Lohr D, Torchia T, Hopper J
Chemistry Department, Arizona State University, Tempe 85287.
J Biol Chem. 1987 Nov 15;262(32):15589-97.
Chromatin in the regions between the upstream activator sequence and the 5' ends of the yeast GAL1 and GAL10 genes has been analyzed by DNase I chromosomal footprinting and micrococcal nuclease digestion using the indirect end-labeling approach. Comparison of wild type chromatin digests to naked DNA digests shows that there are specific regions of these upstream sequences which are strongly protected in chromatin. Comparison to chromatin digests from cells disrupted for the positive regulatory gene, GAL4, or the negative regulatory gene, GAL80, and thus lacking GAL4 or GAL80 function, shows that these regions of protection in wild type chromatin are GAL80-dependent but not GAL4-dependent. The protected regions include DNA lying on (GAL10) or near (GAL1) the respective TATA boxes. These protections are present in both noninduced and induced cells. Both DNA strands are equally protected. Upstream of GAL1 there is a second protected region. This protection shows considerable expression and strand dependence. These observations provide the first evidence that the GAL80 function influences chromatin structure and suggest possible mechanisms by which GAL80 modulates the GAL1 and 10 promoters in induced cells. Micrococcal nuclease digests also suggest a role for GAL80 in a distinctive higher order organization of the intergenic region, perhaps involving multiprotein complexes.
利用间接末端标记法,通过DNase I染色体足迹分析和微球菌核酸酶消化,对酵母GAL1和GAL10基因上游激活序列与5'端之间区域的染色质进行了分析。将野生型染色质消化产物与裸露DNA消化产物进行比较,结果表明这些上游序列存在特定区域,在染色质中受到强烈保护。与因阳性调节基因GAL4或阴性调节基因GAL80缺失而缺乏GAL4或GAL80功能的细胞的染色质消化产物进行比较,结果表明野生型染色质中的这些保护区域依赖于GAL80而非GAL4。受保护区域包括位于各自TATA框上(GAL10)或附近(GAL1)的DNA。这些保护在未诱导和诱导细胞中均存在。两条DNA链受到同等保护。在GAL1上游有第二个受保护区域。这种保护表现出显著的表达和链依赖性。这些观察结果首次证明GAL80功能影响染色质结构,并提示了GAL80在诱导细胞中调节GAL1和GAL10启动子的可能机制。微球菌核酸酶消化还表明GAL80在基因间区域独特的高级组织中发挥作用,可能涉及多蛋白复合物。