Investigating the roles of CREBBP/EP300 and BRD9 in somatic cell reprogramming to pluripotency

Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSC) by overexpressing four transcription factors, OCT4, SOX2, KLF4 and MYC (OSKM). Epigenetic pathways that safeguard somatic cell identity are barriers for somatic cell reprogramming. Chemical or genetic inhibition of such pathways increases the efficiency of reprogramming. In this thesis, I investigated the effects of three chromatin factors CREB (cyclic-AMP response element binding protein) binding protein (CREBBP), E1A binding protein of 300 kDa (EP300) and bromodomain containing 9 (BRD9) during reprogramming using a combination of chemical probes and genetic loss of function experiments. In the first chapter, I investigated barrier function of bromodomain-mediated interaction of CREBBP/EP300 in reprogramming with specific bromodomain inhibitors. RNA-Sequencing showed that CREBBP/EP300 bromodomain inhibition in fibroblasts decreases the expression of genes that are highly expressed in the cells. However, inhibition of the acetyltransferase activity of CREBBP/EP300 by another small molecule, A485, decreased the reprogramming efficiency by downregulating pluripotency-associated genes. Assay for transposase-accessible chromatin (ATAC-Sequencing) and H3K4me1 and H3K27ac chromatin immune-precipitation (ChIP-Sequencing) results indicated that CREBBP/EP300 bromodomain inhibition decreases H3K27ac at accessible chromatin regions and at putative enhancers. Additionally, I functionally showed that continuous expression of PRRX1, a downregulated transcription factor upon CREBBP/EP300 bromodomain inhibition, impairs iPSC generation by suppressing pluripotency-related gene expression. These results uncovered a role for the interaction of CREBBP/EP300 with acetylated lysines in safeguarding somatic cell identity. In the second chapter of my thesis, I performed a SWI/SNF-focused CRISPR-Cas9-mediated knockout screen to identify regulatory subunits of reprogramming. This screen identified non-canonical BRG1- or BRM-associated factors (ncBAF) specific subunits, BRD9 and BRD4 Interacting Chromatin Remodeling Complex Associated Protein (BICRA, GLTSCR1) as barriers to reprogramming. Additionally, I showed that three structurally distinct BRD9 bromodomain inhibitors and a PROteolysis Targeting Chimera (PROTAC) degrader of BRD9 increase reprogramming efficiency and generate iPSCs with only OCT4 and SOX2 expression. BRD9 knockout iPSCs expressed pluripotency markers and short term BRD9 inhibition did not decrease cell proliferation and OCT4-positive cells indicating that BRD9 is dispensable for pluripotency induction and maintenance. However, BRD9 depletion and inhibition impaired mesoderm differentiation of iPSCs. RNA-Seq was performed on fibroblasts and results showed that BRD9 inhibition downregulates highly expressed genes in fibroblasts. ATAC-Seq results showed that chromatin accessibility around putative active enhancers rather than promoters was decreased upon BRD9 inhibition. Additionally, overexpression of MN1 and ZBTB38, which were downregulated upon BRD9 perturbations, decreased reprogramming efficiency. These results indicate a role for BRD9 and ncBAF in maintaining somatic cell identity through maintenance of active enhancers. The findings presented in this thesis show that chromatin factors safeguarding somatic cell identity are barriers to cellular reprogramming and inhibition of such factors can be utilized to increase the low efficiency of human iPSC generation.