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

Hsf1 constitutively binds the HSP104 promoter
Our previous promoter analysis and genetic studies revealed that HSP104
transcriptional induction is mediated by some cooperation between Hsf1 and Msn2/4
[(47); see also Figs. 2 and 3]. The dynamics of promoter occupancy by each of these
activators and the mutual relationship between them are not known. To reveal the
dynamics of promoter occupancy by Hsf1, we employed ChIP assays in wild type and
various mutant strains grown under optimal growth conditions and in response to heat
shock. We found that Hsf1 binding is constitutive under all conditions tested in wild
type cells and in the msn2∆msn4∆, ras2∆ and ras2∆msn2∆msn4∆ strains (Fig. 13A).
Notably, basal binding of Hsf1 to the HSP104 promoter in ras2∆ cells is reduced
compared to its binding in other strains (Figs. 13A and B). In order to rule out the
possibility that lower binding of Hsf1 in ras2∆ cells reflects lower steady state levels
of Hsf1 in these cells, we performed western blot analysis and observed that steady
state levels of Hsf1 are in fact identical in all strains and are not affected by heat
shock (Fig. 13C). The lower Hsf1 promoter occupancy in ras2∆ cells may be
interpreted as if the Ras cascade positively regulates Hsf1's DNA binding ability. We
believe, however, that the weaker Hsf1 binding in ras2∆ cells is a result of
constitutive binding of Msn2/4 to the promoter in this strain [(47); and Fig. 4) that
partially disturbs Hsf1’s binding. Indeed, removal of Msn2/4 from ras2∆ cells
(ras2∆msn2∆msn4∆) results in resumption of efficient binding of Hsf1 to the
promoter (Fig. 13A and B). Also, as shown above in ras2∆ cells, Hsf1 is dispensable
for HSP104 promoter activity as in these cells promoter activity was constitutively
high even after all HSEs were deleted [(47); and Fig. 4].
34