Revealing the Mechanism of HSP104 Transcription Initiation in the ...

is responsible for deacetylating histone H4 it will be necessary to construct many
strains with combined deletions of various HDACs. Many transcriptional activators,
such as Hsf1 (in mammals), Gcn4 and Swi5 have been shown to recruit chromatin
remodeling complexes to promoters in vitro and in vivo (29, 124). Does Hsf1 in yeast
play such a role on heat shock induced promoters? As Hsf1 constitutively binds
HSP104 regardless of stress conditions, perhaps some post-translational modification
of the protein could target chromatin modifying complexes to the HSP104 promoter.
An alternative explanation is that Msn2/4 recruit the nucleosome remodeling
enzymes. The fact that the lower levels of acetylated H3 or H4 are found in ras2∆, in
which Msn2/4 are constitutively active and probably constitutively bound, supports
the notion that Msn2/4 are responsible for these changes.
Studies show that in Drosophila, Hsf1 is responsible for recruiting the
mediator complex to heat shock promoters (95). Hsf1 in S.cerevisiae was also shown
to have a role in recruiting the SRB/MED co-activator complex to Hsf1 dependent
promoters (39). Hsf1 may recruit the SRB/MED complex to the HSP104 since we
find that SRB/MED is involved in transcription initiation of HSP104. The
confirmation of such a hypothesis could be achieved by performing a successive
immunoprecipitation followed by PCR (i.e., double ChIP assays, one
immunoprecipitation against Hsf1 followed by another one against a component of
the SRB/MED complex) as components of the mediator complex have been shown to
be associated with the HSP104 promoter in response to heat shock and to other Hsf1
target genes (39). The Srb10 component of the SRB/MED complex seems to be
involved in transcription of HSP104 (Table 4). We observed that at the level of the
reporter gene activity and, more importantly, at the level of RNA production.
However, our reporter studies demonstrated that the activity of a STRE-LacZ reporter
in srb10∆ was significantly up-regulated even under non-heat shock conditions. This
result is in agreement with studies showing that Msn2 is degraded in a Srb10-
dependent manner (39). Regarding HSP104 transcription, this suggests that other
mechanisms seem to override the loss of Srb10 and that probably Srb10 in the context
of the SRB/MED complex has another function.
Through the genetic screen, we also found that Rpb4, a RNA PolII subunit, is
required for HSP104 as well as for HSP26 and SSA3 transcription [Figs. 14 and 15
and (24)]. Indeed, DNA micro array studies show that 98% of genes are
downregulated in response to heat shock in RPB4 null cells (89). Interestingly and
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