Year 2

The major goal of this study is to determine the role of ERRα in mediating the metabolic switch and epigenetic changes during early reprogramming of human cells. In the human fibroblast, it is ERRα instead of ERRγ that is critical for initiation of reprogramming. Thus we proposed to specifically isolate the ERRα-transiently expressing (tERRα) cells in early reprogramming and examine their properties in detail.
In order to systematically characterize the tERRα cells, we designed and tested various ways to isolate these cells via different reporters. We found that the 1kb sequence immediately upstream of the human ERRα transcription start site is sufficient to recapitulate ERRα activity. As expected, only a small percentage of reprogramming cells (human IMR90 fibroblasts) exhibit high ERRα expression. These cells express ERRα and its downstream targets, many of which are key metabolic enzymes, at a higher level when compared to the ERRα low population. This demonstrates that we have optimized an efficient reprogramming system to allow large-scale isolation for genome-wide studies.
By using time-resolved RNA-Seq on tERRα cells and their counterparts, we successfully characterized the genome-wide transcriptomic dynamics in tERRα cells. Confirming our hypothesis, we found that these cells are undergoing a dramatic mesenchymal-to-epithelial transition, and exhibit a unique metabolic profile. Furthermore, we have charted the promoter and enhancer landscape by investigating the dynamic changes in H3K4me2 (a key transition histone mark) in tERRα cells compared to control cells. We identified significant changes in H3K4me2 levels at genes involved in development, including several key reprogramming genes, suggesting that the tERRα cells are undergoing extensive epigenetic reprogramming. Motif analysis reveals enrichment of optimal binding sites for ERRα, as well as other pluripotency factors, at up-regulated H3K4me2 peaks, suggesting the direct role of ERRα in mediating the epigenetic changes.
We will continue to investigate the dynamics of metabolite changes in tERRα cells. Additional genome-wide assays will also be performed to investigate other epigenetic markers changes in tERRα cells. This should provide valuable insights into the role of ERRα in regulation of metabolic and epigenetic changes required for reprogramming of
human cells.