Ssn6-Tup1 regulates RNR3 by positioning nucleosomes and affecting the chromatin structure at the upstream repression sequence

The DNA damage inducible gene ribonucleotide reductase (RNR3) is regulated by a transcriptional repression mechanism by the recruitment of the Ssn6-Tu p1 corepressor complex to its promoter by the sequence-specific DNA-binding protein Crt1. Ssn6-Tup1 is reported to represses transcription by interfer ing with transcription factors, recruiting histone deacetylases, and positi oning nucleosomes at the promoter of its target genes. Two of the three mec hanisms involve effects on chromatin structure, and therefore, we have deli neated the nucleosomal structure of RNR3 in the repressed and derepressed s tate using multiple nuclease mapping strategies. A regular array of positio ned nucleosomes is detected over the repressed RNR3 promoter that extends i nto the coding sequence. Treating cells with DNA damaging agents or deletin g CRT1, SSN6, or TUP1 derepresses RNR3 transcription, and causes a dramatic disruption of nucleosome positioning over its promoter. Furthermore, derep ression of RNR3 correlated with changes in nuclease sensitivity within the upstream repression sequence (URS) region. Specifically, the loss of a MNas e-hypersensitive site, and the appearance of strong DNase I hypersensitivit y, was observed over the URS. Interestingly, we find that the binding of Cr t1 to the promoter in the absence of Ssn6 or Tup1 is insufficient for nucle osome positioning or regulating chromatin structure at the URS; thus, these two functions are strictly dependent upon Ssn6-Tup1. We propose that RNR3 is regulated by changes in nucleosome positioning and chromatin structure t hat are mediated by Ssn6, Tup1, and CAI.