Studies on transcription activation by the multimeric CCAAT-binding factor CBF.
The CCAAT-binding factor CBF is a heteromeric transcription factor that specifically binds to CCAAT sequences in many eukaryotic genes. CBF consists of three subunits, CBF-A, CBF-B, and CBF-C, all three of which are necessary for DNA binding. In this study we examined the transcription activation function of CBF by two different approaches. We first used a heterologous system in which a series of deletion mutations of CBF-B, fused to the bacterial LexA DNA binding domain, were transfected into HeLa cells together with a reporter gene driven by a minimal promoter containing LexA binding sites. These experiments showed that CBF-B needed both a glutamine-rich domain and an adjacent serine/threonine-rich domain to activate the reporter gene optimally. The glutamine-rich domain by itself activated transcription only modestly. We also set up an in vitro transcription reconstituted system in which trans-activation by CBF occurred through a physiological CCAAT motif. Nuclear extracts from NIH 3T3 cells were first depleted of CBF and then complemented with recombinant CBF-B and a highly purified fraction containing native CBF-A and CBF-C. Recombinant full-length CBF-B together with CBF-A and CBF-C activated transcription of several alpha 2(I) collagen gene promoter constructs. We then tested whether in this system the glutamine- and serine/threonine-rich domains of CBF-B were needed for trans-activation by CBF. We generated a truncated form of CBF-B that was still able to bind DNA in the presence of CBF-A and CBF-C. Even in the absence of the glutamine- and serine/threonine-rich domains of CBF-B, reconstituted CBF did activate transcription, suggesting that CBF transcriptional activation can also be mediated by the other subunits of CBF or by another transcription factor present in the nuclear extracts that interacts with CBF. Taken together our results suggest a model in which CBF has the potential to activate transcription either through the glutamine- and serine/threonine-rich domains of CBF-B or through the other subunits of CBF or through another component recruited by CBF.
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Department of Molecular Genetics, University of Texas, M. D. Anderson Cancer Center, Houston 77030.
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de Crombrugghe
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D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins; D015533:Transcriptional Activation
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins