRNA blastocysts. (D): Micrographs of con (DI) and Brg1 siRNA blastocyst (DII) outgrowths after 96 hrs. Arrows highlight TE cells, and dotted lines outline boundary of TE cell outgrowth. (E): Average outgrowth of blastocysts after 96 hrs of culture. Filled bars, con siRNA blastocysts; open bars, Brg1 siRNA blastocysts. Asterisk denotes statistical significance (p ⬍ .05) between Brg1 siRNA and con siRNA blastocyst outgrowths. Abbreviations: Con, control; hrs, hours; ICM, inner cell mass; siRNA, short interfering RNA; TE, trophectoderm.

genes [39]. These analyses showed several biological processes overrepresented in differentially expressed genes from Brg1 knockdown embryos, including transcription, differentiation, and embryonic development (supporting information Table 1B). Global gene expression profiling and Q-RT-PCR analysis showed that Oct4 expression was upregulated in Brg1 knockdown blastocysts compared with control blastocysts. In normal blastocysts, Oct4 expression is restricted to cells of the ICM and is absent in TE cells. To investigate whether Oct4 protein is upregulated in Brg1 knockdown blastocysts, we evaluated Oct4 immunofluorescence. We confirmed, in control blastocysts, the localization of Oct4 expression in the ICM and absence in the TE (Fig. 2D; supporting information Fig. 2A). On the contrary, in Brg1 knockdown blastocysts, Oct4 expression was widely expressed (Fig. 2D; supporting information Fig. 2B). The majority of Brg1 knockdown blastocysts exhibited widespread Oct4 staining. Based on microarray analysis several TE markers, including Cdx2, Eomes, and Fgfr2, were expressed at similar levels in both control and Brg1 knockdown blastocysts (data not shown), suggesting that knockdown of Brg1 does not block TE formation. Together, these data support a role for Brg1 in blastocyst development by regulating expression of Oct4.

Brg1 Is Required for Maintenance of ES Cell Self-Renewal To investigate the role of Brg1 in maintaining ES cell pluripotency and self-renewal, we used RNAi to target Brg1 transcripts in R1 ES cells. ES cells were (a) stably transfected with Brg1-shRNA or control-shRNA (scrambled) constructs under Zeocin selection for more than 2 weeks (18 days) or (b) transiently transfected with Brg1-shRNA or control constructs every 2 days for up to 10 days (Fig. 3A). In preliminary experiments several Brg1-shRNA and Brg1-miRNA targeting constructs were tested for their ability to knock down Brg1 mRNA (Fig. 3A; supporting information Fig. 3A). Subsequent experiments were carried out using a Brg1shRNA construct that induced the greatest reduction in Brg1 mRNA (⬃60% reduction in Brg1 mRNA relative to controls) (supporting information Fig. 3A–3C) and a control-shRNA vector (scrambled Brg1-shRNA-S7) (Fig. 3A; supporting information Fig. 3B). Normal colony morphology of ES cells was lost in Brg1-shRNA ES cells under stable and transient transfection conditions (Fig. 3B, 3C). Notably, Brg1-shRNA ES cell morphology was flat and lacked tight cell contacts, indicative of differentiation. Phenotypic changes were evident following 6 days of Brg1-shRNA transient transfection

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Figure 2. Global gene expression profiling of day 4 Brg1 siRNA blastocysts. (A): Hierarchical clustering analysis of differentially expressed genes in Brg1 siRNA blastocysts compared with control siRNA blastocysts. Several transcriptional regulators differentially expressed between Brg1 siRNA and control siRNA blastocysts are shown. (B): Quantitative real-time polymerase chain reaction confirmation of pluripotency-related genes differentially expressed in Brg1 siRNA blastocysts. (C): Cellular component and molecular function Gene Ontology term analysis of differentially expressed genes. Cellular components include CYTO, ECS, NUC, PM, and UNK. (D): Confocal immunofluorescence analysis of Oct4 and Brg1 expression in day 4 Brg1 siRNA blastocysts and control siRNA blastocysts. DNA was counterstained with Dapi. Abbreviations: CYTO, cytoplasm; Dapi, 4,6-diamidino-2-phenylindole; ECS, extracellular space; NUC, nucleus; PM, plasma membrane; siRNA, short interfering RNA; UNK, unknown.

(Fig. 3B; supporting information Fig. 4). Alkaline phosphatase staining, a marker of undifferentiated ES cells, decreased in Brg1-shRNA ES cells relative to control ES cells (Fig. 3D). In addition, immunofluorescence analysis demonstrated that Brg1 and Oct4 protein levels were downregulated and Gata4 and Gata6 protein levels were upregulated in Brg1shRNA ES cells relative to control ES cells, suggesting a loss of pluripotency and differentiation (Fig. 3E–3H). To further study the effect of Brg1 silencing on expression of pluripotency and differentiation genes, we analyzed mRNA expression levels of Brg1-shRNA ES cells at 6, 8, and 10 days (Fig. 3I–3K). To identify differentially expressed genes, we performed Q-RT-PCR using primers specific to a panel of pluripotency and differentiation markers [40]. Following 6 days of Brg1 knockdown, we observed downregulation of a number of pluripotency-related and stemness genes [40] comwww.StemCells.com

pared with control ES cells, including Oct4, Sox2, Sall4, Fgf5, T, Gbx2, Klf5, Il6st, Rest, Phc1, Eed, Lifr, Kit, and Nog (Fig. 3J). We also observed upregulation of differentiation markers in Brg1-shRNA ES cells, including Fzd5, Fzd8, Gata3, Gdf5, Stat1, Nkx3–2, Nodal, and Wnt6 (Fig. 3K), suggesting a loss of pluripotency and self-renewal. Expression analysis of Brg1-shRNA ES cells at 6, 8, and 10 days further showed that Brg1 mRNA knockdown induces downregulation of pluripotency genes and upregulation of differentiation markers (Fig. 3I–3K). Key pluripotency-related genes Oct4, Sox2, Sall4, and Rest were downregulated ⬃50% in Brg1-shRNA ES cells. Consistent with the loss of other SWI/SNF subunits (e.g., BAF250a and BAF250b), a reduction of Brg1 transcripts in ES cells disrupts self-renewal and leads to differentiation.

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Figure 3. Brg1 is required for the maintenance of embryonic stem (ES) cell self-renewal. (A): Brg1-shRNA and scrambled control-shRNA (Brg1-shRNA-3-S3, -S5, -S7) oligonucleotide sequences and shRNA expression vector used to deplete Brg1 mRNA in ES cells. Red arrows indicate shRNA sequences, and gray regions represent scrambled sequences. Three Brg1-shRNA oligonucleotide sequences were tested in ES cells (Brg1-shRNA-1, -2, -3), of which the Brg1-shRNA-3 sequence (ⴱ) produced the greatest Brg1 mRNA knockdown as determined by quantitative real-time polymerase chain reaction (Q-RT-PCR) expression of Brg1 mRNA in ES cells 48 hours postnucleofection of Brg1-shRNA constructs. (B): Brg1-shRNA ES cells transiently transfected every 2 d for 8 d. Flattened ES cells were evident in Brg1-shRNA ES cell cultures, whereas control ES cells maintained their three-dimensional colony structure. (C): Stably transfected Brg1-shRNA ES cells and control-shRNA ES cells under Zeocin selection for 2 weeks. Brg1-shRNA ES cells were flattened and differentiated compared with control-shRNA ES cells. (D): Alkaline phosphatase staining of Brg1-shRNA and control-shRNA ES cells. Control ES cells are highly positive for alkaline phosphatase staining, whereas Brg1-shRNA ES cells exhibit reduced staining and a flattened morphology. (E–H): Brg1 (E), Oct4 (F), Gata4 (G), and Gata6 (H) immunostaining of control-shRNA ES cells and Brg1-shRNA ES cells. Brg1-shRNA ES cells have decreased Brg1 and Oct4 expression and increased Gata4 and Gata6 expression relative to control-shRNA ES cells. Nuclei were stained with Hoechst 33258. (I): Global gene expression changes in Brg1-shRNA ES cells at 6, 8, and 10 d. Expression levels of a panel of pluripotency and differentiation markers are altered in Brg1-shRNA ES cells over time. Real-time Q-RT-PCR was performed on Brg1-shRNA mRNA. Fold change relative expression levels were mean-centered and clustered according to gene expression over time. Data were normalized to Eef1a1 as an internal control. (J): Q-RT-PCR was used to compare expression of Brg1-shRNA ES cells with control ES cells. Expression of pluripotency genes were downregulated and the expression of differentiation genes (K) was upregulated in Brg1-shRNA ES cells at 6, 8, and 10 d. The horizontal line (J) indicates 50% mRNA expression relative to the control. Data were normalized to Eef1a1 as an internal control and then to the expression level of control ES cells at 8 d. Abbreviations: d, days; shRNA, short hairpin RNA.

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Figure 4. Genome-wide location analysis of Brg1 in embryonic stem cells. (A): Distribution of Brg1 binding regions relative to the closest TSS of known genes. (B): Length of Brg1-enriched DNA sequences. (C): GC content composition of Brg1 and RNA polymerase II chromatin immunoprecipitation (ChIP)-enriched sequences. (D): Gene Ontology (GO) functional annotation of Brg1-bound genes was performed using Ingenuity Pathway Analysis (IPA). The 20 most significant biological process GO terms are shown. (E): Cellular component and molecular function GO terms of annotated Brg1 ChIP-enriched sequences identified using IPA. Abbreviations: bp, base pairs; TSS, transcription start site.

Genome-Wide Brg1 Promoter Binding in ES Cells Because Brg1 is required for ES cell self-renewal, we investigated the role of Brg1 promoter occupancy in regulating expression of pluripotency-related genes. To identify genome-wide Brg1 promoter binding in ES cells, we combined chromatin immunoprecipitation and DNA microarrays. We used high-density DNA tiling arrays containing probes covering a distance from ⫺6 kilobases (kb) to ⫹2.5 kb relative to the TSS for 28,000 mouse promoter regions. A Brg1 polyclonal antibody was used to immunoprecipitate Brg1-occupied promoter regions in ES cells. As a negative control for the specificity of the Brg1 antibody a nonimmune IgG antibody was used. Brg1 and control ChIP-enriched regions were amplified, labeled, and hybridized to DNA tiling arrays (Materials and Methods). Brg1-bound regions were identified by detecting peaks and annotating those peaks with the nearest gene (Materials and Methods) [33]. Brg1 was found to bind 1,596 promoter regions of known protein-coding genes (supporting information Table 2). Binding locations of Brg1 are located near the TSS of genes, where the majority of bound regions are located within 5 kb of TSSs (1,187 genes) (Fig. 4A). Brg1 binding regions are located upstream, downstream, and within coding and noncoding regions of genes. Brg1-bound DNA regions have an average length of 250 base pairs (Fig. 4B), with a GC-dinucleotide composition (46.3%), slightly higher than genome-wide GC-dinucleotide composition (41%) (Fig. 4C). Brg1 targets were functionally annotated using IPA. Overrepresented biological processes in Brg1 targets included gene expression, cell growth and proliferation, and development (Fig. 4D, 4E). www.StemCells.com

To ensure our that dataset included high-quality protein-DNA interactions, we performed ChIP-chip analysis using a monoclonal RNA polymerase II (RNApolII) antibody to identify genome-wide promoter binding of actively transcribed genes in ES cells. Consistent with previous ChIP-chip studies, RNApolII binding was greatly enriched at the TSSs of genes that are highly expressed in ES cells (Fig. 5A), whereas transcriptionally repressed genes were not significantly bound by RNApolII (data not shown). Genes whose transcripts are enriched in ES cells also bound by RNApolII include genes such as Oct4, Sox2, Nanog, Fgf4, Dnmt3l, Fbx15, Tdgf1, and Sall4. Genes not significantly bound by RNApolII in ES cells include trophectodermal genes Cdx2 and Hand1 and genes expressed in endoderm, such as Gata4, Gata6, Sox7, and Sox11. Evaluation of RNApolII binding in ES cells revealed a 40% overlap with Brg1-enriched genes. These results, in the context of other ChIP-chip studies performed in ES cells, confirmed that our Brg1 dataset was of high quality.

Brg1 Occupies Key ES Cell Enriched Genes The results of our ChIP-chip analysis revealed that Brg1 binds genes encoding key transcriptional regulators that promote ES cell pluripotency and self-renewal, such as Pou5f1 (Oct4), Sox2, Nanog, Sall4, FoxD3, Klf2, Klf5, Klf6, Id1, Id2, and Mycn (supporting information Table 2; Fig. 5A–5C; supporting information Fig. 5). Brg1 also binds genes highly enriched in ES cells whose role in maintaining an undifferentiated state is less clear, such as Dnmt3l, Dppa2, Fbx15, and Tdgf1 [5, 41, 42]. Interestingly, Brg1 binds genes encoding epigenetic regulators, such as de novo DNA methyltransferases (Dnmt3a and Dnmt3b), Dnmt3l, and histone

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Figure 5. Brg1 genomic binding site analysis. (A): Brg1 and RNAPII genomic binding locations of embryonic stem cell-enriched genes. Brg1 binding is enriched near the DE of Pou5f1 (Oct4), whereas RNAPII binds near the transcription start site (TSS). Brg1 peak binding sites are located upstream relative to the TSS (Pou5f1, Sox2, Nanog, Dnmt3l, Sall4, and Tdgf1), and within intragenic sequences relative to the TSS (Sall4). Brg1 and RNAPII FC and MA enrichment values converted to log2 are shown. Chr number, sequence position, and Cons are also indicated on the plot. (B): Validation of Brg1-chromatin immunoprecipitation (ChIP)-enriched genomic binding regions by quantitative real-time polymerase chain reaction (Q-RT-PCR). Results are shown as percentage of relative enrichment relative to input. (C): ChIP and Q-RT-PCR analysis of Brg1 binding to the Oct4 DE. A panel of primers spanning the 5⬘ upstream region of the Oct4 promoter is shown. Abbreviations: Cons, conservation; chr, chromosome; DE, distal enhancer; FC, fold change; MA, moving average; PE, proximal enhancer; PP, proximal promoter; RNAPII, RNA polymerase II.

Kidder, Palmer, Knott et al. deactylases (Hdac1, Hdac3, and Hdac4). Quantitative PCR was used to confirm Brg1-enriched regions (Fig. 5B, 5C). Brg1 binding to epigenetic regulators suggests a role of Brg1 in regulating chromatin structure in ES cells. Because Brg1 binds promoters of core regulatory transcription factors Oct4, Sox2, and Nanog, we examined whether there is a correlation between Brg1 binding and Oct4, Sox2, and Nanog binding throughout the genome. ES cell-enriched transcription factors Oct4, Sox2, and Nanog regulate pluripotency by co-occupying many genes and activating or suppressing transcription [1, 2]. Using data from mouse ES cells [2] we observed 79 genes cobound by Brg1/Oct4/Nanog, 137 genes cobound by Brg1/Oct4 (14% of Oct4 targets), and 274 genes cobound by Brg1/Nanog (11% of Nanog targets) (Fig. 6A). Examples of genes cobound by Brg1/ Oct4/Nanog include Oct4, Sox2, Nanog, Hes1, and Rest. Using data from human ES cells we observed 95 genes cobound by Brg1/Sox2 (7% of Sox2 targets) (data not shown). These data suggest significant co-occupancy between Oct4/Sox2/Nanog and Brg1 targets, implicating a role for Brg1 in regulating transcription of a subset of Oct4/Sox2/Nanog targets.

Brg1 Binds Active and Inactive Genes Brg1 is known to associate with multiprotein coactivator and corepressor complexes to positively or negatively regulate transcription [43]. To understand the expression state of Brg1 targets in ES cells, we compared genes bound by Brg1 with gene expression data from undifferentiated ES cells and EB differentiated ES cells [35]. These results showed that approximately half (54%) of Brg1 targets were expressed at higher levels in ES cells compared with EBs (Fig. 6B). Actively transcribed genes bound by Brg1 include genes such as Oct4, Sox2, and Nanog that are essential for ES cell pluripotency, suggesting that Brg1 occupancy of pluripotency-related gene promoters positively regulates transcription and promotes ES cell self-renewal. Genes whose expression is elevated in EBs also bound by Brg1 include genes involved in differentiation of trophectoderm (e.g., Eomes, Tead4), ectoderm (e.g., Otx2, Stat1, Tbx3), mesoderm (e.g., Acvr1, Bmpr1a, brachyury/T, Nodal, Myog), and endoderm (e.g., Foxp1, Sox11, Wnt9a) (supporting information Fig. 6). We further evaluated the expression profile of Brg1 target genes using gene set enrichment analysis (GSEA) [44] (Fig. 6C). Many target genes were expressed highly in ES cells or EBs, demonstrating that Brg1 binds active and inactive or repressed genes in ES cells. Together, these results implicate Brg1 in promoting expression of self-renewal genes and repression of lineage-specific genes in ES cells. Brg1 was enriched at promoters of polycomb complex genes Phc1, Rnf2, and Eed, core components of PRC1 and PRC2 repressive complexes, which have recently been shown to maintain ES cell pluripotency by repressing genes that regulate development [11]. Moreover, disruption of Brg1 expression in ES cells resulted in reduced expression of Phc1 and Eed, suggesting that Brg1 regulates expression of polycomb complex proteins (Fig. 3J). To examine whether Brg1 co-occupies inactive or repressed genes also bound by PcG proteins Phc1, Rnf2, Suz12, and Eed in ES cells, we compared our list of Brg1-bound genes with a published ChIP-chip study in mouse ES cells [11]. PRC1 proteins Phc1 and Rnf2 were cobound by Brg1 at 57 genes, including Col28a1, Igfbp5, Nkx6 –2, Sox11, Tbx3, and Dmrt1, whereas PRC2 proteins Suz12 and Eed were cobound by Brg1 at 42 genes, including Col27a1, Hoxb2, and Wnt9b (Fig. 6A). Cobinding of PRC1 (Phc1, Rnf2), PRC2 (Suz12, Eed), and Brg1 was found at 34 genes, including Cxcl27a1, Col4a1, Foxb2, Mafb, Gata3, Nkx2–2, Eomes, and Fzd2. Gata3 was among those genes upregulated in Brg1-shRNA ES cells also bound by Brg1/PRC1/PRC2 proteins (Fig. 3K). These data suggest that association of Brg1 with transcriptionally repressed ES cell chromatin coincides in part with PcG protein binding. www.StemCells.com

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We further investigated the relationship between Brg1 and bivalent chromatin by analyzing genes in ES cells that contain histone marks H3K4me3 and H3K27me3 [45, 46]. We found 127 genes bound by Brg1 that contain these histone marks, including Wnt6, Wnt9b, Fzd2, Fzd8, Sfrp1, Cxcl12, Npas3, Foxj1, Otx2, and Tbx3 (Fig. 6A), further suggesting an association between Brg1 and gene repression. In addition, RNAi-mediated knockdown of Brg1 in ES cells resulted in increased expression of several of these genes, including Fzd8 and Wnt6, implicating a role for Brg1 in maintaining transcriptional repression of a subset of genes containing these repressive histone marks (Fig. 3K).

Brg1 Occupies Genes Expressed in Various Tissues To understand the expression pattern of Brg1-occupied genes in different tissues where Brg1 has previously been shown to be important, we compared Brg1-bound genes with public gene expression data from 61 mouse cell types and clustered those genes according to similar expression patterns (Fig. 6E) [47]. We observed clusters of genes bound by Brg1, without RNApolII binding, in several lineages, including neural, myeloid, and lymphoid, suggesting that Brg1 functions in a variety of cell types to promote developmental processes such as differentiation. Many genes bound by Brg1 in ES cells exhibit lineage-specific gene expression, suggesting Brg1 inhibits expression of these genes in ES cells. For example, Brg1 occupies genes expressed in myeloid and lymphoid cells, such as Cnr2 (macrophages), CD52 (lymphocytes), and Nfatc1 (T cells). This is in agreement with previous work showing that Brg1 is required for myeloid differentiation into granulocytes [23]. Likewise, Brg1 has an essential role in neural differentiation [48], which is consistent with Brg1 occupancy of genes in ES cells that are highly expressed in differentiated neural tissues, such as Ckb (brain), Nsg2, Gng7, Mobp (oligodendrocytes), Ank2, Mlc1, Ina, Syt11, Ncdn, and Syngr3. The relatively low expression of these Brg1-bound genes in ES cells compared with differentiated cells suggests that Brg1 inhibits transcription of these target genes in ES cells and promotes transcription of these genes in differentiated cells. We further evaluated the expression of Brg1 targets during preimplantation development by comparing Brg1 targets with gene expression data from unfertilized oocytes, one-cell, twocell, four-cell, and eight-cell embryos, morula, and blastocysts (Fig. 6B) [49]. GSEA was used to evaluate the expression profile of Brg1 targets during preimplantation development (Fig. 6D). Brg1 targets were differentially expressed during these stages. Specifically, more Brg1 targets were expressed in four-cell to blastocyst stage embryos than in unfertilized oocytes and one-cell embryos. These results suggest that Brg1 may regulate target gene expression temporally and in a cell typespecific manner in oocytes and preimplantation embryos.

DISCUSSION Results presented here reveal a previously unknown role of the SWI/ SNF-Brg1 protein in ES cell pluripotency and self-renewal. To date little is known about the role of Brg1 during early mammalian development. Our results in early embryos and ES cells show that (a) Brg1 is required for proper blastocyst development, (b) Brg1 is required for maintenance of ES self-renewal and pluripotency, and (c) Brg1 occupies key pluripotency genes, lineage-specific genes, and epigenetic modifier genes. Therefore, we propose a model whereby Brg1 acts in a hierarchy, upstream of core transcription factor networks, to promote blastocyst development and ES cell pluripotency. In the present study we used ChIP-chip analysis to map genomic binding sites of Brg1 in ES cells. Using this approach we identified a number of Brg1 target genes in ES cells that have

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Figure 6. Expression profile of Brg1 occupied genes. (A): Relationship between Brg1-bound genes and Oct4/Nanog-bound genes, polycomb repressive complex 1 (Rnf2/Phc1)-bound genes, polycomb repressive complex 2 (Suz12/Eed)-bound genes, genes with histone marks H3K4/27me3, and genes differentially expressed between BAF250b⫺/⫺ and wild-type ES cells. (B): Expression profile of Brg1 targets in undifferentiated ES cells and differentiated EBs over 14 days (Affymetrix), and unfertilized oocytes and preimplantation-stage embryos (Agilent). Differentially expressed genes (False Discovery Rate less than 5%) were compared with Brg1-bound genes. (C): Brg1 targets are active and inactive in ES cells. Shown is gene set enrichment analysis (GSEA) of Brg1 targets in ES cells and differentiated EBs. Red indicates active genes in ES cells and blue indicates active genes in EBs. (D): Brg1 targets are expressed in oocytes and preimplantation-stage embryos. GSEA of Brg1 targets in oocytes and preimplantation embryos. Red indicates active genes in oocytes and one-cell/two-cell embryos. Blue indicates active genes in four-cell and eight-cell embryos, morulae, and blastocysts. (E): Expression profile of Brg1-bound targets in 61 tissues (GNF SymAtlas). Differentially expressed genes (p ⬍ 5%) were compared with Brg1-bound genes (analysis is described in Materials and Methods). Red indicates high expression, and green indicates low expression. Abbreviations: EB, embryoid body, ES, embryonic stem; h, hours.

Kidder, Palmer, Knott et al. important roles in preimplantation development and ES cell pluripotency. For example, Brg1 binds to the promoters of genes essential for maintaining ES cell pluripotency, such as Oct4, Sox2, Nanog, Sall4, and Rest. Oct4, Sox2, and Nanog are part of the core ES cell transcriptional circuitry and maintain pluripotency by activating self-renewal genes and suppressing lineage-specific genes [1, 2]. Brg1 also binds to a subset of Oct4, Sox2, and Nanog target genes and a subset of PcG protein (Rnf2, Phc1, Eed, and Suz12) target genes. We also found enrichment of Brg1 at a number of genes important for epigenetic regulation of chromatin, including Hdac1, Hdac3, Hdac4, Dnmt3a, Dnmt3b, Dnmt3l, and PADi4. Using RNAi to knock down Brg1 transcripts in ES cells, we observed downregulation of Oct4, Sox2, Sall4, Rest, and Phc1, and upregulation of a number of lineage-specific genes. Therefore, these data suggest a hierarchy within the ES cell transcriptional network, where Brg1 is upstream of pluripotency-related genes, such as Oct4, Sox2, and Nanog. Our data also provide additional insight into the role of Brg1 during preimplantation development, when pluripotency is established. Transcriptome analysis of Brg1 knockdown blastocysts revealed a number of Brg1-regulated genes important in transcription, differentiation, and development. In particular, the levels of Oct4 and Nanog mRNA were upregulated approximately twofold. Using confocal microscopy, we found that Oct4 was widely expressed in Brg1 knockdown blastocysts. The inability of Brg1-deficient blastocysts to form outgrowths and give rise to ES cell colonies may be caused by misexpression of Oct4. Recent studies have established a relationship between the level of Oct4 protein in mouse blastocysts and the frequency of ES cell derivation [50, 51]. Although we do not have direct evidence for Brg1 binding to the Oct4 promoter in blastocysts, our genome-wide promoter analysis in ES cells revealed binding of Brg1 to the distal enhancer of Oct4, which is important in driving expression of Oct4 in ES cells and ICM cells [52, 53]. It will be important to evaluate Brg1 function in trophoblast stem cells to determine whether Brg1 is necessary for developmental repression of Oct4. Altogether, these results suggest a dynamic role for Brg1 in promoting early embryonic development and ES pluripotency by regulating transcription of key pluripotency-related genes. Brg1 functions as a transcriptional activator and repressor in different tissues and cell types. By comparing our Brg1 ChIP targets with public gene expression data from oocytes, embryos, ES cells, and various somatic cell types we found that Brg1 associates with both active and inactive genes. One likely mechanism is that Brg1 recruits different coactivators and corepressors to target gene promoters to activate and repress gene expression. For example, Brg1 can associate with complexes containing histone-modifying enzymes such as coactivator-associated arginine methyltransferase-1 (CARM1) in the nucleosome methylation activation complex (NUMAC) to activate transcription [54]. Brg1 also associates with components of the nuclear receptor corepressors-1 complex (NcoR-1), such as histone deacetylase 3 (Hdac3) and the transcriptional corepressor Kap-1, to repress transcription [55–57]. Furthermore, Brg1 interacting proteins were identified, including proteins that are involved in transcriptional activation, such as ␤-catenin, p53,

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Smad3, Stat1, and Stat2, and proteins that are involved in transcriptional repression, such as Hdac1/2, Hp1, Mbd3, Mi-2␤, mSin3A, and Rest [43]. Interestingly, Liang et al. recently reported that Nanog associates with repressors Nurd, Sin3A, and SWI/SNF-Brg1 [58]. In the present study Brg1 was found to bind 274 Nanog target genes. These data suggest that Brg1 may coregulate a subset of Nanog target genes in ES cells to maintain self-renewal and pluripotency. Altogether, these data support a dynamic role for Brg1 in positively and negatively regulating ES and somatic cell gene expression programs. Recent studies have shown that overexpression of four transcription factors (Oct4, Sox2, Klf4, and c-Myc or Mycn) induces pluripotency in human and mouse somatic cells [7–10, 59]. Our genome-wide data showed that Brg1 binds three of the four main reprogramming factors (Oct4, Sox2, and Mycn). Although we did not observe binding to Klf4, Brg1 occupies two genes functionally redundant to Klf4 (Klf2 and Klf5) [13]. Because the efficiency of deriving iPS cells from somatic cells is extremely low (1/5,000 to 1/10,000 cells), it will be interesting to determine whether coexpression of Brg1 or other epigenetic modifiers in addition to Oct4, Sox2, Klf4, and c-Myc improves the efficiency of reprogramming in somatic cells.

CONCLUSION Results reported here provide strong evidence that Brg1 is required for ES cell self-renewal and pluripotency. Consistent with our results, two recent studies showed that SWI/SNF-Brg1 subunits, BAF250a and BAF250b, are required for ES self-renewal and pluripotency [25, 26]. Moreover, ⬃64% of Brg1 targets (Fig. 6A) identified in this study were differentially expressed in BAF250b⫺/⫺ ES cells [26]. Because our data include several known pluripotency regulators (e.g., Oct4, Sox2, Nanog, Sall4, Rest), it is likely that other Brg1 targets included in this dataset that have not yet been studied in ES cells have essential roles in maintaining ES cell pluripotency. Our results provide a foundation for further examination of these mechanisms.

ACKNOWLEDGMENTS We thank Dr. Eric Grund and Catherine Wilson for technical assistance with the Q-RT-PCR analysis and confocal microscopy. Drs. George Smith and Luca Magnani are thanked for critically reading the manuscript. We also acknowledge Dr. Andras Nagy at Mount Sinai Hospital for license of his R1 ES cell line. This work was funded by an internal grant at EMD Serono Research Institute and a reproductive and developmental sciences program grant at Michigan State University (to J.G.K.).

DISCLOSURE

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CONFLICTS

The authors indicate no potential conflicts of interest.

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