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Molecular and Cellular Biology, August 2005, p. 6772-6788, Vol. 25, No. 15
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.15.6772-6788.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Components of the ESCRT Pathway, DFG16, and YGR122w Are Required for Rim101 To Act as a Corepressor with Nrg1 at the Negative Regulatory Element of the DIT1 Gene of Saccharomyces cerevisiae

Karen Rothfels,1 Jason C. Tanny,2,{dagger} Enikö Molnar,2 Helena Friesen,2 Cosimo Commisso,2 and Jacqueline Segall1,2*

Department of Biochemistry,1 Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada2

Received 21 December 2004/ Returned for modification 22 February 2005/ Accepted 4 May 2005

The divergently transcribed DIT1 and DIT2 genes of Saccharomyces cerevisiae, which belong to the mid-late class of sporulation-specific genes, are subject to Ssn6-Tup1-mediated repression in mitotic cells. The Ssn6-Tup1 complex, which is required for repression of diverse sets of coordinately regulated genes, is known to be recruited to target genes by promoter-specific DNA-binding proteins. In this study, we show that a 42-bp negative regulatory element (NRE) present in the DIT1-DIT2 intergenic region consists of two distinct subsites and that a multimer of each subsite supports efficient Ssn6-Tup1-dependent repression of a CYC1-lacZ reporter gene. By genetic screening procedures, we identified DFG16, YGR122w, VPS36, and the DNA-binding proteins Rim101 and Nrg1 as potential mediators of NRE-directed repression. We show that Nrg1 and Rim101 bind simultaneously to adjacent target sites within the NRE in vitro and act as corepressors in vivo. We have found that the ability of Rim101 to be proteolytically processed to its active form and mediate NRE-directed repression not only depends on the previously characterized RIM signaling pathway but also requires Dfg16, Ygr122w, and components of the ESCRT trafficking pathway. Interestingly, Rim101 was processed in bro1 and doa4 strains but was unable to mediate efficient repression.

* Corresponding author. Mailing address: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada. Phone: (416) 978-4981. Fax: (416) 978-8548. E-mail: j.segall{at}utoronto.ca.

{dagger} Present address: Laboratory of Chromatin Biology, Rockefeller University, New York, NY 10021.

Molecular and Cellular Biology, August 2005, p. 6772-6788, Vol. 25, No. 15
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.15.6772-6788.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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