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

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Summary All living organisms are continuously exposed to sub-optimal growth conditions and have therefore developed appropriate responses in order to survive. The response to many of these stressful conditions is mostly regulated at the level of transcription, leading to up-regulation of the expression of many stress-related genes. Many genes are upregulated in response to any given stress (the ‘general stress response’) and some of these genes are induced most strongly in response to a specific stress (the ‘specific stress response’). Important aspects of the co-regulation of many genes by the general response and of specific regulation by the specific stress responses have been revealed. Yet, the relationship between these two responses is not understood. In this thesis we study the transcriptional regulation of HSP104 of the yeast S.cerevisiae, a gene highly expressed in response to heat shock. An early analysis of the HSP104 promoter region, through its analysis via 5’deletion, mutagenesis and heterologous studies, showed that two different families of transcriptional activators regulate the expression of this gene. i) Hsf1, which upon activation by heat shock binds Heat Shock Elements (HSEs). ii) Msn2/4, which upon activation by a broad range of stresses, bind STress Response Elements (STREs) and are negatively regulated by the Ras/cAMP/PKA pathway. This analysis also showed that the promoter displays a highly flexible mode of activation. It is maximally activated in the presence of both sets of transcriptional activators but can also be activated by either one alone. We were also able to map the elements in the promoter responsible for basal activity (the sequence between -334 to -300) and those essential for heat shock-regulated activity (47). These results were obtained primarily during the course of my M.Sc. studies. In this thesis, I describe the continuation of the effort to reveal the mechanism of transcriptional activation of the HSP104 promoter. I describe four experimental routes. One, continuing the approach used in the studies described above, we proceeded with additional 5’ deletions of the HSP104 promoter (particularly the fragment between -334 and -300) attempting to obtain a finely tuned map of the sequence(s) responsible for the basal activity of HSP104 and for other unexpected properties of the sequence between - 334 to -300. Two, using chromatin immunoprecipitation (ChIP), we monitored some of the major changes occurring in vivo on the promoter following stress. Three, using a i
genetic approach, we identified components of the basal transcription machinery that are important for HSP104 promoter activity. Four, using a combination of ChIP experiments and a genetic approach, we sought possible regulators of Hsf1. Through the deletion analysis, we found that important part of the properties of the 34bp between -334 and -300 could be accounted for by a short HSE-like sequence residing in -305. Using ChIP assays we show that under optimal growth conditions nucleosomes on the HSP104 promoter contain mostly acetylated H3 and H4. However, following heat shock there is a rapid, but transient, decrease in the concentration of acetylated histones on the promoter which seems to be partly mediated by Msn2/4. Namely, the Ras/PKA pathway controls H3 and H4 acetylation state via Msn2/4, thereby governing induction of the promoter. We further show that the decrease in acetylated H3 and H4 on the promoter occurs via two distinct mechanisms. Finally, we show that Hsf1 binding to the promoter is constitutive regardless of stress conditions, but is reduced in ras2Δ cells. Using the genetic approach, we found that Rpb4, components of the SRB/MED coactivator complex, or of the SAGA and SWI/SNF complexes are critical for proper HSP104 transcription. We also identified components of the basal transcription machinery (primarily of the SAGA complex) that are critical for Hsf1 activity. These four approaches combined allow the establishment of a model describing the series of molecular events occurring on the HSP104 promoter before and after heat shock. ii
Page 1 and 2: Revealing the Mechanism of HSP104 T
Page 3: This thesis is dedicated to my fami
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 and 56:
to stress. This change in nucleosom
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perhaps not surprisingly, overexpre
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21. Chandy, M., J. L. Gutierrez, P.
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56. Hietakangas, V., J. K. Ahlskog,
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89. Miyao, T., J. D. Barnett, and N
Page 65 and 66:
121. Stanhill, A., N. Schick, and D
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