Nuclear Proteins Nut1p and Nut2p Cooperate To Negatively Regulate a Swi4p-Dependent lacZ Reporter Gene in Saccharomyces cerevisiae

ABSTRACT

The URS2 region of the Saccharomyces cerevisiae HOupstream region contains 10 binding sites for the Swi4p/Swi6p transcription factor and confers Swi4p dependence for transcription. Using a hybrid promoter, UAS _GAL (upstream activation sequence of GAL1)-URS2R, in which the GAL1-10 regulatory region is fused to the proximal 360 bp of URS2, we isolated mutants in which Swi4p is no longer required for transcription. Mutations of SIN4, ROX3,SRB8, SRB9, SRB10,SRB11, and two novel genes, NUT1 and NUT2, relieve the requirement of Swi4p for expression of this reporter. We found that NUT1 (open reading frame [ORF] YGL151w) is a nonessential gene, that NUT2 (ORF YPR168w) is essential, and that both Nut1p and Nut2p encode nuclear proteins. Deletion of NUT1 causes a constitutive, Swi4p-independent phenotype only in combination with the nut2-1 allele or an allele of CCR4. In contrast, inactivation of a temperature-sensitive allele ofNUT2, nut2-ts70, alone causes constitutivity.nut1Δ nut2-1 cells and sin4Δ cells exhibit Swi4p-independent expression of an ho-lacZ reporter but not of an intact ho gene. Likewise, a pPHO5-lacZconstruct is constitutively expressed in nut1 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.

ACKNOWLEDGMENTS

We are particularly grateful to Meghan Sharp for isolating the Nut⁻ mutants and to Sally Horne for characterization of the NUT2-like genes, during their rotations in the laboratory, and to Sandy Johnson and Megan Grether for improvements on the manuscript. We thank Marian Carlson, Wolfram Hörz, David Stillman, Madhu Wahi, Richard Young, and their colleagues for generously providing plasmids, and Megan Grether, Carol Gross, Wolfram Hörz, Sandy Johnson, Hay-Oak Park, Sylvia Sanders, Shai Shaham, and Anita Sil for numerous discussions during the course of this work.

This work was supported by NIH research grant AI18738 to I.H., a Howard Hughes predoctoral fellowship to R.K.T., and grants from the Markey Foundation and the Herbert W. Boyer Fund.

ADDENDUM IN PROOF

We have recently learned that Nut2p may be a component of the mediator complex (Claes Gustafsson, personal communication; Young-Joon Kim, personal communication). The mediator complex is required for yeast transcriptional activa- tion in vitro and also contains Sin4p, Rox3p, and Rgr1p (see references 18 and 28).