Genomic footprinting of the yeast zinc finger protein Rme1p and its roles in repression of the meiotic activator IME1.
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.
Shimizu
M
M
School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. shimizum@ps.tokyo.ac.jp
Li
W
W
Covitz
P A
PA
Hara
M
M
Shindo
H
H
Mitchell
A P
AP
D001665:Binding Sites; D018983:DNA Footprinting; D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D056892:Mediator Complex; D008540:Meiosis / Q000235:genetics*; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics*; D012331:RNA, Fungal / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins / Q000235:genetics / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D016335:Zinc Fingers
D004271:DNA, Fungal; D005656:Fungal Proteins; C080326:IME1 protein, S cerevisiae; D056892:Mediator Complex; D009687:Nuclear Proteins; C095061:RGR1 protein, S cerevisiae; C071594:RME1 protein, S cerevisiae; D012331:RNA, Fungal; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C076878:SIN4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors