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(Received for publication, May 29, 1997, and in revised form, August 8, 1997)
From the Department of Molecular Genetics, The University of Texas
M. D. Anderson Cancer Center, Houston, Texas 77030
CBF is a heterotrimeric protein that binds to DNA
containing CCAAT motifs. Here we have analyzed interactions of
recombinant CBF with DNA using hydroxyl radical footprinting and
methylation interference assays. In the CBF-DNA complex, three separate
DNA regions are protected from hydroxyl radical cleavage, one located over and immediately adjacent to the CCAAT motif itself and the other
two located on both sides of the CCAAT motif. The methylation interference assay showed, however, that only in the CCAAT motif region
methylation of bases was able to interfere with the formation of a
CBF-DNA complex, suggesting that CBF makes sequence-specific contacts
only in the CCAAT motif region. To further determine the specific DNA
sequences necessary for CBF binding, we employed a polymerase chain
reaction-mediated random binding site selection method. This analysis
showed that CBF binding to DNA requires the CCAAT sequence and other
specific sequences immediately flanking both ends of the CCAAT motif.
We also showed that the nature of the flanking nucleotide sequences
affects the affinity of CBF for DNA. Interestingly, most of the CCAAT
motifs present in various higher eukaryotic promoters correspond to the
CBF binding sites that were selected, consistent with the hypothesis
that these motifs are binding sites for CBF and, hence, that CBF could
regulate transcription of numerous eukaryotic genes.
Volume 272, Number 42,
Issue of October 17, 1997
pp. 26562-26572
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
DNA Binding Specificity of the CCAAT-binding Factor CBF/NF-Y
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