UHRF1 ubiquitin E3 ligase activity in DNA methylation and skin stem cell homeostasis

Abstract

UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1), a multi-domain epigenetic factor, plays critical roles in a variety of biological processes, including DNA methylation, histone modifications, stem cells maintenance, embryonic development and tumorigenesis. C-terminal RING domain confers UHRF1 an E3 ubiquitin ligase activity and UHRF1-mediated histone H3 ubiquitination is an essential step in the maintenance of DNA methylation. Non-histone substrates of UHRF1 include DNMT3A and PML. The ubiquitination of PML tumor suppressor promotes tumorigenesis. Previous studies revealed that UHRF1 can be post-translationally modified by phosphorylation, ubiquitination, and methylation. Work from our laboratory suggested that UHRF1 can also be acetylated by histone acetyltransferase Mof on lysine residues Lys667, Lys668, and Lys670 located in the pre-RING linker region. In this work, I found that Mof-mediated UHRF1 acetylation resulted in increased E3 ligase activity and was more stable against β-TrCP1-mediated ubiquitination degradation. UHRF1 acetylation promoted UHRF1-mediated histone H3 ubiquitination, which in turn facilitated DNMT1 recruitment for more efficient DNA methylation maintenance. I also identified HDAC1 to act as a counterpart of Mof to deacetylate UHRF1. Besides, I showed, for the first time, that Mof knockdown in mouse ES cells led to a reduction of global DNA methylation. In this study, KLF4 (Kruppel-like factor 4) was identified as a novel ubiquitination substrate of UHRF1. UHRF1-dependent KLF4 ubiquitination was found to be critical in regulating mouse ES cells' pluripotency and ectodermal differentiation. By crossing K5-Cre mice with Uhrf1flox/flox mice, Uhrf1 was specifically depleted in K5 expressing cells. K5-Cre; Uhrf1flox/flox mice gradually developed SCC-like hyperplasia in epidermal basal layers and lost hair follicles after postnatal ten days. Uhrf1-mediated Klf4 ubiquitination was also found in the conditional knockout mice where increased Klf4 levels were observed in the basal layer. FACS sorting analyses revealed the exhaustion of hair follicle bulge stem cells, enhanced Tnf-α mediated inflammatory response in the epidermal stem cells IFE and inhibition of Wnt/β-catenin signaling in K5-Cre; Uhrf1flox/flox mice skin. These data suggested that UHRF1 is critical for the homeostasis of epidermal and hair follicle stem cells. Upon UHRF1 depletion in the epidermis, the elevated KLF4, increased inflammatory response, and reduced Wnt/β-catenin signaling together contributed to the dysregulated skin stem cell renewal and differentiation. Taken together, this work revealed a novel mechanism underlying the regulation of UHRF1 E3 ligase activity through Mof/HDAC1-mediated acetylation. I also demonstrated the essential role for UHRF1 in skin stem cell homeostasis as an E3 ligase via KLF4 ubiquitination independent of its function in DNA methylation, emphasizing the importance of the previously less studied post-translational modifications either on UHRF1 itself or by UHRF1.