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

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.

Vitamin D receptor cross-talk with p63 signaling promotes epidermal cell fate.

The vitamin D receptor with its ligand 1,25 dihydroxy vitamin D3 (1,25D3) regulates epidermal stem cell fate, such that VDR removal from Krt14 expressing keratinocytes delays re-epithelialization of epidermis after wound injury in mice. In this study we deleted Vdr from Lrig1 expressing stem cells in the isthmus of the hair follicle then used lineage tracing to evaluate the impact on re-epithelialization following injury. We showed that Vdr deletion from these cells prevents their migration to and regeneration of the interfollicular epidermis without impairing their ability to repopulate the sebaceous gland. To pursue the molecular basis for these effects of VDR, we performed genome wide transcriptional analysis of keratinocytes from Vdr cKO and control littermate mice. Ingenuity Pathway analysis (IPA) pointed us to the TP53 family including p63 as a partner with VDR, a transcriptional factor that is essential for proliferation and differentiation of epidermal keratinocytes. Epigenetic studies on epidermal keratinocytes derived from interfollicular epidermis showed that VDR is colocalized with p63 within the specific regulatory region of MED1 containing super-enhancers of epidermal fate driven transcription factor genes such as Fos and Jun. Gene ontology analysis further implicated that Vdr and p63 associated genomic regions regulate genes involving stem cell fate and epidermal differentiation. To demonstrate the functional interaction between VDR and p63, we evaluated the response to 1,25(OH)2D3 of keratinocytes lacking p63 and noted a reduction in epidermal cell fate determining transcription factors such as Fos, Jun. We conclude that VDR is required for the epidermal stem cell fate orientation towards interfollicular epidermis. We propose that this role of VDR involves cross-talk with the epidermal master regulator p63 through super-enhancer mediated epigenetic dynamics.

Vitamin D Receptor Promotes Global Nucleotide Excision Repair by Facilitating XPC Dissociation from Damaged DNA.

Vitamin D receptor (VDR) is important for normal DNA repair, although the mechanism by which it acts is unclear. After focal UV irradiation to create subnuclear spots of DNA damage, epidermal keratinocytes from VDR-null mice as well as human epidermal keratinocytes depleted of VDR with small interfering RNA removed pyrimidine-pyrimidone (6-4) photoproducts more slowly than control cells. Costaining with antibodies to XPC, the DNA damage recognition sensor that initiates nucleotide excision repair, showed that XPC rapidly accumulated at spots of damage and gradually faded in control human keratinocytes. In VDR-depleted keratinocytes, XPC associated with DNA damage with comparable efficiency; however, XPC's dissociation dynamics were altered so that significantly more XPC was bound and retained over time than in control cells. The XPF endonuclease, which acts subsequently in nucleotide excision repair, bound and dissociated with comparable kinetics in control and VDR-depleted cells, but the extent of binding was reduced in the latter. These results as well as kinetic modeling of the data suggest that VDR's importance in the repair of UV-induced DNA damage is mediated in part by its ability to facilitate the dissociation of XPC from damaged DNA for the normal recruitment and assembly of other repair proteins to proceed.