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

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1 1 A) B) units 200 180 160 140 120 100 80 60 40 20 0 SP1 30 0 C 39 0 C +334-CYC1min -334-CYC1min +305-CYC1min units 700 600 500 400 300 200 100 0 ras2∆ +334-CYC1min +305-CYC1min 30 0 C 39 0 C Figure 7. Sequences between -334 and -160bp of the HSP104 promoter contain all elements responsible for the heat shock response and the Ras response. A) β-galactosidase activity of the chimeric -334HSP104-CYC1-LacZ and -305HSP104-CYC1-LacZ constructs were assayed in the wild type strain SP1 at 30 o C and at 39 o C. ‘-‘ is the inverted and ‘+’ is the native orientation of the HSP104 insert with respect to the CYC1 promoter. B) Same constructs as in A) assayed in ras2∆ cells under the same experimental conditions. To summarize the results obtained (see Table 1), we showed, through a combination of a genetic approach and a molecular approach (5’deletions of the HSP104 promoter, point mutations, fusion to a heterologous promoter and the use of several yeast mutants) that the HSE/Hsf1 and the STRE/Msn2/4 systems cooperate to achieve maximal inducible expression. However, in the absence of one set of factors (e.g., in msn2∆msn4∆ cells or in constructs lacking HSEs) proper induction of HSP104 promoter is achieved exclusively through the other. We also showed that HSP104 is constitutively derepressed in ras2 cells. This derepression is evoked exclusively via STREs with no role for HSEs. Strikingly, in ras2∆msn2∆msn4∆ cells, we observed that the HSP104 promoter is also derepressed via the upstream 34bp. Thus, appropriate transcription of HSP104 is usually obtained through cooperation between the STRE/Msn2/4 and HSE/Hsf1 systems, but each factor could activate the promoter on its own, backing up the other. Transcription control of HSP104 is therefore adaptive and robust, ensuring proper expression under extreme conditions and in various mutants. Finally, we identified a 34bp fragment residing upstream to the HSP104 enhancer that is critical for the promoter basal activity. This fragment is highly specific to this promoter and can acquire, under particular conditions, Ras responsive properties (i.e., in ras2∆msn2∆msn4∆ cells). 17
Table 1. Summary of the role of various fragments in HSP104 promoter -300 to -285 Goals of Study In this thesis, I describe the continuation of the effort to reveal the mechanism of transcriptional activation of the HSP104 promoter. The overall goal is to obtain sufficient data that will allow the establishment of a global model of the molecular events leading to the activation of the HSP104 promoter. To achieve this goal, I undertook 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 identify the sequence(s) responsible for the basal reporter activity of HSP104 and the unexpected activity observed in the ras2∆msn2∆msn4∆ strain. Two, using chromatin immunoprecipitation (ChIP), we monitored some of the major changes occurring in vivo on the promoter following stress. Three, using a 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 properties of the 34bp between -334 and -300 could be accounted to 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. It seems that 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 ras2∆ cells. Using the genetic approach, we found that Rpb4, components 18
Page 1 and 2: Revealing the Mechanism of HSP104 T
Page 3 and 4: 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: 1 1 A) B) 700 600 500 ras2∆msn2
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
Page 57 and 58: perhaps not surprisingly, overexpre
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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