Transcriptional regulation of the Sox2:Oct-3/4 gene regulatory network in embryonal carcinoma and embryonic stem cells.

摘要

The Sox2:Oct-3/4 gene regulatory network is comprised of a set of genes critical for embryogenesis as well as stem cell pluripotency and self-renewal. Sox2 and Oct-3/4 bind to an HMG/POU cassette within the enhancers of their target genes. Given the ability of Sox2 and Oct-3/4 to strongly stimulate their own transcription, it is essential that negative feedback control exists to limit overexpression of Sox2, Oct-3/4, and their target genes. It is becoming increasingly clear that precise expression levels of Sox2:Oct3/4 target genes are essential for normal development and cell fate. The work in this dissertation examines how Sox2:Oct-3/4 target gene expression is precisely controlled through the following mechanisms: (1) positive and negative cis-regulatory elements, (2) context-dependent cis-regulatory element function, (3) differential transcription factor and cofactor function, and (4) positive and negative feedback control. We demonstrate that transcription of the Sox2:Oct-3/4 target gene, Nanog, is controlled by multiple positive and negative context-dependent cis-elements that contribute to its expression in embryonal carcinoma (EC) and embryonic stem (ES) cells. We also examine the differential expression of another Sox2:Oct-3/4 target gene, FGF-4, in human and mouse cells. The results argue that the lower level of FGF-4 expression in human ES cells is due to the lack of a functional GT box in the human FGF-4 enhancer. Our work also delves into the function of Sox2:Oct-3/4 target gene HMG/POU cassettes and demonstrates that six known HMG/POU cassettes are equally functional in F9 and P19 EC cells. Moreover, despite previous predictions, the differential expression of FGF-4 between F9 and P 19 EC cells is due to lower FGF-4 enhancer activity in P19 EC cells and not a difference in HMG/POU cassette function. Lastly, we examined the mechanisms that limit Sox2:Oct-3/4 target gene expression. We demonstrate that increasing levels of Sox2 leads to a reduction in the expression of all Sox2:Oct-3/4 target genes; whereas, Oct-3/4 and Nanog only have a negative feedback effect on their own promoters. Collectively, the work in this dissertation provides significant insights into the expression of a network of critical developmental regulators and how their levels are precisely controlled through a variety of mechanisms.