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

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In response to heat shock, acetylated H3 histones dissociate from the promoter whereas acetylated H4 histones undergo deacetylation The genetic and molecular evidence obtained so far show that Hsf1 and Msn2/4 are responsible for both basal and inducible activities of the HSP104 promoter [Figs. 2, 3, 5 and further details in (47)]. We do not know, however, how these transcriptional activators directly affect the promoter to render it active. As the major effect of transcriptional activators is remodeling of the chromatin, particularly imposing histone acetylation or deacetylation, we monitored the acetylation state of histones H3 and H4 residing on the HSP104 promoter. We performed ChIP assays that allow in vivo monitoring of the promoter-bound histones. We first tested the status of H3 histones (Fig. 10). Using anti-acetylated H3 antibodies in the ChIP assay, we observed that under optimal growth conditions acetylated H3 molecules occupy the promoter in all strains tested (Fig. 10A). Importantly, however, the HSP104 promoter in ras2∆ cells is less occupied with acetylated H3 under non-heat shock conditions (Fig. 10A). Next, we measured the status of H3 histones following stress. ChIP analysis shows that in all strains tested, acetylated H3 histones are scarcely detectable on the promoter, five to seven minutes following heat shock (Fig. 10B). The decrease observed in acetylated histone H3 on the promoter was very short lived in wild type and in ras2∆ cells (Fig. 10B). Fifteen minutes after the beginning of heat shock, acetylated histone H3 was again occupying the promoter at the same level as before heat shock. In strains with deleted MSN2 and MSN4, acetylated histones did not reoccupy the promoter indicating that Msn2/4 may have a role in the process (Fig. 10B). The loss of acetylated H3 from the HSP104 promoter shortly after heat shock could be achieved by either of the following mechanisms: 1) Histone deacetylation. 2) Specific removal of acetylated histones from nucleosomes. 3) Total disassembly of nucleosomes. To distinguish between these possibilities, we performed a ChIP assay using anti H3 antibodies. As shown in Figure 10C, we observed partial reduction in H3 promoter occupancy following heat shock. At the same time, acetylated H3 molecules are not detected (Figs. 10B and 10C). The results strongly suggest that acetylated H3 is specifically removed from the promoter in response to heat shock whereas non-acetylated H3 remains bound. Namely, there seems to be partial 29
nucleosome disassembly involving specifically the removal of only acetylated H3 molecules from the HSP104 promoter in response to heat shock. A) Strain W.T. msn2∆msn4∆ ras2∆ ras2∆msn2∆msn4∆ RAS2 val19 IP:ace H3 WCE B) C) Strain W.T. msn2∆msn4∆ Heat Shock - 7’ 10’ 15’ - 5’ 10’ 15’ SP1 IP: ace H3 IP: ace H3 Heat Shock - 5’ 10’ 15’ WCE WCE IP:totH3 IP:aceH3 Strain ras2∆ ras2∆msn2∆msn4∆ WCE Heat Shock - 5’ 10’ 15’ - 5’ 10’ 15’ IP: ace H3 IP: ace H3 WCE WCE Figure 10. Under non-heat shock conditions, some histone H3 molecules are acetylated on the HSP104 promoter and in response to heat shock, acetylated H3 histones dissociate from nucleosomes. A) ChIP analysis using anti-acetylated H3 antibodies, to monitor the presence of the protein on the HSP104 promoter of the indicated strains, grown under optimal growth conditions. B) Acetylated state of histones H3 was monitored via ChIP assays on the HSP104 promoter of wild type, ras2∆, msn2∆msn4∆ and ras2∆msn2∆msn4∆ cells in response to heat shock. Cross-linking and further processes of the ChIP analysis were performed at the indicated time points. C) Histone H3 undergoes transient remodeling in response to heat shock. Antibody recognizing total histone H3 was used in ChIP assays as well as antibody recognizing acetylated histone H3. WCE= whole cell extract. 30
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 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: 1 A) units 100.00 90.00 80.00 70.00
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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