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

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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 51
perhaps not surprisingly, overexpression of Msn2 could suppress the growth phenotype observed for Rpb4, but only at 34 o C. It would be interesting to test whether in rpb4∆ cells, Hsf1 binds the HSP104 promoter and whether the modifications of histones H3 and H4 still take place. The study presented in this thesis allows establishment of a model describing the molecular events leading to the transcriptional activation of the HSP104 promoter: under optimal growth conditions, the promoter is occupied by Hsf1 and acetylated histones. Under these conditions, the promoter is only partially active via the 34bp (most probably via the HSE at -304 to -300), but also partially via downstream STREs (because lower basal levels are measured in msn2∆msn4∆ cells or in constructs lacking internal STREs, the ∆78 constructs). Upon lowering cAMP levels, permanently, such as in ras∆2 cells or under stress conditions, cells remove the negative regulation imposed by the Ras/cAMP/PKA pathway on Msn2/4. This enables the nuclear localization of the STRE-binding transcriptional activators which subsequently leads to the transcriptional activation of STRE-containing genes. In parallel, upon heat shock (which also activates Msn2/4), HSE containing promoters are transcriptionally activated through the binding of Hsf1. In the case of HSP104, the binding of both transcriptional activators leads to proper transcriptional activation of this gene via the recruitment of chromatin modifying complexes (in an Msn2/4 dependent manner) which promote histone H4 deacetylation and disassembly of acetylated H3 histones from nucleosomes. In addition to these chromatin modifications, proper transcriptional induction could be enhanced through the recruitment of the SRB/MED complex [perhaps via Hsf1 (39)]. Finally, the RNA PolII is recruited, but it must be in its holoenzyme form (i.e., containing all subunits including Rpb4) Thus, we now have a comprehensive working model that describes the major molecular steps leading to the transcriptional activation of the HSP104 gene. REFERENCES 1. Aalfs, J. D., and R. E. Kingston. 2000. What does 'chromatin remodeling' mean? Trends Biochem Sci 25:548-55. 2. Agalioti, T., S. Lomvardas, B. Parekh, J. Yie, T. Maniatis, and D. Thanos. 2000. Ordered recruitment of chromatin modifying and general transcription factors to the IFN-beta promoter. Cell 103:667-78. 52
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Revealing the Mechanism of HSP104 T
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This thesis is dedicated to my fami
Page 5 and 6: genetic approach, we identified com
Page 7 and 8: INTRODUCTION The cellular stress re
Page 9 and 10: for their transcription, some 16% o
Page 11 and 12: Transcription under stress in S.cer
Page 13 and 14: In mammalian cells, Hsf1 is a monom
Page 15 and 16: Hsf1 and Msn2/4 can exclusively or
Page 17 and 18: A) 5’-XhoI -713 BamHI-3’ ATG La
Page 19 and 20: 1 We first noticed that the activit
Page 21 and 22: 1 1 A) B) 700 600 500 ras2∆msn2
Page 23 and 24: Table 1. Summary of the role of var
Page 25 and 26: same enzymes. BS-HA-HSF was obtaine
Page 27 and 28: β-Galactosidase assay Overnight cu
Page 29 and 30: to the fact that the HSEs are prese
Page 31 and 32: The systematic 5’ deletion analys
Page 33 and 34: 1 A) units 100.00 90.00 80.00 70.00
Page 35 and 36: nucleosome disassembly involving sp
Page 37 and 38: A) Strain W.T. msn2∆msn4∆ ras2
Page 39 and 40: Hsf1 constitutively binds the HSP10
Page 41 and 42: Next, we attempted to directly meas
Page 43 and 44: Table 3. List of mutants in which r
Page 45 and 46: Table 4. Strains required for posit
Page 47 and 48: Overall, the results nevertheless s
Page 49 and 50: Regulation of Hsf1 As explained in
Page 51 and 52: Table 7. Comparison between HSE-Lac
Page 53 and 54: promoter. III) The identification o
Page 55: to stress. This change in nucleosom
Page 59 and 60: 21. Chandy, M., J. L. Gutierrez, P.
Page 61 and 62: 56. Hietakangas, V., J. K. Ahlskog,
Page 63 and 64: 89. Miyao, T., J. D. Barnett, and N
Page 65 and 66: 121. Stanhill, A., N. Schick, and D
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