Self-renewal and pluripotency are defining properties of an embryonic stem cell (ESC). ESCs must maintain an undifferentiated, but poised state when only upon receipt of the proper stimuli does it begin the process of differentiation and commitment, giving rise to the diverse cell types and tissues that make up an organism. Stem cell identity is largely dictated by an intricate transcriptional circuit controlled by the master regulators Oct4, Sox2 and Nanog. However, the binding of these transcriptional factors is in turn highly dependent on the accessibility of the chromatin landscape. To access the underlying genomic information, histone proteins must be repositioned or removed, a function that is performed by ATP-dependent chromatin remodeling complexes. In ESCs, a specialized BAF complex, esBAF exists, which is essential for maintaining self-renewal and pluripotency. esBAF consists of the defining subunits - BRG1, BAF155, ARID1A, BAF57, BAF47 and six others. Recently, this applicant discovered a novel, non-canonical BAF complex in ESCs that contains the Bromodomain-containing protein 9 (BRD9), but excludes ARID1A and BAF47. Thus far, studies on esBAF have been done by performing deletion or knockdown experiments of BRG1 or BAF155, both of which are also present in the BRD9-containing BAF complex, or BRD9-BAF. Thus, the specific function of BRD9-BAF in ESC biology remains completely unexplored. Indeed, we find that inhibition of BRD9 results in loss of ESC self renewal. This proposal hypothesizes that BRD9-BAF?s distinct complex composition gives it unique function and targeting critical for the specific regulation of transcriptional programs in ESCs.
The specific aims of this project are 1. to identify the complex composition of BRD9-BAF and to characterize its in vitro ATPase and chromatin remodeling activities and 2. to elucidate the role of BRD9 in BRD9-BAF?s genomic localization and function in ESCs. The composition and biochemical activities of BRD9-BAF will be defined using proteomics and in vitro ATPase and chromatin remodeling assays. The role of BRD9-BAF in ESC pluripotency will be elucidated by assessing ES cell growth and differentiation potential upon loss of BRD9 function using both small molecule and genetic perturbations. Furthermore, the genomic localization and activity of BRD9-BAF will be established using the following genome-wide experiments: chromatin immunoprecipitation (ChIP)-seq, RNA-seq, and Assay for Transposase- Accessible Chromatin (ATAC)-seq. These studies will establish BRD9?s role in ESC biology and aid in our understanding of how BAF complex heterogeneity contributes to the precise control of the ESC transcription program.
Embryonic stem cells give rise to all the different cell types that make up an organism, a property that has been useful in modeling diseases and studying mammalian development in vitro. This proposal aims to define the role of a chromatin remodeling complex that contains BRD9 in regulating the expression of stem-cell specific genes. The proposed research will further enhance our knowledge of the mechanisms that control stem cell identity, which will be key in the efforts towards advancing regenerative medicine.
|Gatchalian, Jovylyn; Malik, Shivani; Ho, Josephine et al. (2018) A non-canonical BRD9-containing BAF chromatin remodeling complex regulates naive pluripotency in mouse embryonic stem cells. Nat Commun 9:5139|