C/EBPalpha expression is partially regulated by C/EBPbeta in response to DNA damage and C/EBPalpha-deficient fibroblasts display an impaired G1 checkpoint.
We observed that CCAAT/enhancer-binding protein (C/EBP)alpha is highly inducible in primary 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/EBPalpha (C/EBPalpha(-/-)) display an impaired G1 checkpoint as evidenced by an inappropriate entry into the S-phase in response to DNA damage, and these cells also display an enhanced G1/S transition in response to mitogens. The induction of C/EBPalpha by DNA damage in fibroblasts does not require p53. Electrophoretic mobility shift assay (EMSA) analysis of nuclear extracts prepared from ultraviolet B (UVB)- and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated fibroblasts showed increased binding of C/EBPbeta to a C/EBP consensus sequence and chromatin immunoprecipitation (ChIP) analysis also showed increased C/EBPbeta binding to the C/EBPalpha promoter. To determine whether C/EBPbeta has a function in the regulation of C/EBPalpha, we treated C/EBPbeta(-/-) fibroblasts with UVB or MNNG. We observed that C/EBPalpha induction was impaired in both UVB- and MNNG-treated C/EBPbeta(-/-) fibroblasts. Our study shows a novel function for C/EBPbeta in the regulation of C/EBPalpha in response to DNA damage and provides definitive genetic evidence that C/EBPalpha has a critical role in the DNA damage G1 checkpoint.
Ranjan
R
R
Cell Signaling and Cancer Group, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC 27695-7633, USA.
Thompson
E A
EA
Yoon
K
K
Smart
R C
RC
D000818:Animals; D015153:Blotting, Western; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000235:genetics / Q000378:metabolism*; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D005434:Flow Cytometry; D016193:G1 Phase; D015870:Gene Expression; D008297:Male; D008769:Methylnitronitrosoguanidine / Q000633:toxicity; D051379:Mice; D008810:Mice, Inbred C57BL; D008815:Mice, Inbred Strains; D018345:Mice, Knockout; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016196:S Phase; D013997:Time Factors; D014466:Ultraviolet Rays
D022763:CCAAT-Enhancer-Binding Protein-alpha; D022782:CCAAT-Enhancer-Binding Protein-beta; D008769:Methylnitronitrosoguanidine