Recruitment of the Tup1-Ssn6 general repression complex and its
effects on HEM13 .
Lee Klinkenberg, Richard Zitomer
Biological Sciences, University at Albany/SUNY, 1400 Washington Ave,
Albany, NY 12222, USA
The Tup1-Ssn6 general repression complex represses a number of regulons
in S. cerevisiae. It is recruited to target genes by regulon
specific DNA binding proteins, such as Rox1 for the hypoxic regulon. We
have explored a number of features of Rox1 recruitment of Tup1-Ssn6.
First, repression at some hypoxic genes, such as ANB1, is
partially dependent on another DNA binding protein, Mot3. To determine
whether Mot3 aids Rox1 binding through Tup1-Ssn6 recruitment, I fused
the DNA binding domain of ROX1 to the SSN6 coding region.
If Mot3 functions by recruiting Ssn6 to the DNA, then this construct
should eliminate Mot3 enhancement of repression. Second, it has been
proposed that Ssn6 interacts with the regulon specific repressors while
Tup1 provides the repression activity. This model suggests that Ssn6 can
be recruited to hypoxic genes in the absence of Tup1, but the converse
should not be correct. ChIP assays are being used to examine Ssn6
recruitment in vivo. I explored the effects of deletions of Rox1,
Mot3, and Tup1 on the recruitment of Ssn6 to native strongly repressed
ANB1 and weakly repressed HEM13. Third, like ANB1,
the control region of HEM13 is made up of four Rox1 binding
sites, but is only weakly repressed. The effects on repression of
targeted deletions of these sites was determined. Also, I explored the
effects of deletions of the N-terminal tail of histone H3 and histone H4
on expression of a HEM13 reporter gene and nucleosome positioning
at the HEM13 promoter.
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