Role and Regulation of CIEBPa in Response to DNA Damage

DNA damage;Cell cycle regulation;C/EBP

Ranjan, Rakesh ; Robert C. Smart , Committee Chair,Ranjan, Rakesh ; Robert C. Smart ; Committee Chair

University:North Carolina State University

关键词: DNA damage;    Cell cycle regulation;    C/EBP;   

Others  :  https://repository.lib.ncsu.edu/bitstream/handle/1840.16/3096/etd.pdf?sequence=1&isAllowed=y

美国|英语

来源: null

【 摘 要 】

C/EBPa and C/EBPβ are members of basic leucine zipper (bZIP) class of transcription factors.They are abundantly expressed in epidermis.C/EBPa expression is diminished in mouse and human squamous cell as well as basal cell carcinomas.Recently, C/EBPa has been shown to be an epithelial tumor suppressor gene in genetically engineered mouse model.siRNA experiment has suggested a role for C/EBPα in DNA damage G1 checkpoint response.But definitive genetic evidence is lacking.Here, we report that C/EBPα is highly inducible in primary dermal fibroblasts by DNA damaging agents that induce strand breaks, alkylate and crosslink DNA as well as those that produce bulky DNA lesions.Fibroblasts deficient in C/EBPα (C/EBPα-/-) display an impaired G1 checkpoint as evidenced by inappropriate entry into S-phase in response to DNA damage and these cells also display an enhanced G1 to S transition in response to mitogens. The induction of C/EBPα by DNA damage in fibroblasts does not require p53.EMSA analysis of nuclear extracts prepared from UVB- and MNNG-treated fibroblasts revealed increased binding of C/EBPβ to a C/EBP consensus sequence and ChIP analysis revealed increased C/EBPβ binding to the C/EBPα promoter.To determine whether C/EBPβ has a role in the regulation of C/EBPα we treated C/EBPβ-/-fibroblasts with UVB or MNNG.We observed C/EBPα induction was impaired in both UVB- and MNNG- treated C/EBPβ-/- fibroblasts.Our study reveals a novel role for C/EBPβ in the regulation of C/EBPα in response to DNA damage and provides definitive genetic evidence that C/EBPα has a critical role in the DNA damage G1 checkpoint.Since, the evidence for C/EBPa as a tumor suppressor gene in human skin is mounting; we decided to further study the signaling pathway of C/EBPa induction in response to DNA damage in keratinocytes.C/EBPα has been shown to be induced by DNA damage in human and mouse skin, and in primary and immortalized keratinocyte cell lines.In keratinocytes, the induction of C/EBPa requires p53; p53 directly binds to C/EBPa promoter and is responsible for increases in C/EBPa mRNA expression in response to DNA damage.We show that GSK3β inhibitors block C/EBPa protein and message induction in response to DNA damage without altering p53 protein levels.Further, we found that GSK3β interaction with p53 increased in response to DNA damage.In addition, UVB treatment of keratinocytes resulted in post-translation modification of C/EBPa protein.Our results suggest that GSK3β regulates C/EBPa expression, interacts with p53, and that C/EBPa protein undergoes post-translational modification in response to DNA damage.Hence, from these two studies we have provided evidence thati)C/EBPa has a role in DNA damage G1 checkpoint response and in mitogen induced G1/S transition.ii)C/EBPa is induced in response to various DNA damage in different cell types and regulation of C/EBPa is cell type specific.iii)GSK3β might have a role in C/EBPa induction in response to DNA damage in keratinocytes.