|dc.description.abstract||Embryonic stem cells are pluripotent; they have the ability to form any cell type of the developing embryo. Due to their pluripotent nature there is a strong interest in understanding the biology of embryonic stem cells. This interest stems not only from furthering understanding of early events in human development, but also because pluripotent stem cells have potential applications in regenerative medicine and cancer biology.
Transcription programmes and cell cycle dynamics delicately balance the transition between pluripotency and differentiation. However, the molecular switches that govern whether embryonic stem cells self-renew and maintain their pluripotent state, or differentiate are poorly understood. The protein complex cohesin has distinct roles in both the cell cycle and in the regulation of gene expression. Because cohesin links cell proliferation and cell differentiation pathways it is a candidate for a molecular switch that governs the fate of embryonic stem cells.
Research conducted prior to this study revealed that myca (zebrafish c-Myc) expression is downregulated in zebrafish embryos harbouring a null mutation in the rad21 subunit of cohesin. c-Myc is a transcription factor that plays a key role in maintaining the pluripotent state, therefore this finding linked cohesin to pluripotency. Following on from this, my studies focused on investigating the transcriptional role of cohesin and its potential links with pluripotency, using the zebrafish as a model. My first objective was to determine if cohesin directly regulated the expression of myca, and to understand the mechanism of this regulation. Using chromatin immunoprecipitation (ChIP) coupled with quantitative PCR (qPCR) I showed that cohesin bound to the myca locus, supporting a direct role for cohesin in regulating myca expression. Furthermore, analysis of published data revealed that the positive regulation of c-Myc by cohesin is conserved in Drosophila, mouse and human. In zebrafish, cohesin bound 1.27 kb upstream of the transcriptional start site of myca and at the transcriptional start site itself. These cohesin binding sites are conserved at the mammalian c-MYC locus, suggesting that the mechanism by which cohesin regulates c-Myc is also conserved. To investigate what this mechanism could be I tested the hypothesis that cohesin binding upstream of myca functioned as a chromatin barrier, preventing the spread of repressive chromatin into the myca locus, to allow myca expression. According to this hypothesis, the absence of cohesin would compromise chromatin barrier activity, allowing repressive chromatin marks to spread into the myca locus, preventing myca transcription. However, ChIP-qPCR experiments revealed no spreading of repressive chromatin marks into the myca locus in the absence of Rad21, ruling out this chromatin barrier model of how cohesin regulates myca expression. While the mechanism of cohesin-dependent regulation of c-Myc remains enigmatic, results suggest that chromatin changes at the transcriptional start site may be important.
Having confirmed that myca is directly regulated by cohesin, I then went on to determine if there were broader transcriptional links between cohesin and pluripotency. To investigate this possibility, ChIP coupled with high throughput sequencing (ChIP-seq) was performed to identify genes bound by, and therefore potentially regulated by, cohesin during pluripotency. This genome-wide analysis revealed that cohesin bound the regulatory regions of a number of genes encoding transcription factors (including POU domain, class 5 transcription factor 1 (pou5f1), SRY-box containing gene (sox2), Nanog homeobox (nanog)) and chromatin regulators (including polycomb group complex members bmi1 and enhancer of polycomb homologue 2 (epc2), and the histone-modifying enzyme SET domain containing 1B a (setd1ba)), all of which are involved in maintaining the pluripotent state. Preliminary studies suggest this binding has functional consequences, with Rad21 knockdown affecting the expression of the genes it binds.
These studies have revealed a strong potential for cohesin to play an important role in pluripotency, by regulating the expression of genes that maintain the pluripotent state. Together with published data, the findings suggest that cohesin may function as a master transcriptional facilitator in pluripotency, and through this role may determine the fate of embryonic stem cells.||