Year 3

            Our goal was to determine the role of ERRa during early reprogramming of human cells. In human fibroblasts, it is ERRa that is critical for initiation of reprogramming. Thus we proposed to specifically isolate the ERRa-transiently expressing (tERRa) cells in early reprogramming and examine their properties in detail. We designed and tested various ways to isolate these cells via different reporters. We found that the 1kb sequence immediately upstream of the human ERRa transcription start site is sufficient to recapitulate ERRa activity. As expected, only a small percentage of reprogramming cells (human IMR90 fibroblasts) exhibit high ERRa expression. These cells express ERRa and its downstream targets, many of which are key metabolic enzymes, at a higher level when compared to the ERRa low population. This indicated that we had optimized an efficient reprogramming system to allow large-scale isolation for genome-wide studies. Using time-resolved RNA-Seq, we successfully characterized the genome-wide transcriptomic dynamics in tERRa cells. We found that these cells are undergoing a dramatic mesenchymal-to-epithelial transition, and exhibit a unique metabolic profile. We also charted the promoter and enhancer landscape by investigating the dynamic changes in H3K4me2 (a key transition histone mark regulated by a-ketoglutarate) in tERRa 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 tERRa cells are undergoing extensive epigenetic reprogramming. Motif analysis reveals enrichment of optimal binding sites for ERRa, as well as other pluripotency factors, at up-regulated H3K4me2 peaks, suggesting the direct role of ERRa in mediating the epigenetic changes.  Our characterization of the ERRa-a-ketoglutarate-H3K4Me2 axis should help to shed light on the regulation of cell fate and plasticity during early reprogramming of human cells.