Transcriptional control of pluripotency requires coordinated action of key transcription factors such as Nanog, Oct4, and Sox2 in conjunction with many epigenetic regulators including histone modifying enzymes such as H3K9 methyltransferases G9a/GLP and DNA (de)methylation enzymes such as DNMT1/3a/3b and TET1/2/3 family proteins. TET family proteins have gained wide popularity due to their intrinsic enzymatic activities leading to active and passive DNA demethylation. Intensive studies have been performed and much knowledge gained in understanding how TET-mediated conversions of 5mC into 5hmC and 5fC/5caC contribute to Nanog/Oct4/Sox2 functions in promoting stem cell pluripotency. In contrast, while catalytic activity independent functions of TET proteins in transcriptional repression of target genes in ESCs are well established, little mechanistic insights are available to explain their catalytic activity independent functions in controlling retrotransposon silencing and Polycomb target repression, two epigenetic regulatory pathways that are functionally and evolutionally significant for stem cell pluripotency and early development. The goal of this exploratory grant proposal is to dissect the molecular mechanism by which Tet1 and Tet2 repress endogenous retrovirus (ERV) elements and a subset of developmental regulators in maintaining stem cell pluripotency. We will document an RNA binding protein Pspc1 as a novel partner protein of Tet1/2 and its functional contribution to TET and Polycomb functions in transcriptional repression. Our proposed studies encompass two Specific Aims: 1) Define functional significance of the Tet1-Pspc1 partnership in regulating ERVIII and 2C genes for stem cell pluripotency; 2) Explore novel Tet1 functions in regulating RNA targets for pluripotency of ESCs. Specifically, we will establish the physical and functional relationship among Pspc1, Tet1/2, and histone modifying complex G9a/GLP in ERVIII and 2C gene regulation for pluripotency control. Our studies will fill in a major knowledge gap in our understanding of how TET proteins control retroelements in the pluripotent genome of ESCs and how Tet1 and Polycomb functionally cooperate without direct physical association in repressing developmental regulators and maintaining pluripotency. This exploratory project will reveal novel Tet1 functions in regulating retroelements and other non-coding RNA targets for pluripotency of ESCs, and also have an overarching impact in our understanding of retrotransposon-mediated genome evolution and Polycomb-shaped epigenome in pluripotency and early development.
Pluripotent stem cells have enormous therapeutic and regenerative potential in medicine. Elucidation of the molecular mechanisms by which transcriptional regulators and epigenetic cofactors control pluripotency will facilitate therapeuti applications of pluripotent stem cells in medicine. Understanding the novel functions of TET family proteins in transcriptional repression of transposable elements and developmental genes in pluripotent cells will not only improve our knowledge on gene regulation in early human development and disease progression but also help develop safe stem cell medicines.
