Investigating the role of OCT4 isoforms in human embryonic stem cell self-renewal and differentiation.

摘要

The focus of our study is on Oct4, a POU domain transcription factor and critical regulator of pluripotency whose levels are precisely controlled in mouse embryonic stem cells (mESC). In contrast to the single murine Oct4 isoform, which is better understood and more widely studied, three alternatively spliced isoforms exist in humans---OCT4A, OCT4B, and OCT4B1.;In this thesis, we first examine the functional role of the OCT4A isoform in hESC by using an inducible lentiviral overexpression and knockdown strategy to manipulate OCT4A above or below physiologic levels. Additionally, we demonstrate the utility of an effective and efficient method to achieve conditional gene expression in hESC. We show that specific knockdown of OCT4A results in hESC differentiation, as indicated by changes in morphology, cell surface antigen expression, and upregulation of ectodermal genes. In contrast, inducible overexpression of OCT4A in hESC leads to a transient instability of the hESC phenotype, as indicated by changes in morphology, cell surface antigen expression, and transcriptional profile that returns to baseline within 5 days. In addition, sustained expression of OCT4A enhances the cloning efficiency of hESC, suggesting that higher levels of OCT4A are beneficial for hESC self-renewal. Our results demonstrate that OCT4A dysregulation in hESC does not result in the same differentiation patterns seen in mESC and highlight the importance of conducting isoform-specific studies for human OCT4.;We next examine a mechanism for regulating self-renewal and differentiation of hESC that involves the core transcriptional regulators NANOG, SOX2, and OCT4A. These proteins occupy and transactivate the promoters of genes expressed during self-renewal and repress the promoters of developmental genes required for differentiation. Nuclear localization signals (NLS) have previously been identified in mouse Oct4 and Sox2, and both NLS and nuclear export signal (NES) sequences were recently identified in human NANOG. In this study, we hypothesize that nucleocytoplasmic shuttling of core ESC factors contributes to regulation of self-renewal and differentiation. We determine that amino acids (aa) 190-204 in the POUB domain of human OCT4A and aa 71-84 in the HMG domain of human SOX2 contain functional NES sequences. Furthermore, these NES from OCT4A and SOX2 depend on the CRM1 nuclear export pathway. Using an endoderm differentiation protocol of H9 hESC, we investigate whether localization of core hESC factors changes during differentiation. As predicted, NANOG, OCT4A, and SOX2 are found in the nucleus during the undifferentiated, self-renewal state, but four days after endoderm induction, differentiating cells at colony edges demonstrate pan-cellular distribution of NANOG, OCT4 and SOX2. By day 5, the core hESC factors are undetectable in differentiating cells. In summary, we show that the core hESC transcription factors have functional NES and propose that such signals function to rapidly remove core factors from the nucleus at the time of lineage commitment, thereby halting the self-renewal program and allowing differentiation to proceed.;Finally, in the last section of this thesis, we describe our studies examining the function of OCT4B in hESC. Analysis of OCT4B expression in hESC shows that OCT4B transcript is expressed at low levels in hESC and human teratocarcinoma cell lines but that endogenous OCT4B protein cannot be detected by western blotting. In addition, OCT4B transcript levels do not change as hESC differentiate into embryoid bodies over the course of five weeks. Transient transfection of siRNA oligos specifically targeting the unique N-terminus of OCT4B do not result in efficient OCT4B knockdown, although siRNA targeting the C-terminus is effective. There is no change in hESC phenotype by morphology or cell surface marker expression after OCT4B downregulation. Inducible overexpression of V5-tagged OCT4B in hESC results in low levels of transgene transcript and undetectable levels of V5-tagged protein by western blotting and immunofluorescence staining. In contrast, inducible overexpression of OCT4B-V5 in 293A cells results in abundant OCT4B-V5 protein, suggesting a silencing mechanism specific to hESC. Lastly, heat shock assays fail to confirm a role for OCT4B as a stress response protein in hESC, contrary to previously reported results. (Abstract shortened by UMI.)