Investigating molecular and regulatory boundaries of the pluripotent state
Sindhu-Bertrand, Camille Anne
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AbstractAlthough progressively restricted to specialized functions during ontogeny, differentiated somatic cell nuclei can be experimentally directed to other cell types, including those with complete developmental potential. The technical challenges inherent to initial nuclear reprogramming methods such as somatic cell nuclear transfer and cell fusion pose significant hurdles to precisely dissecting the regulatory programs governing cell identity. The discovery of reprogramming via ectopic delivery of a defined set of transcription factors provided a tractable platform to uncover molecular characteristics of cellular specification and differentiation, cell type stability, and pluripotency. Despite ongoing progress, a detailed molecular understanding of the terminal events that coordinate the reprogramming from somatic to pluripotent cell remains elusive. To better understand how transcription factors mediate such a dramatic change in cell state, we optimized a time course-based model where differentiating cells are systematically challenged to reacquire pluripotency. Using this approach, we identify a transient period of time after pluripotency exit where cells are developmentally determined, yet respond to induction of exogenous reprogramming factors by reverting to the pluripotent state in a near-deterministic fashion. This brief period is followed by a rapid decline into somatic-like reprogramming kinetics and efficiency. By investigating transcriptional, epigenetic, and transcription factor dynamics on either side of this window, we find several key molecular parameters that prescribe the regulatory boundary between non-pluripotent and pluripotent identities. We show that the route to pluripotency is directed through OCT4 engagement at a distinguishable subset of pluripotent-state enhancers that are uniquely regulated during differentiation in vitro and in vivo and encode a distinctive combination of cis-regulatory sequences. From these data, we present a model where delayed silencing of these enhancers predisposes them to function as primary genetic targets for the final, deterministic transition into molecular pluripotency.
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