ES cell pluripotency and germ-layer formation require the SWI/SNF chromatin remodeling component BAF250a

Abstract

ATP-dependent chromatin remodeling complexes are a notable group of epigenetic modifiers that use the energy of ATP hydrolysis to change the structure of chromatin, thereby altering its accessibility to nuclear factors. BAF250a (ARID1a) is a unique and defining subunit of the BAF chromatin remodeling complex with the potential to facilitate chromosome alterations critical during development. Our studies show that ablation of BAF250a in early mouse embryos results in developmental arrest (about embryonic day 6.5) and absence of the mesodermal layer, indicating its critical role in early germ-layer formation. Moreover, BAF250a deficiency compromises ES cell pluripotency, severely inhibits self-renewal, and promotes differentiation into primitive endoderm-like cells under normal feeder-free culture conditions. Interestingly, this phenotype can be partially rescued by the presence of embryonic fibroblast cells. DNA microarray, immunostaining, and RNA analyses revealed that BAF250a-mediated chromatin remodeling contributes to the proper expression of numerous genes involved in ES cell self-renewal, including Sox2, Utf1, and Oct4. Furthermore, the pluripotency defects in BAF250a mutant ES cells appear to be cell lineage-specific. For example, embryoid body-based analyses demonstrated that BAF250a-ablated stem cells are defective in differentiating into fully functional mesoderm-derived cardiomyocytes and adipocytes but are capable of differentiating into ectoderm-derived neurons. Our results suggest that BAF250a is a key component of the gene regulatory machinery in ES cells controlling self-renewal, differentiation, and cell lineage decisions.

Fig. 1.

Relative expression of BAF250a in early stage embryos and ES cells. (A) qRT-PCR analysis of relative expression of BAF250a, Oct4, Nanog, and GAPDH. (B) Immunostaining of BAF250a and Oct4 in blastocyst stage embryos. (C) Immunostaining of BAF250a expression in ES cells. *, not detectable expression; arrows, nuclei of fibroblast feeder cells. (Scale bar in B, 50 μm.)

Fig. 2.

Strategies to generate BAF250a conditional knockout embryos and ES cell lines. (A) Diagram of the strategy for homologous recombination. (B) Derivation of BAF250a homozygous ES cells from WT ES cells. (C) Derivation of BAF250a homozygous ES cells from embryos. See Materials and Methods for details. (D) Southern (E) RT-PCR and (F) Western blot analyses confirmed homologous recombination and indicated that BAF250a is absent in BAF250a homozygous ES cells. NE, nuclear extract; f, flox allele (reverted WT allele); H1, BamH I; Pro, protamine; *, a nonspecific band.

Fig. 3.

Ablation of BAF250a causes defects in early embryogenesis. (A) BAF250a^−/− embryos failed to develop beyond E6.5, and to form mesoderm. (B) BAF250a^−/− embryos failed to express Brachyury T. Embryos were serially sectioned and stained, and only one section is shown here. (Scale bar, 100 μm.)

Fig. 4.

BAF250a ablation severely inhibits normal proliferation and promotes differentiation of ES cells. (A) Generation of BAF250 KO ES cells in vitro. LacZ staining indicates deletion of β-geo (KO allele) and conversion of BAF250a^+/− to BAF250a^−/− cells after tamoxifen induced Cre activation. Impaired growth and altered morphology are apparent within 76 h of drug addition. (B) Slow growth of BAF250a (^−/−) ES cells. The plot represents the average of three separate experiments. (C) Lack of BAF250a promotes ES cell differentiation. The BAF250a Het (^+/−, Left) ES cells grow and appear normal under feeder free culturing conditions, whereas Homo (^−/−) ES cells form clusters, and differentiate into primitive endoderm-like cells (Right). (D) Morphology of embryo-derived BAF250a^−/− ES cells at early passages with feeder cells. (E) Immunostaining of ES markers in BAF250a^−/− ES cells.

Fig. 5.

Genes affected in BAF250a deficient ES cells. (A) ES cell markers Oct4, Sox2, and Utf1 are down-regulated in BAF250a-deficient cells. (B) GATA-4, GATA-6, and VAMLC are up-regulated in BAF250a-deficient cells. (C) Expression of other BAF subunits in BAF250a-deficient cells. (D) Western blot analysis of BAF subunits in BAF250a^f/+, BAF250a^+/−, and BAF250a^−/− ES cells. VAMLC, ventricular alkaline myosin light chain.

Fig. 6.

Lineage-specific effect of BAF250a on ES cell differentiation. Differentiation potential of BAF250a^−/− ES cells into cardiomyocytes, adipocytes, myocytes, and neurons. Cardiomyocyte, myocyte, and neuron formation are indicated by immunostaining against TnT, MF20, and Tuj1 and TH, respectively, whereas adipocyte formation is indicated by Oil red O staining.