Dissecting The Functional Differences Of Distinct SWI/SNF Components In Human Embryonic Stem Cells

The SWI/SNF chromatin remodeling complex are evolutionarily conserved multimeric enzymatic machinaries. These complex contain either BRG1(SMARCA4) or BRM (SMARCA2) as ATP-dependent motors to change the nucleosomal structure using energy from ATP hydrolysis. At least three distinct SWI/SNF-like complexes have been identified in mammalian cells:BAF250a (ARID1A)-associated complex (termed as BAF-A; BAF refers to BRG1or BRM-associated factors), BAF250b (ARID1B)-associated complex (termed as BAF-B), and BAF180/200(PBRM1)-associated complex (termed as PBAF). These SWI/SNF complex consist of11-15common BRG1or BRM-associated factors(BAFs), including BAF170(SMARCC2), BAF155(SMARCC1), BAF60A(SMARCD1), BAF57(SMARCE1), BAF53A(ACTL6A), ACTIN and BAF47(SMARCB1). SWI/SNF complex participate in many basic celluar activities, such as transcription replication and chromatin repair. The key features of embryonic stem (ES) cells are their self-renewal ability to maintain undifferentiated states and pluripotency to differentiate into essentially all cell lineages. Both processes are ultimately achieved through the re-organization of global chromatin structures to reprogram gene expression patterns. Several reports demonstrated a requirement of SWI/SNF complex in mouse embryonic stem cells(MES), but few are about human embryonic stem cells(HES). Our previous work revealed that BRG1deficiency could lead to loss of stem cell characters in HES.In this study, to understand the function of SWI/SNF complex in HES, I knocked down several subunits of this complex in MES and HES by RNA interference. I first confirmed that depletion of BRG1or BAF155in MES, rather than BAF170, led to loss of the pluripotency feature. I next demonstrated that depletion of BAF170, rather than BAF155, resulted in loss of stem cell morphology in HES. Consistent with this result, the alkaline phosphatase staining and the expression of cell surface markers Tra-1-60and Tra-1-81were obviously reduced in BAF170depleted cells. In addition, the phenotypical changes in BAF170upon BAF170deficiency could be rescued by ectopic expression of BAF170, but couldn’t by BAF155overexpression. Although BAF155and BAF170share a high homology in mammalian cells, their apparently can’t replace each other in HES. Depletion of BAF170could led HES to lose the pluripotency, but deletion of BAF155I only found the morphology of the knockdown clones were smaller than the normal ones. This result showed us that the expression of cadherin proteins was different between the RNA interference HES of BAF155and the normal HES. At the same time, combined with previous studies I concluded that BAF170and BRG1play the same role in HES. Further experiments demonstrated that depletion of BRM, BAF200, BAF250a and BAF250b couldn’t lead to differentiation in HES.To further investigate the molecular mechanism how different SWI/SNF components play distinct role in HES, whole genome-wide expression arrays were performed. Interestingly, I observed that BRG1and BRM co-regulate only a small number of genes. For example, most altered genes upon BRG1depletion were upregulated, while they remain unchanged in BRM deficient HES. In addition, BRG1appears to downregulate certain genes at a greater degree than BRM. These results suggest that BRG1and BRM co-regulate certain biological process, but they distinctly different, and BRG1plays an more significant role than BRM in HES. I further performed co-immunoprecipitation(Co-iP) experiment by BRG1antibody and mass spectrometry analyses. Several proteins involved in HES self-renewal, differentiation and transcriptional regulation were identified. Among them, DNMT1, a DNA methytransferase, were confirmed to be associated with BRG1by Co-iP experiment, suggesting that BRG1regulates the pluripotency of HES through methylation. Additional interacting partners of BRG1will be further confirmed in the future.Accumulating evidence suggest that SWI/SNF chromatin remodeling complex also plays an important role during embryo development. BRG1null mutation led to death at peri-implantation. I thus established an embryonic stem cell line in which BRG1deletion can be induced by4OH tamoxifen. Priliminary experiments demonstrated that this ES cell line contains normal karyotype, and no mycoplasma contamination was detected in these cells. In addition, BRG1expression was largely diminished upon induction by4OH-TM. The embryoid body formation experiment showed that BRG1deletion resulted in smaller EB formation and reduced AP+embryonic germ cell colony development. Further investigation using this tool will help us understand the biological requirement and underlying mechanism of SWI/SNF complex during embryonic development in vitro.