Brahma-related gene 1 (BRG1), a chromatin remodeling ATPase, is known to function as a key regulator of gene expression eliciting both activator and repressor functions within different cell types. In pluripotent embryonic stem cells (ESCs), BRG1 is found at key regulatory elements of pluripotency genes and functions as a negative regulator to govern lineage determination. However, the underlying mechanisms by which BRG1 regulates pluripotency genes in ESCs and the trophoblast lineage are largely unknown. To elucidate the BRG1-dependent mechanisms that regulate pluripotency during early embryonic development I used a combination of mouse preimplantation embryos and CDX2-inducible ESCs that transdifferentiate into trophoblast-like cells. The cell line allowed for biochemical experiments to be performed that required large amounts of biological material to uncover mechanisms that could then be verified in the embryo.In the first experimental study of my dissertation I demonstrated that a series of dynamic transcriptional and epigenetic changes occurred at the Nanog and Oct4/Pou5f1 proximal and distal enhancer regions during trophoblast lineage development. Initially, CDX2 was recruited to Nanog and Oct4 enhancers and colocalized with BRG1. Next, OCT4 and RNA polymerase II (RNAPII) were lost and major changes in chromatin structure occurred. Histone H3 lysine 9 and lysine 14 acetylation (H3K9/14Ac) were significantly reduced and p300 and histone deacetylase 1 (HDAC1) were lost at these genes. These changes were accompanied by an increase in nucleosome occupancy as assayed by chromatin accessibility and total histone H3 chromatin immunoprecipitation (ChIP) experiments. Lastly, I showed that DNA methylation at these regulatory regions was a final step accompanying Nanog and Oct4 silencing in the trophoblast lineage. The results of these early experiments provided an epigenetic framework for subsequent functional experiments that resolved the role of BRG1 in pluripotency and trophoblast lineage development.In the second experimental study of my dissertation I examined the biological role of BRG1 in pluripotency gene regulation and trophoblast lineage development. To accomplish this a series of experiments were performed in preimplantation embryos and CDX2-inducible ESCs. First, I demonstrated that BRG1 antagonizes histone H3K9/14 acetylation at the Nanog proximal enhancer in both pluripotent ESCs and the trophoblast lineage. To understand how BRG1 regulates H3K9/14 acetylation a series of biochemical experiments were performed. I discovered that BRG1 forms a functional deacetylation complex with histone deacetylase 1 (HDAC1) in ESCs and preimplantation embryos. An important observation obtained from the embryo study was that the interaction of BRG1 with HDAC1 occurred at a higher frequency in the trophoblast lineage than in the inner cell mass (ICM). In agreement with a role in transcriptional repression, inhibition of HDAC1 resulted in an increase in Nanog expression in ESCs and failure to repress Nanog during trophoblast lineage development. Importantly, disruption of HDAC1 phenocopied BRG1 depleted ESCs and embryos, suggesting that HDAC1 cooperates with BRG1 to govern Nanog expression. Lastly, I provide nucleosome-mapping data that supports a dual role for BRG1 in histone deacetylation and chromatin remodeling during early embryonic development. Collectively, the results of these experiments provide novel information on the underlying mechanisms by which BRG1 converges with the pluripotency gene network to modulate pluripotency gene expression and support development of the trophoblast lineage.
Read
