Pluripotent stem cells are a renewable cell source with the capacity to differentiate into any specialized cell in the adult body. These traits make pluripotent cells a tractable tool for studying normal and dysfunctional biological networks in the context of development. Therefore, pluripotent cells have the potential to greatly impact human health through drug testing, disease modeling, tissue engineering, and regenerative medicine. Harnessing this potential requires a detailed understanding of the biology that governs pluripotency and directs differentiation. The mechanisms that guide these processes have been studied extensively at the protein and RNA level; however, the role of epigenetic regulation has only recently come into focus. A largely unexplored aspect of epigenetic regulation is the spatial organization of genes relative to distant genomic regions and regulatory elements. It is our central hypothesis that genomic regions important for pluripotency associate in the nucleus and change upon induction of differentiation and reprogramming. Charting DNA interaction and its dynamics will resolve this regulatory system in pluripotent cells and may provide a means to manipulate these cells for research and therapeutic purposes.
Specific Aims :
The first aim i n this proposal will map long-range DNA interaction for key pluripotency genes in pluripotent cells.
The second aim tracks chromatin interactions at various stages of development and also explores the dynamics of DNA interaction during reprogramming.
The third aim i dentifies novel protein components that mediate chromatin interaction and applies biochemistry to characterize these proteins. Study Design: We propose research that combines expertise in stem cell biology with emerging genomic technology. Specifically, we will use modified circular chromosome conformation capture (m4C) to examine DNA interaction for key pluripotency genes Oct4 and Sox2 in pluripotent and multipotent stem cells. We will also track chromatin organization following the induction of pluripotency by sampling cells at intermediate stages of reprogramming. By characterizing proteins that mediate chromatin structure, we will determine key epigenetic regulators. Through targeted knockdown of these regulators, we will establish their functional role in pluripotency. These data, coupled to existing gene expression, chromatin modification, and ChIP results will reveal in unparalleled detail the epigenetic regulatory mechanisms at play during pluripotency, differentiation, and reprogramming. The sponsoring laboratory has numerous resources available to accomplish these goals, including instrumentation and technical training for m4C and deep sequencing. Resources are also available through the host institution for computational support and large-scale data analysis. The training program includes numerous opportunities for career development through cutting edge research, grant writing, and student mentoring.
Pluripotent stem cells hold enormous promise for regenerative medicine, tissue engineering, disease modeling, and drug testing. Understanding the regulatory networks that direct these cells to self-renew or differentiate to specialized lineages is paramount to realizing this potential. To meet this need, the research proposed here will characterize long-range DNA interaction and its dynamics in the context of pluripotency, differentiation and reprogramming.
| Brumbaugh, Justin; Di Stefano, Bruno; Wang, Xiuye et al. (2018) Nudt21 Controls Cell Fate by Connecting Alternative Polyadenylation to Chromatin Signaling. Cell 172:106-120.e21 |
| Choi, Jiho; Clement, Kendell; Huebner, Aaron J et al. (2017) DUSP9 Modulates DNA Hypomethylation in Female Mouse Pluripotent Stem Cells. Cell Stem Cell 20:706-719.e7 |
| Borkent, Marti; Bennett, Brian D; Lackford, Brad et al. (2016) A Serial shRNA Screen for Roadblocks to Reprogramming Identifies the Protein Modifier SUMO2. Stem Cell Reports 6:704-716 |
| Bar-Nur, Ori; Verheul, Cassandra; Sommer, Andreia G et al. (2015) Lineage conversion induced by pluripotency factors involves transient passage through an iPSC stage. Nat Biotechnol 33:761-8 |
| Cheloufi, Sihem; Elling, Ulrich; Hopfgartner, Barbara et al. (2015) The histone chaperone CAF-1 safeguards somatic cell identity. Nature 528:218-24 |
| Bar-Nur, Ori; Brumbaugh, Justin; Verheul, Cassandra et al. (2014) Small molecules facilitate rapid and synchronous iPSC generation. Nat Methods 11:1170-6 |
