Rupa Sridharan, Ph.D. - US grants

? DESCRIPTION (provided by applicant): Pluripotent stem cells (PSCs) have the remarkable properties of self-renewal and the capacity to generate differentiated cell types upon exposure to the correct stimulus. PSCs can be derived from the embryo (embryonic stem cells - ESCs) or by overexpression of transcription factors from somatic cells (induced pluripotent stem cells- iPSCs). The process of reprogramming to the iPSC state is slow- taking about 2-3 weeks to complete and inefficient - with only about a maximum of 5% of the starting population completing the process. The properties of PSCs are maintained extrinsically by controlling signaling pathways that prevent their differentiation. Intrinsically there is an auto regulatory lop of core transcription factors, which interacts with the epigenome to maintain the pluripotent state. While in general it is known that modifying the epigenome impacts reprogramming, how specific chromatin modifiers and signaling pathways mechanistically engage with the pluripotency regulatory network is largely unknown. We have found that in reprogramming intermediates, the combined action of a chromatin regulator and signaling modulator synergistically allowed the acquisition of an iPSC state at a very high efficiency. Using this system we have determined that, temporal erasure of key epigenetic marks occurs concomitant with both the transcriptional activation of pluripotency genes and down regulation of key growth factor signaling genes. In this proposal we will investigate the mechanism of interplay between the epigenome and signaling during the acquisition of pluripotency with the following aims: 1) To elucidate the mechanism of differential contribution of histone demethylases to pluripotency 2) To determine the interdependence of epigenetic marks during the acquisition of pluripotency and 3) To define the mechanistic contribution of gene repression during reprogramming. In this proposal we will gain a significant understanding of the mechanism of reprogramming and the barriers that have to be overcome to reach the iPSC state. This information is essential for expediting the process and increasing the efficiency and will therefore be highly impactful in translating the use of iPSCs for therapeutic purposes.

ABSTRACT Functional specialization in a multicellular organism arises when cell fate is established by a specific gene expression pattern. During development from a totipotent cell this is accomplished by the synthesis of spatial and signaling cues that result in epigenetic modifications to elicit unipotent gene expression. Once established, such gene expression patterns are stable unless disrupted by disease or injury. Remarkably the over expression of a few proteins can result in reprogramming of an established cell fate to generate induced pluripotent stem cells (iPSCs) that have the potential to develop into any of the cells of an embryo just like embryonic stem cells (ESCs). iPSCs are the ideal starting point for regenerative therapy since they overcome the ethical and practical concerns of using ESCs. Thus reprogramming provides an ideal model system to mechanistically define cell identity safeguards. However a critical barrier to studying reprogramming is the low efficiency (~3%) and differential kinetics (2-3 weeks) of obtaining iPSCs, so that heterogeneous transcriptional changes are masked in population based studies. We have generated a high efficiency system that combines epigenetic and signaling regulators. Using this system, in this proposal we will determine the most parsimonious route of reprogramming to iPSCs and elucidate the chromatin transitions in cells that become reprogrammed.