Regulation of XPC Binding Dynamics and Global Nucleotide Excision Repair by p63 and Vitamin D Receptor.

Abstract

p63 and the vitamin D receptor (VDR) play important roles in epidermal development and differentiation, but their roles and relationship in the response to ultraviolet (UV) radiation are less clear. Using TERT-immortalized human keratinocytes expressing shRNA targeting p63 in concert with exogenously applied siRNA targeting VDR, we assessed p63 and VDR's separate and combined effect on nucleotide excision repair (NER) of UV-induced 6-4 photoproducts (6-4PP). Knockdown of p63 reduced VDR and XPC expression relative to nontargeting controls, while knockdown of VDR had no effect on p63 and XPC protein expression, though alone it modestly reduced XPC mRNA. Upon UV irradiation through filters with 3 μm pores to create spatially discrete spots of DNA damage, keratinocytes depleted of p63 or VDR exhibited slower removal of 6-4PP than control cells over the first 30 min. Costaining of control cells with antibodies to XPC revealed that XPC accumulated at DNA damage foci, peaking within 15 min and gradually fading over 90 min as NER proceeded. In either p63- or VDR-depleted keratinocytes, XPC overaccumulated at spots of DNA damage so that 50% more XPC was retained at 15 min and 100% more XPC was retained at 30 min than in control cells, suggesting dissociation of XPC after binding was also delayed. Concurrent knockdown of VDR and p63 resulted in similar impairment of 6-4PP repair and XPC overaccumulation but even slower release of XPC from DNA damage sites such that 200% more XPC was retained relative to controls at 30 min post-UV. These results suggest that VDR accounts for some of p63's effects in delaying 6-4PP repair associated with overaccumulation and slower dissociation of XPC, though p63's regulation of basal XPC expression appears to be VDR-independent. The results are consistent with a model where XPC dissociation is an important step during NER and that failure to do so may inhibit subsequent repair steps. This work further links two important regulators of epidermal growth and differentiation to the DNA repair response to UV.