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Regulation and Biological Consequence of Retrotransposon Activation in Human Pluripotency

  • Author(s): Martin, Joseph William
  • Advisor(s): He, Lin
  • et al.
No data is associated with this publication.
Abstract

Mammalian genomes contain millions of transposable element sequences, but the overall impact they have on host biological processes remains poorly characterized. Retrotransposon insertions are known to actively shape embryonic development of model organisms, mainly by influencing transcription of host genes or by contributing functional protein or RNA products. Retrotransposons are also transcribed in human preimplantation embryos, and characterization of exapted retrotransposon families will help explain how human embryogenesis differs from other organisms. The second chapter of this dissertation presents studies on Human Endogenous Retrovirus-H (HERVH), which is a primate specific retrotransposon family functional in human embryonic stem cells (hESCs). The human genome contains over 2000 HERVH insertions and is associated with four distinct promoters, complicating efforts to understand host transcriptional regulation. Also, while HERVH RNA is clearly essential to maintain stemness, the mechanism of its action is unknown. To investigate the regulation of HERVH, we utilize bioinformatic prediction and reporter assays to identify transcription factor binding sites unique to the LTR7Y subfamily. A major finding of this dissertation is that HERVH regulation is recapitulated in naive and primed hESC culture, and the LTR7Y can be used to mark the naive state. We also find evidence that the primary mechanism of the HERVH RNA is in trans. We use genome engineering to delete an individual HERVH insertion and find it does not phenocopy family-wide HERVH knockdown or effect adjacent genes. Furthermore, ectopic overexpression of HERVH RNA effects the dynamics of BMP4/LY294002 induced mesendoderm differentiation, establishing a trans role for HERVH RNA in the postimplantation primitive streak. HERVH is the most active endogenous retrovirus (ERV) in preimplantation embryos, but we find other retrotransposons classes are more likely to modify adjacent cellular genes. In the third chapter of this dissertation, we identify a short interspersed element (SINE) insertion within ZBTB16, a developmentally important zinc-finger transcription factor. This SINE element serves as an alternative promoter, generating a transcript isoform found in the human oocyte that is capable of producing a truncated protein product. Using in vitro cell culture assays, we find the truncated ZBTB16 protein retains its function as a cell cycle regulator but shows altered subcellular localization and is resistant to post translational degradation, implying it may act to slow the cell cycle of early human embryos. The studies in this dissertation serve to clarify the complex regulation and mechanisms of function for retrotransposon families that are essential for human development. These findings advance the field of hESC biology by establishing clear markers of different pluripotent cell types, and ultimately add substantially to our understanding of how retrotransposons have shaped human pluripotency.

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This item is under embargo until March 10, 2023.