| Abstract |
In Saccharomyces cerevisiae, Mac1 is a copper-regulated. Mac1 recognizes and binds
to specific DNA sequences and activates genes involved in copper uptake (CTR1, CTR3,
FRE1, FRE7). Previous evidence suggested that, in addition to copper (copper ions
negatively regulate Mac1), protein interactions could modulate Mac1 function (Voutsina
et al., 2001). In order to identify such proteins, a genomic expression library was
screened using the two yeast hybrid technology (Bilsland et al., 2004; Voutsina et al.,
2005). Among the potentially Mac1-interacting proteins, the nuclear phosphoprotein
Rad9 and the histone chaperone Hir1were identified.
Rad9 is a prototype DNA-damage checkpoint protein required for the DNA checkpoint
pathway when DNA damage occurs (double strand break) by X-ray irradiation through
out the cell cycle. In this study, we present evidence indicating a new direct role of Rad9
in RNA polymerase II-dependent transcription that is independent of cell cycle
checkpoint. We found Rad9 to associate directly with the copper-regulated transcriptional
activator Mac1 and exert a negative effect on both its DNA binding and transactivation
functions. We also found Rad9 to be recruited on Mac1-regulated genes in a Mac1-
dependent manner. It was not only associated with CTR1 promoter but also along the
CTR1 protein coding region. This Rad9 localization, coincided with transcriptional
induction, correlated with the characteristic quantitative association pattern of RNA
polymerase II and we also found that components of the Mac1-dependent transcriptional
initiation complex were exclusively responsible for Rad9 localization in the coding
region of CTR1 since we were able to transfer Rad9 in the coding region of ACT1 (were
Rad9 is not normally localized) artificially fused downstream to the CTR1 promoter in
the genome. We also found that Rad9 localization in the coding region was partly
dependent on the Hir1 histone chaperone which is involved in transcription initiation and
elongation of CTR1 (see below). On the contrary, histone H3 dimethylation of K79
known to be recognized by Rad9 under DNA damage conditions (Huyen et al., 2004) was
irrelevant to Rad9 localization in the CTR1 coding region. We also identified new genetic
and physical associations of Rad9 with elongating factors (e.g. Hir1), Mac1 interacting
proteins and Rad9 dependent localization of DNA damage effectors on CTR1 gene (e.g. Rad53 kinase). Our data, in combination with the fact that CTR1 (and FRE1
«neighborhood») belongs to a group of genes that are characterized as hotspots for
meiotic recombination (which is initiated by the formation of DSB) point to a new role
for Rad9 mediating a crosstalk between transcription and DNA repair under
physiological unchallenged conditions. When meiotic recombination begins, components
of the transcriptional machinery (mainly chromatin remodelers) are already present ready
to be used by the DNA repair machinery under the surveillance of Rad9.
In parallel, we further investigated the role of Hir1 which as we mentioned before
interacts with Rad9, on Mac1 regulated transcription Our genetic and biochemical
evidence suggests that Hir1 facilitates transcriptional activation of induced CTR1
expression by affecting Snf2 recruitment since we demonstrated that Snf2 and Hir1
partially depend on each other for their localization on CTR1 promoter and both associate
with the protein coding region of CTR1. We also showed that Hir1 association follows
that of the elongating RNA polymerase II but Snf2 does not. Moreover, we found that
Hir1 acts in combination with the Spt16 (component of the yFACT complex) on the
induced CTR1 transcription. Thus we demonstrated a novel activating role for Hir1
protein on a new category of RNA polymerase II genes, those responding to copper
depletion. Its role was revealed by distinct interacting partners in the Mac1-dependent
CTR1 transcriptional initiation and elongation.
In light of the above data we performed protein complex indentification experiments by
immunoprecipitation and mass spectrometry analysis (in cooperation with Dr.Rodriguez–
Navarro / Institute of Principe Philippe, Valencia, Spain) and we expect them to reveal
novel protein associations between regulators of transcription, metalloregulated
transcription and DNA damage surveying and response pathways. We believe that this
approach will help us in analysing and understanding the cross-talking mechanisms
governing environmentally induced signalling pathways.
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