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

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Next we tested the status of H4 histones on the HSP104 promoter (Fig. 11). Just like we observed in the case of histone H3, acetylated H4 molecules occupy the HSP104 promoter under optimal growth conditions (Fig. 11A). Also, we detected that less acetylated H4 histones occupy the HSP104 promoter in ras2∆ cells. These results strongly indicate a role for Ras2 in promoting acetylated histone occupancy on the HSP104 promoter. We next monitored the changes occurring to acetylated H4 molecules on the promoter of HSP104 following heat shock (Fig. 11B). Similarly to acetylated H3 histones, we also observed a transient decrease in acetylated H4 molecules on the promoter in response to heat shock. In wild type cells, following a fifteen minutes heat shock, acetylated H4 molecules reoccupied the promoter, but not to the same level as before heat shock. Very little acetylated H4 reoccupied HSP104 promoter in strains with deleted MSN2 and MSN4, just like the case for H3 histones (Fig. 10B). In an attempt to identify the mechanism leading to the decrease in acetylated H4 histones in response to heat shock, we measured the presence of nonacetylated histone H4 on the promoter in response to heat shock. As shown in Figure 11C, we could not measure non-acetylated H4 molecules in wild type cells not exposed to heat shock (Fig. 11C, left lane). However, five minutes following heat shock, this promoter becomes occupied with non-acetylated H4. This result is a clear mirror image to that in Figure 11B, showing the disappearance of acetylated H4 from the promoter. We therefore conclude that in response to heat shock, histones H4 remain associated with the promoter but undergo extensive deacetylation. A similar mirror image is observed for the ras2∆ strain. The HSP104 promoter in this strain is occupied with low levels of acetylated H4 under optimal conditions (Fig. 11A) and in contrast contains increased levels of non-acetylated H4 (Fig. 11C, lane 5). As in this strain the promoter is spontaneously active (47), it seems that reduced levels of acetylated H4 and increased levels of non-acetylated H4 is important for promoter activity. In summary, H3 and H4 population on the promoter is modified via two different mechanisms. Acetylated H4 molecules undergo deacetylation whereas acetylated H3 are removed altogether. These changes that occur in response to heat shock and in ras2∆ cells correlate with promoter activity. 31
A) Strain W.T. msn2∆msn4∆ ras2∆ ras2∆msn2∆msn4∆ RAS2 val19 IP:ace H4 WCE B) Strain W.T. msn2∆msn4∆ Heat Shock - 7’ 10’ 15’ - 5’ 10’ 15’ 15’ IP: ace H4 IP: ace H4 WCE WCE Strain ras2∆ ras2∆msn2∆msn4∆ Heat Shock - 5’ 10’ 15’ - 5’ 10’ 15’ IP: ace H4 IP: ace H4 WCE WCE C) Strain Heat Shock msn2∆msn4∆ ras2∆ ras2∆msn2∆msn4∆ wt - 5’ - 5’ - 5’ 10’ 15’ - 5’ 10’ 15’ IP:nonace H4 WCE Figure 11. Under non-heat shock conditions, all histone H4 molecules are acetylated on the HSP104 promoter and in response to heat shock, acetylated H4 histones are deacetylated. A) ChIP analysis using anti-acetylated H4 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 H4 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 performe at the indicated time points. C) Histone H4 undergoes extensive deacetylation in response to heat shock. Antibody recognizing non-acetylated histone H4 was used for ChIP assays as well as antibody recognizing acetylated histone H4. 32
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 and 34: 1 A) units 100.00 90.00 80.00 70.00
Page 35: nucleosome disassembly involving sp
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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