Action of a RAP1 carboxy-terminal silencing domain reveals an underlying competition between HMR and telomeres in yeast.
RAP1 is a sequence-specific DNA-binding protein in yeast that can either repress or activate transcription. Previous studies have demonstrated a direct role for RAP1 in silencing at HM mating-type loci and telomeres. Here, we show that a small carboxy-terminal domain of RAP1 is sufficient to establish repression when fused to the GAL4 DNA-binding domain (GBD) and targeted to mutated HMR silencers containing GAL4 DNA-binding sites. Silencing by GBD/RAP1 hybrids, like normal silencing at HMR, requires the trans-acting factors SIR2, SIR3, and SIR4. However, GBD/RAP1-mediated silencing is independent of SIR1, whose product is normally required for the establishment of repression at HMR. Targeted silencing also displays an unusual response to silencing-defective rap1s mutations. The incorporation of a rap1s missense mutation into GBD/RAP1 hybrids can improve targeted silencing, yet wild-type GBD/RAP1 hybrids fail to establish repression in strains in which the endogenous RAP1 locus carries a rap1s mutation. In addition, we find that telomeric silencing is increased in rap1s strains. We propose that the rap1s mutation creates an HMR-specific silencing defect by shifting a balance between silencing at HMR and telomeres in favor of telomeric silencing. This balance is regulated by telomere length and by interactions between the RAP1 carboxyl terminus and both RIF1 and SIR4 proteins. In support of this model, we show that abnormally long telomeres antagonize silencing at HMR and a rap1s hybrid protein displays a strengthened interaction with SIR4 in a two-hybrid assay.
Buck
S W
SW
Department of Microbiology, College of Physicians and Surgeons of Columbia University, New York, New York 10032.
Shore
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D001483:Base Sequence; D002874:Chromosome Mapping; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D016615:Telomere; D014157:Transcription Factors / Q000235:genetics*
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D014157:Transcription Factors