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Theses Doctoral

Regulation of Proviral Expression and Post-Translational Modifications in Embryonic Cells

Lee, Andreia H.

Moloney Murine Leukemia Virus (M-MLV) proviral DNA is transcriptionally silenced in mouse embryonic cells by a repressor complex containing tripartite-motif-containing 28 (Trim28). Trim28 depends on post-translational modifications, such as sumoylation and phosphorylation, and its interactions with several co-repressor proteins to regulate its repressive activity. YY1 is one such Trim28 co-repressor protein, recently found to tether the Trim28 silencing complex to the M-MLV promoter. Here, we investigated the biochemical interaction of Trim28 and YY1, and the role of sumoylation and phosphorylation of Trim28 in mediating M-MLV silencing. Experiments probing the binding of YY1 and Trim28 in vitro suggested that their interaction occurs indirectly. Mutational studies demonstrated that the RBCC domain of Trim28 is sufficient for interaction with YY1 while the acidic region 1 and zinc fingers of YY1 were necessary and sufficient for its interaction with Trim28. Additionally, we found that the K779 residue was critical for Trim28-mediated silencing of M-MLV in embryonic cells.
The repressor complex that silences M-MLV is very large and likely consists of many protein subunits. A few proteins contained in the repressor complex have been identified, including Trim28, but the identity of most of the components forming the repressor complex are unknown. We detected a new form of the complex that is of even high molecular weight and likely contains additional associated cofactors. We reported an approach for purifying this larger repressor complex and identified new candidates for cofactors that may potentially function in the silencing of M-MLV.
We also examined the regulation of sumoylation in embryonic cells. Sumoylation conjugation is a post-translational modification that affects a diverse range of processes and is important for embryo survival. Overall inhibition of the SUMO pathways results in embryonic lethality demonstrating the importance of the SUMO pathways for embryonic viability; however, our understanding of SUMO function in embryos at the cellular and molecular level is still greatly lacking. We demonstrated that SUMO1 cannot be overexpressed in embryonic carcinoma and embryonic stem cells and that SUMO1 overexpression is prevented at a post-transcriptional level. This occurred specifically for SUMO1 and not for SUMO2 overexpression. Furthermore, blocking conjugation or increasing the deconjugation of SUMO1 to substrates significantly improved SUMO1 overexpression. The results indicate that the overexpression of SUMO1 protein, in itself, is tolerated in embryonic cells, but the accumulation of substrate(s) modified by SUMO1 appears to be strongly prevented by an embryonic-specific post-transcriptional mechanism.

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More About This Work

Academic Units
Biological Sciences
Thesis Advisors
Goff, Stephen P.
Degree
Ph.D., Columbia University
Published Here
May 11, 2017
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