Stable expression of a dominant negative mutant of CCAAT binding factor/NF-Y in mouse fibroblast cells resulting in retardation of cell growth and inhibition of transcription of various cellular genes.
The heterotrimeric CCAAT-binding factor CBF specifically interacts with the CCAAT motif present in the proximal promoters of numerous mammalian genes. To understand the in vivo function of CBF, a dominant negative mutant of CBF-B subunit that inhibits DNA binding of wild type CBF was stably expressed in mouse fibroblast cells under control of tetracycline-responsive promoter. Expression of the mutant CBF-B but not the wild-type CBF-B resulted in retardation of fibroblast cell growth. The analysis of cell growth using bromodeoxyuridine labeling showed that expression of the mutant CBF-B decreased the number of cells entering into S phase, and also delayed induction of S phase in the quiescent cells after serum stimulation, thus indicating that the inhibition of CBF binding prolonged the progression of S phase in fibroblasts. These results provide direct evidence for the first time that CBF is an important regulator of fibroblast growth. The inhibition of CBF binding reduced expression of various cellular genes including the alpha2(1) collagen, E2F1, and topoisomerase IIalpha genes which promoters contain the CBF-binding site. This result implied that expression of many other genes which promoters contain CBF-binding site was also decreased by the inhibition of CBF binding, and that the decreased expression of multiple cellular genes possibly caused the retardation of fibroblast cell growth.
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Department of Molecular Genetics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
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D000818:Animals; D001973:Bromodeoxyuridine / Q000378:metabolism; D022762:CCAAT-Enhancer-Binding Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000235:genetics*; D003094:Collagen / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D005347:Fibroblasts; D005786:Gene Expression Regulation / Q000187:drug effects; D051379:Mice; D009154:Mutation; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D011485:Protein Binding / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D056465:Retinoblastoma-Binding Protein 1; D016196:S Phase / Q000235:genetics; D013752:Tetracycline / Q000494:pharmacology; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics*; D014162:Transfection; D051840:Y-Box-Binding Protein 1
C533431:Arid4a protein, mouse; D022762:CCAAT-Enhancer-Binding Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493754:E2f1 protein, mouse; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D012333:RNA, Messenger; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D003094:Collagen; D004250:DNA Topoisomerases, Type II; D013752:Tetracycline; D001973:Bromodeoxyuridine