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

Qianghua Hu,Sankar N Maity

doi:10.1074/jbc.275.6.4435

Abstract

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.

Highlights

Eukaryotic RNA polymerase II promoters consist of multiple DNA elements that together control the transcriptional activity of the promoters
Those studies indicated that the mutant CBF-B acted as a competitive inhibitor of interaction between wild-type CBF-B and CBF-A/CBF-C and formed an inactive complex with wild-type CBF-A/CBF-C [35, 36]
Identification of Genes whose Expression Is Altered as a Consequence of Inhibition of CBF Binding—Previously using an in vitro reconstituted transcription system we demonstrated that recombinant CBF activates transcription of the ␣2(1) collagen promoter which contained a CBF-binding site, and that mutation in the CBF-binding site of the promoter abolished the CBF-dependent transcription activation [16], indicating that the CBF binding to the promoter is required for the in vitro transcription activation of this promoter

Summary

Introduction

Eukaryotic RNA polymerase II promoters consist of multiple DNA elements that together control the transcriptional activity of the promoters. The conserved segment of CBF-B that is needed for formation of the CBF-DNA complex shows no homology with the histone-fold motif or with any of the known dimerization motifs found in other heteromeric DNA-binding proteins [15]. This analysis classified CBF as a unique DNA-binding protein. The specific DNA sequences that are required for CBF binding have been defined by using a PCR-mediated random binding selection method This showed that specific sequences flanking to the CCAAT are required for high-affinity CBF binding and that the CCAAT motifs of various mammalian promoters are very similar to the high-affinity CBF-binding site [17]. The CBF-binding sites in promoters activator; CMV, cytomegalovirus; TRE, tetracycline responsive; BrdUrd, 5Ј-bromo-2Ј-deoxyuridine; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; DDRT, differential display reverse transcription

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Results

Conclusion