Mechanisms of Nucleosome Positioning and Transcription Regulation of Yeast DNA Damage Inducible Genes
Open Access
- Author:
- Zhang, Zhengjian
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 04, 2004
- Committee Members:
- Joseph C. Reese, Committee Chair/Co-Chair
Jerry L Workman, Committee Member
David Scott Gilmour, Committee Member
Wesley Tsoekjen Ng, Committee Member
Liwang Cui, Committee Member - Keywords:
- DNA damage
imitation switch (ISWI)
transcription repression
Ssn6-Tup1
nucleosome positioning
chromatin - Abstract:
- The induction of repair genes upon DNA damage is conserved among all the living organisms examined to date. In the yeast Saccharomyces cerevesiae, these genes are repressed by Crt1, which recognizes the DNA damage response elements (DREs) and recruits the global co-repressor complex Ssn6-Tup1. Nucleosome positioning has been known to be important for gene repression for decades, but how it is achieved and maintained in vivo is still elusive. One factor, Ssn6-Tup1 has been identified and is believed to cause nucleosome positioning by binding to the tails of histones and spreading across the repressive chromatin domain. This is called the “extended scaffold” model. Previous work from our lab has shown that Ssn6-Tup1 is responsible for nucleosome positioning at the DNA damage inducible gene RNR3. In an attempt to determine the mechanism of nucleosome positioning at RNR3, we first thoroughly characterizated the chromatin structure of the RNR3 locus using nuclease mapping strategies. We found that the entire RNR3 coding sequence and the upstream regulatory sequence (URS) (2.9 kb in length) is embedded in 19 well positioned nucleosomes, and DNA damage or deletion of CRT1 or TUP1 cause identical disruption of the positioning. Surprisingly, we, and others, have shown that Ssn6-Tup1 crosslinking is restricted to the upstream regulatory sequence (URS), yet nucleosome positioning extends far into the coding region. Thus, the extended scaffold model cannot explain how Ssn6-Tup1 positions nucleosomes at RNR3. We investigated the role of known yeast chromatin remodeling factors in regulating the chromatin structure at RNR3 and found that the imitation SWItch (ISWI) family member ISW2 is required for the nucleosome positioning. In comparison with TUP1 deletion, ISW2 mutation causes equal nucleosome disruption from the TATA box to about +2 kb into the coding sequence, but the regions further upstream (where Crt1-Ssn6-Tup1 binds) or downstream (~+2.0 to +2.7 kb) of that resembles the wild type and are disrupted upon induction. We determined that Isw2p can be specifically crosslinked across the RNR3 gene using the ChIP assay. Interestingly, the crosslinking of Ssn6-Tup1 and Isw2 are independent of each other, suggesting collaboration of these two factors is necessary for the maintenance of the repressive chromatin structure. The Imitation SWItch (ISWI) chromatin remodeling factors have been implicated in nucleosome positioning in vivo. In vitro, they can mobilize nucleosomes bi-directionally, making it difficult to envision how they can establish precise translational positioning of nucleosomes in vivo. It has been proposed they require other cellular factors to do so, but none have been identified thus far. By showing the dependency on Tup1 for ISW2 mediated nucleosome positioning at a subset of promoters, we revealed a novel collaboration between two nucleosome positioning activities in vivo. In contrast to the dramatic increase in transcription observed in Dcrt1 or Dtup1 strains, only slight derepression was detected in an ISW2 deletion mutant, although the nucleosomes embedding the TATA box and the coding sequence were disrupted. Chromatin immunoprecipitation (ChIP) experiments demonstrated lack of TBP recruitment and preinitiation complex (PIC) formation. Thus, Tup1, which is localized to the URS independently of ISW2, can block PIC formation and repress transcription in the absence of nucleosome positioning. In addition to nucleosome positioning, HDAC recruitment and direct interference with activator or Mediator have been proposed for Ssn6-Tup1 repression. A clear analysis of the contribution of each repression pathway has not been described, however, mainly due to the challenge of separating the nucleosome positioning activity of Tup1 from the others. Our finding that in the absence of a functional ISW2 complex (and effective nucleosome positioning), Ssn6-Tup1 maintains the majority of the repression function over the DNA damage inducible genes, made it possible to specifically disrupt the nucleosome positioning ability of Ssn6-Tup1. We showed the remaining repression is mediated by histone deacetylase Hda1 and the Mediator complex subunits. Similar to ISW2 deletion, mutation of HDA1 or various Mediator subunit genes individually only slightly increased the transcription of the DNA damage inducible genes. Only in triple mutants simultaneously disrupted in ISW2, HDA1 and Mediator genes could significantly high levels of transcription be observed. We confirmed the association of Ssn6-Tup1 to the promoters in the various mutants. Thus multiple, redundant mechanisms are utilized by Ssn6-Tup1 in the regulation of the DNA damage inducible genes. Redundancy was also observed at other Ssn6-Tup1 target genes, with relative contributions of each mechanism varying. We propose that Ssn6-Tup1 has developed multiple mechanisms in order to function as a "global" co-repressor, and different groups of genes have developed different strategies to utilize Ssn6/Tup1 in repression. We further analyzed the function of the sequence specific DNA binding protein Crt1 in the regulation of the DNA damage inducible genes. Recruitment of Ssn6-Tup1 is mediated through a Crt1 N-terminal domain, which also interacts with the TFIID coactivator. The function of Crt1 C-terminus was unknown. We identified a Crt1 C-terminal repression domain which is, in contrast to the N-terminal domain, independent of Ssn6-Tup1 and histone deacetylases. We also further mapped the N-terminal repression domain and distinguished it from that required for TFIID interaction. Crt1 mutants were then constructed in an attempt to disrupt TFIID interaction but preserve Ssn6-Tup1 recruitment. All of the mutants repressed the DNA damage inducible genes, but most of them, called “derepression-defective” mutants, failed to release the repression upon DNA damage. Further characterization of the derepression defective mutants indicated that they are specifically blocked after corepressor release but before or during coactivator recruitment. These results imply a two-step activation model of the DNA damage inducible genes. The implications of this and the Crt1 conservation to its higher eukaryotic homologues, is discussed.