Year 1

The major goal of this study is to determine the role of a nuclear hormone receptor, the estrogen-related receptor alpha (ERRα) in mediating the metabolic and epigenetic changes required during early reprogramming of human cells. Our recent findings have shown that in mice, its homologue ERRγ, is essential in mediating reprogramming. Gene expression analysis performed in human fibroblast cells indicated that in human cells, it is ERRα instead of ERRγ that is critical for initiation of reprogramming. Thus we proposed to specifically isolate the ERR-transiently expressing (tERRα) cells in early reprogramming and examine their properties in detail.

The first step towards achieving our goals is to overcome the difficulty in identifying and isolating sufficient quantities of tERRα cells for our proposed experiments. To do this, we first designed and tested various ways to isolate these cells using different reporter systems. We identified sequences in the human ERRα promoter region that allowed us to detect ERRα activity in vivo. After optimization of our reporter system, we confirmed that only a small percentage of reprogramming cells (in this case human fibroblasts) exhibit high ERRα expression. This small subpopulation of cells exhibits a detectably higher level of ERRα and its downstream targets, many of which are key metabolic enzymes, than the remaining population.

This demonstrates that we have optimized an efficient reprogramming system that will allow large-scale isolation of tERRα cells for our proposed genome-wide studies.
Our next steps will be to focus on the characterization of these tERRα cells. We will perform time-course RNA-Seq on tERRα cells and their counterparts, to determine the gene expression signatures of these cells. We will also examine whether these cells have a higher reprogramming efficiency compared to the rest of the cells. Furthermore, we will determine the epigenomic landscape of these cells by performing chromatin immunoprecipitation with massively parallel DNA sequencing (ChIP-Seq) on isolated tERRα cells and compare this to the rest of the reprogramming population. This should provide valuable insights into the role of ERRα in regulation of metabolic and epigenetic changes required for reprogramming of human cells.