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Molecular and Cellular Biology, March 2001, p. 2057-2069, Vol. 21, No. 6
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.6.2057-2069.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Identification of the Sin3-Binding Site in Ume6 Defines
a Two-Step Process for Conversion of Ume6 from a Transcriptional
Repressor to an Activator in Yeast
Brian K.
Washburn and
Rochelle Easton
Esposito*
Department of Molecular Genetics and Cell
Biology, University of Chicago, Chicago, Illinois 60637
Received 21 September 2000/Returned for modification 1 November
2000/Accepted 26 December 2000
The DNA-binding protein Ume6 is required for both
repression and activation of meiosis-specific genes, through
interaction with the Sin3 corepressor and Rpd3 histone deacetylase and
the meiotic activator Ime1. Here we show that fusion of a heterologous activation domain to Ume6 is unable to convert it into a constitutive activator of early meiotic gene transcription, indicating that an
additional function is needed to overcome repression at these promoters. Mutations in UME6 allowing the fusion to
activate lie in a predicted amphipathic alpha helix and
specifically disrupt interaction with Sin3 but not with Teal, an
activator of Ty transcription also found to interact with Ume6 in a
two-hybrid screen. The mutations cause a loss of repression by Ume6 and
precisely identify the Ume6 Sin3-binding domain, which we show
interacts with the paired amphipathic helix 2 region of Sin3. Analysis
of these mutants indicates that conversion of Ume6 to an activator
involves two genetically distinct steps that act to relieve
Sin3-mediated repression and provide an activation domain to Ume6. The
mutants further demonstrate that premature expression and lack of
subsequent rerepression of Ume6-Sin3-regulated genes are not
deleterious to meiotic progression and suggest that the essential role
of Sin3 in meiosis is independent of Ume6. The model for Ume6 function
arising from these studies indicates that Ume6 is similar in many
respects to metazoan regulators that utilize Sin3, such as the
Myc-Mad-Max system and nuclear hormone receptors, and provides new
insights into the control of transcriptional repression and activation
by the Ume6-URS1 regulatory complex in yeast.
*
Corresponding author. Mailing address: Department of
Molecular Genetics and Cell Biology, University of Chicago, 920 E. 58th St., Chicago, IL 60637. Phone: (773) 702-8046. Fax: (773)
702-8093. E-mail: re-esposito{at}uchicago.edu.
Molecular and Cellular Biology, March 2001, p. 2057-2069, Vol. 21, No. 6
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.6.2057-2069.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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