Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Pharmacology and Toxicology

Supervisor

Dr. Lina Dagnino

Abstract

The E2F1 transcription factor regulates the expression of key genes involved in cell proliferation and differentiation to maintain skin homeostasis. The expression of E2F1 is tightly regulated during cell cycle progression and when cells are committed to differentiate, as well as in response to DNA damage. In keratinocytes, E2F1 protein and transcript levels increase following UV-induced DNA damage, whereas, in response to Ca2+-induced differentiation, both E2F1 protein and transcript levels decrease. In this thesis, I examined in detail the mechanism that modulates E2F1 stability following DNA damage and during keratinocyte differentiation. I show that E2F1 associates with hHR23 and together these proteins participate in the nucleotide excision repair (NER) pathway. hHR23A stabilizes E2F1 by inhibiting its proteasomal degradation. Moreover, E2F1 regulates hHR23A subcellular localization and recruits hHR23A to sites of DNA photodamage to facilitate DNA repair. These results unveil a novel mechanism involving E2F1 and hHR23A to enhance the repair of damaged DNA, contributing to the maintenance of genomic stability following UV-induced DNA damage in epidermal cells. In contrast to DNA damage, proper keratinocyte differentiation occurs when E2F1 is down regulated. Although the regulation of E2F1 that leads to its nuclear export and degradation has been reported, the exact mechanisms are poorly characterized. When E2F1 Ser403 and/or Thr433 are substituted to an alanine (E2F1 ST/A), a significant increase in the half-life of this mutant in differentiated keratinocytes is observed. Furthermore, the E2F1 ST/A mutant does not form appreciable levels of K11- or K48-linked polyubiquitylation, which is associated with degradation. Moreover, I show that iii Cdh1, an activator protein of the anaphase-promoting complex/cyclosome (APC/C) complex, interacts with E2F1 in keratinocytes, and its inactivation prevents the turnover of E2F1 following Ca2+-induced differentiation. Collectively, these results reveal a new mechanism involving E2F1 and Cdh1 in coordinating its degradation through post-translational modification and nuclear export in differentiated keratinocytes. By elucidating the appropriate events required for the turnover of E2F1, we can better understand and treat various epidermal diseases such as non-melanoma skin carcinoma.

Available for download on Sunday, November 11, 2018


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