Shimizu M, Li W, Covitz P A, Hara M, Shindo H, Mitchell A P
School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Nucleic Acids Res. 1998 May 15;26(10):2329-36. doi: 10.1093/nar/26.10.2329.
The zinc finger protein Rme1p is a negative regulator of the meiotic activator IME1 in Saccharomyces cerevisiae . Prior studies have shown that Rme1p binds in vitro to a site near nt -2030 in the IME1 upstream region, but a genomic mutation in that site has little effect on repression of IME1 . To identify Rme1p binding sites in vivo , we have examined the binding of Rme1p to genomic sites through in vivo footprinting. We show that Rme1p binds to two sites in the IME1 upstream region, near nt -1950 and -2030. Mutations in both binding sites abolish repression of chromosomal IME1 by Rme1p, whereas a mutation in either single site causes partial derepression. Therefore, both Rme1p binding sites are essential for repression of IME1 . Prior studies have shown that repression by Rme1p depends upon RGR1 and SIN4 , which specify RNA polymerase II mediator subunits that are required for normal nucleosome density. We find that RGR1 and SIN4 are not simply required for Rme1p to bind to DNA in vivo . These results suggest that Rme1p functions directly as a repressor of IME1 and that Rgr1p and Sin4p are required for DNA-bound Rme1p to exert repression.
锌指蛋白Rme1p是酿酒酵母中减数分裂激活因子IME1的负调控因子。先前的研究表明，Rme1p在体外与IME1上游区域中靠近核苷酸-2030的位点结合，但该位点的基因组突变对IME1的抑制作用影响很小。为了在体内鉴定Rme1p的结合位点，我们通过体内足迹法检测了Rme1p与基因组位点的结合情况。我们发现Rme1p与IME1上游区域中靠近核苷酸-1950和-2030的两个位点结合。两个结合位点的突变都会消除Rme1p对染色体IME1的抑制作用，而单个位点的突变会导致部分去抑制。因此，两个Rme1p结合位点对于IME1的抑制都是必不可少的。先前的研究表明，Rme1p的抑制作用依赖于RGR1和SIN4，它们指定了正常核小体密度所需的RNA聚合酶II中介亚基。我们发现RGR1和SIN4不仅仅是Rme1p在体内与DNA结合所必需的。这些结果表明，Rme1p直接作为IME1的抑制因子发挥作用，并且DNA结合的Rme1p发挥抑制作用需要Rgr1p和Sin4p。
