Bi W, Wu L, Coustry F, de Crombrugghe B, Maity S N
Department of Molecular Genetics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
J Biol Chem. 1997 Oct 17;272(42):26562-72. doi: 10.1074/jbc.272.42.26562.
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.
CBF是一种异源三聚体蛋白，可与含有CCAAT基序的DNA结合。在此，我们使用羟基自由基足迹法和甲基化干扰试验分析了重组CBF与DNA的相互作用。在CBF-DNA复合物中，有三个独立的DNA区域受到保护，免受羟基自由基切割，一个位于CCAAT基序本身之上并与其紧邻，另外两个位于CCAAT基序的两侧。然而，甲基化干扰试验表明，只有在CCAAT基序区域碱基的甲基化才能干扰CBF-DNA复合物的形成，这表明CBF仅在CCAAT基序区域进行序列特异性接触。为了进一步确定CBF结合所需的特定DNA序列，我们采用了聚合酶链反应介导的随机结合位点选择方法。该分析表明，CBF与DNA的结合需要CCAAT序列以及紧邻CCAAT基序两端的其他特定序列。我们还表明，侧翼核苷酸序列的性质会影响CBF对DNA的亲和力。有趣的是，各种高等真核生物启动子中存在的大多数CCAAT基序与所选的CBF结合位点相对应，这与这些基序是CBF的结合位点这一假设一致，因此，CBF可能调节众多真核基因的转录。
