Nuclear Proteins Nut1p and Nut2p Cooperate To Negatively Regulate a Swi4p-DependentlacZReporter Gene inSaccharomyces cerevisiae

The URS2 region of theSaccharomyces cerevisiae HO upstream region contains 10 binding sites for the Swi4p/Swi6p transcription factor and confers Swi4p dependence for transcription. Using a hybrid promoter, UASGAL (upstream activation sequence ofGAL1 )-URS2R, in which theGAL1-10 regulatory region is fused to the proximal 360 bp of URS2, we isolated mutants in which Swi4p is no longer required for transcription. Mutations ofSIN4 ,ROX3 ,SRB8 ,SRB9 ,SRB10 ,SRB11 , and two novel genes,NUT1 andNUT2 , relieve the requirement of Swi4p for expression of this reporter. We found thatNUT1 (open reading frame [ORF] YGL151w) is a nonessential gene, thatNUT2 (ORF YPR168w) is essential, and that both Nut1p and Nut2p encode nuclear proteins. Deletion ofNUT1 causes a constitutive, Swi4p-independent phenotype only in combination with thenut2-1 allele or an allele ofCCR4 . In contrast, inactivation of a temperature-sensitive allele ofNUT2 ,nut2-ts70 , alone causes constitutivity.nut1Δ nut2-1 cells andsin4Δ cells exhibit Swi4p-independent expression of anho-lacZ reporter but not of an intactho gene. Likewise, apPHO5-lacZ construct is constitutively expressed innut1 nut2 mutants relative to their wild-type counterparts. These results suggest that Nut1p, Nut2p, Sin4p, and Ccr4p define a group of proteins that negatively regulate transcription in a subtle manner which is revealed by artificial reporter genes.