Transcriptional regulation of pluripotency in human embryonic stem cells

All of the diverse cells in the human body contain the same genetic information, and originally arose from a single cell, a fertilized egg. Embryogenesis is a result of cell division followed by differential gene expression, to selectively activate only the genes needed for development of each specialized cell type. By understanding the multiple gene activities required to either maintain stem cell pluripotency or effect cell specific differentiation, it should be possible to define conditions under which undifferentiated stem cells may be grown in large volume in culture, or induced to become mature cell types of therapeutic interest. The experiments described in this proposal are directed at understanding the regulation of gene expression in stem cells as they self-renew, and in the conversion of adult cells back to a stem cell-like state. During this reporting period we discovered mutations in SCC-A have been linked to dyskeratosis congenital (DC), a genetic disorder characterized by proliferative defect in stem cells. We were also able to purify SCC-B to homogeneity which means this could greatly enhance iPSC generation efficiency. SCCs are potential targets for drugs aimed at increasing or decreasing the ability of stem cells to divide. SCCs could also facilitate the production of high quality induced pluripotent stem cells for regenerative medicine and tissue replacement therapy.

All of the diverse cells in the human body contain the same genetic information, and originally arose from a single cell, a fertilized egg. Embryogenesis is a result of cell division followed by differential gene expression, to selectively activate only the genes needed for development of each specialized cell type. By understanding the multiple gene activities required to either maintain stem cell pluripotency or effect cell specific differentiation, it should be possible to define conditions under which undifferentiated stem cells may be grown in large volume in culture, or induced to become mature cell types of therapeutic interest. The experiments described in this proposal are directed at understanding the regulation of gene expression in stem cells as they self-renew, and in the conversion of adult cells back to a stem cell-like state. During this reporting period we discovered mutations in SCC-A have been linked to dyskeratosis congenital (DC), a genetic disorder characterized by proliferative defect in stem cells. We were also able to purify SCC-B to homogeneity which means this could greatly enhance iPSC generation efficiency. SCCs are potential targets for drugs aimed at increasing or decreasing the ability of stem cells to divide. SCCs could also facilitate the production of high quality induced pluripotent stem cells for regenerative medicine and tissue replacement therapy.

The experiments described in this proposal are directed at understanding the regulation of gene expression in stem cells as they self-renew, and in the conversion of adult cells back to a stem cell-like state. Our laboratory has identified two protein complexes, SCC and SCC-A, as well a third protein, SCC-B, that interact and are required for the activity of genes needed for stem cell pluripotency. A major goal of this project was to isolate and characterize these critical regulators of stem cell function. Purification and characterization of SCC-A and SCC revealed that they consist of nucleic acid-interacting proteins that had been recognized as playing critical roles in DNA repair and RNA processing, but had not previously been associated with pluripotency. SSC-B is linked to immune function, but had likewise not been previously associated with the regulation of gene expression. The studies during the current reporting period focused on the mechanisms of interactions between these co-activators, and on determining the structure of key components of the complexes, in particular SCC component XPC. XPC was shown to be essential for the recruitment of SCC to pluripotency genes by stem cell transcription factors Oct4 and Sox 2. XPC was also shown to enhance the activity of demethylating enzyme TDG in reprogramming, and the regions of both proteins required for their interaction was determined. Additionally, the structure of the human holo-XPC (SCC) complex was determined by single-particle electron microscopy.