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Molecular and Cellular Biology, October 2000, p. 7088-7098, Vol. 20, No. 19
Department of Biological Sciences, University
at Albany/SUNY, Albany, New York 12222
Received 10 May 2000/Returned for modification 8 June 2000/Accepted 3 July 2000
The hypoxic genes of Saccharomyces cerevisiae are
repressed by a complex consisting of the aerobically expressed,
sequence-specific DNA-binding protein Rox1 and the Tup1-Ssn6 general
repressors. The regulatory region of one well-studied hypoxic gene,
ANB1, is comprised of two operators, OpA and OpB, each of
which has two strong Rox1 binding sites, yet OpA represses
transcription almost 10 times more effectively than OpB. We show here
that this difference is due to the presence of a Mot3 binding site in
OpA. Mutations in this site reduced OpA repression to OpB levels, and the addition of a Mot3 binding site to OpB enhanced repression. Deletion of the mot3 gene also resulted in reduced
repression of ANB1. Repression of two other hypoxic genes
in which Mot3 sites were associated with Rox1 sites was reduced in the
deletion strain, but other hypoxic genes were unaffected. In addition,
the mot3
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Roles of Transcription Factor Mot3 and Chromatin in Repression
of the Hypoxic Gene ANB1 in Yeast
and
mutation caused a partial derepression of the
Mig1-Tup1-Ssn6-repressed SUC2 gene, but not the
2-Mcm1-Tup1-Ssn6-repressed STE2 gene. The Mot3 protein
was demonstrated to bind to the ANB1 OpA in vitro. Competition experiments indicated that there was no interaction between
Rox1 and Mot3, indicating that Mot3 functions either in Tup1-Ssn6
recruitment or directly in repression. A great deal of evidence
has accumulated suggesting that the Tup1-Ssn6 complex represses
transcription through both nucleosome positioning and a direct
interaction with the basal transcriptional machinery. We
demonstrate here that under repressed conditions a nucleosome is
positioned over the TATA box in the wild-type ANB1
promoter. This nucleosome was absent in cells carrying a
rox1, tup1, or mot3 deletion, all
of which cause some degree of derepression. Interestingly, however,
this positioned nucleosome was also lost in a cell carrying a deletion
of the N-terminal coding region of histone H4, yet ANB1
expression remained fully repressed. A similar deletion in the gene for
histone H3, which had no effect on repression, had only a minor effect
on the positioned nucleosome. These results indicate that the
nucleosome phasing on the ANB1 promoter caused by the
Rox1-Mot3-Tup1-Ssn6 complex is either completely redundant with a
chromatin-independent repression mechanism or, less likely, plays no
role in repression at all.
*
Corresponding author. Mailing address: Department of
Biological Sciences, University at Albany/SUNY, Albany, NY 12222. Phone: (518) 442-4385. Fax: (518) 442-4767. E-mail:
rz144{at}csc.albany.edu.
Permanent address: Department de Biologia Celular y Molecular,
Univ. de la Coruna, Campus de La Zapateira sln, 15071 La Coruna, Spain.
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