Year 3

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.