Abstract
The E2F1 transcription factor plays key roles in skin homeostasis. In the epidermis, E2F1 expression is essential for normal proliferation of undifferentiated keratinocytes, regeneration after injury and DNA repair following UV radiation-induced photodamage. Abnormal E2F1 expression promotes nonmelanoma skin carcinoma. In addition, E2F1 must be downregulated for proper keratinocyte differentiation, but the relevant mechanisms involved remain poorly understood. We show that differentiation signals induce a series of post-translational modifications in E2F1 that are jointly required for its downregulation. Analysis of the structural determinants that govern these processes revealed a central role for S403 and T433. In particular, substitution of these two amino acid residues with non-phosphorylatable alanine (E2F1 ST/A) interferes with E2F1 nuclear export, K11- and K48-linked polyubiquitylation and degradation in differentiated keratinocytes. In contrast, replacement of S403 and T433 with phosphomimetic aspartic acid to generate a pseudophosphorylated E2F1 mutant protein (E2F1 ST/D) generates a protein that is regulated in a manner indistinguishable from that of wild type E2F1. Cdh1 is an activating cofactor that interacts with the anaphase-promoting complex/cyclosome (APC/C) ubiquitin E3 ligase, promoting proteasomal degradation of various substrates. We found that Cdh1 associates with E2F1 in keratinocytes. Inhibition or RNAi-mediated silencing of Cdh1 prevents E2F1 degradation in response to differentiation signals. Our results reveal novel regulatory mechanisms that jointly modulate post-translational modifications and downregulation of E2F1, which are necessary for proper epidermal keratinocyte differentiation.
Highlights
The E2F family of transcription factors plays important roles in myriad physiological processes
Our studies describe a multifactorial mechanism of E2F1 degradation during keratinocyte differentiation that involves coordinate changes in subcellular localization and ubiquitylation patterns, and in which S403 and T433 play key modulatory roles
E2F1 participates in repair of DNA photodamage induced by UV radiation through various mechanisms, including association with hHR23 proteins involved in nuclear excision repair [9]
Summary
The E2F family of transcription factors plays important roles in myriad physiological processes. Physiological activation of keratinocytes to regenerate the wounded epidermis is associated with E2F1 upregulation, and is severely impaired in the absence of this transcription factor [8]. Due to their chronic exposure to solar UV radiation, epidermal keratinocytes have developed efficient mechanisms for DNA repair to www.impactjournals.com/oncotarget avoid carcinogenic transformation, which rely on E2F1 and various other factors [9, 10]
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