Date of Award
Doctor of Philosophy (PhD)
de Lange Laboratory
Shelterin is an essential telomeric protein complex that prevents DNA damage signaling and DNA repair in a compartmentalized manner. We assessed contributions of the conserved shelterin component, Rap1, to telomere endprotection. Rap1 was first discovered in budding yeast as a transcription factor and was later shown to bind directly to telomeres. Two important functions of Rap1 in yeast are: the negative regulation of telomere length, and the inhibition of the double-strand break (DSB) repair pathway non homologous end-joining (NHEJ). Mammalian Rap1 interacts with shelterin factor TRF2, to localize to telomeres, and human Rap1 is implicated in repressing NHEJ. Surprisingly, removal of Rap1 from telomeres revealed that mouse Rap1 was not required to inhibit NHEJ, but instead was critical for the repression of the DSB repair pathway homology directed repair (HDR). We showed that complex formation of Rap1 and TRF2 was most likely necessary to repress HDR, although the mechanism of how it does so remains to be determined. Two discrepancies exist between mouse and human Rap1 regarding repression of NHEJ and regulation of telomere length. Human Rap1 was proposed to inhibit NHEJ, but we observed no evidence of telomere fusions in the mouse Rap1 knockout. Similarly, telomere elongation was observed upon knockdown of human Rap1, but no telomere length phenotypes were observed in Rap1-deficient mice. With the advent of new genome-editing technologies that facilitate targeting in human cells, we constructed TALEN-mediated knockouts of human Rap1 in numerous cell lines. Loss of human Rap1 did not lead to an induction of NHEJ, or show consistent changes in telomere length, indicating that similar to mouse Rap1, human Rap1 does not have an important function in protection or length regulation of human telomeres. Instead, we found that mammalian Rap1, like its unicellular orthologs, affects gene expression. Therefore, perhaps the conservation of Rap1 reflects its role in transcriptional regulation rather than a function at telomeres. Organismal discrepancies regarding the function of shelterin components, other than Rap1, also exist. Targeting of human shelterin component POT1 (POT1a/b in the mouse) with shRNAs shows a reduction in 3’ telomere overhangs and a mild induction of DNA damage signaling. However, deletion of mouse POT1a and –b results in extended 3’ overhangs and a massive induction of the DNA damage response. To understand the role of human POT1 in telomere protection, we used TALENs to generate human knockout cell lines lacking POT1. We found that similar to mouse POT1, deletion of human POT1 elicited significant DNA damage signaling. Strikingly, the amount of 3’ singlestranded DNA remained unchanged upon loss of POT1, highlighting a potential difference in overhang regulation between mice and humans.
Kabir, Shaheen, "Investigating Mechanisms of Telomere End-Protection" (2015). Student Theses and Dissertations. 269.