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Examining the Functions of Master Regulators in Maintaining Pluripotency and Inducing Reprogramming

  • Author(s): Lo, Hung-Hao
  • Advisor(s): Smale, Stephen T
  • et al.
Abstract

Pluripotency factors Oct4, Sox2, and Nanog orchestrate an elaborate hierarchy of gene regulation governing embryonic stem cell (ESC) identity. The capability of differentiating into cell from any of the three germ layers offers a potential solution and path toward the discovery of novel therapies for devastating diseases. The differentiation of ESCs requires three fundamentally distinct transitions in the transcriptional state: 1) the activation of silent genes in ESCs that are transcribed in differentiated states, 2) the silencing of ESC-transcribed genes that are entirely inactive in the differentiated cells, 3) the modulation of transcription for genes that are expressed in both populations. Deciphering the mechanisms regulating the transitions of these transcriptional states will illuminate how the pluripotent state is established and maintained by the regulation of pluripotency factors. Because Oct4 and Sox2 are the core regulators of the transcriptional regulatory network for pluripotency, we focused our studies on mechanisms regulated by Oct4 and Sox2 in ESCs.

Oct4 and Sox2 bind to thousands of enhancer composite sites at pluripotency genes and differentiation-promoting genes. These Oct4/Sox2 target genes exhibit distinct transitions of transcriptional states for the establishment of pluripotency. As distinct mechanisms are likely to regulate different transcriptional states, a critical step toward a mechanistic understanding of pluripotency is to delineate the genes with well-defined transcription characteristic. Because most studies have relied on low stringency criteria to define differential expression to infer regulatory mechanisms, they did not rigorously evaluate the selective functions of Oct4/Sox2 composite binding critical for establishing pluripotency. To scrutinize how Oct4 and Sox2 account for possible differences in the regulatory mechanisms necessary for distinct transcriptional states, we refined the gene classification and interrogated the role of Oct4/Sox2 binding at enhancer composite motifs at distinct gene classes. By combining RNA-seq, ChIP-seq, and ATAC-seq with functional validation employed by CRISPR/Cas9 mutagenesis, we discovered that Oct4/Sox2 function differently across gene groups with various transcriptional states. In addition to a role in transcriptional activation at ESC-specific and Dynamic genes, my data suggest that Oct4/Sox2 motifs at silent genes may mediate the transcriptional repression in ESCs. In Chapter 3, we also employed a gene-centric approach to quantitatively compare transcription factor co-binding, histone modifications, chromatin accessibility, enhancer properties, and the genomic context of transcriptional regulation by Oct4 and Sox2 in ESCs. Together, we extended our understanding of the critical role played by Oct4 and Sox2 in the establishment of pluripotency.

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