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

More documents

Recommendations

Info

A) Strain W.T. Strain ras2∆ Integrated vector HSFp-HA-HSF Integrated vector empty HSFp-HA-HSF Heat Shock - 5’ 10’ 15’ 15’ Heat Shock - 15’ - 5’ 10’ 15’ IP: HA-HSF IP: HA-HSF WCE WCE Strain msn2∆msn4∆ Strain ras2∆msn2∆msn4∆ Integrated vector empty HSFp-HA-HSF Integrated vector empty HSFp-HA-HSF Heat Shock - 15’ 15’ - 5’ 10’ 15’ 15’ Heat Shock - 15’ - 5’ 10’ 15’ IP: HA-HSF IP: HA-HSF WCE WCE B) C) Strain Integrated vector Heat Shock W.T. msn2∆msn4∆ HSFp-HA-HSF ras2∆ - - - - ras2∆msn2∆msn4∆ Strain Integrated vector Heat Shock IB: α-HA empty HSFp-HA-HSF - - W.T. 5’ 5’ 10’ 15’ 10’ 15’ msn2∆msn4∆ empty HSFp-HA-HSF - - 5’ 10’ 15’ IP: HA-HSF Strain ras2∆ ras2∆ msn2∆msn4∆ WCE Integrated vector Heat Shock empty HSFp-HA-HSF - - 5’ 10’ 15’ empty HSFp-HA-HSF - - 5’ 10’ 15’ IB: α-HA Figure 13. HSF constitutively binds to the HSP104 promoter. A) Wild type, ras2∆, msn2∆msn4∆ and ras2∆msn2∆msn4∆ strains were crosslinked at the indicated time points following heat shock and ChIP was performed on HA tagged Hsf1. B) Samples of ChIP experiments shown in A (from non-heat shocked cells) were loaded on the same gel to compare HSF binding under basal conditions in different strains. C) Western analysis on HA-tagged Hsf1 showing similar protein level in all strains and under all conditions. 35
Next, we attempted to directly measure the occupancy of Msn2 and Msn4 on the promoter. Similarly to Hsf1, we constructed Ha-tagged proteins and inserted them in the yeast genome at the URA3 locus. We were unfortunately unsuccessful in detecting either Msn2 or Msn4 on the HSP104 promoter under normal or heat shock conditions. Furthermore, we could not measure via ChIP assays Msn2/4 binding in ras2∆ cells in which these two transcriptional activators are believed to constitutively bind STREs (data not shown). We tried different variations of tagging Msn2 (either at the N-terminus or C-terminus) and we also tried to over-express Msn2 under the Alcohol Dehydrogenase 1 (ADH1) promoter known to be a strong constitutive promoter. We also tried growing cells in minimal media (YNB) as opposed to rich media (YPD). In all cases, DNA binding of the proteins was not detected although the Msn2/4 proteins were confirmed to be expressed and active. It seems that the inability to measure Msn2/4 binding via ChIP is not specific to our laboratory. There is no report in the literature on successful ChIP on Msn2 or Msn4. Some reports (part of all genome binding assay) do report on Msn2/4 binding to some promoter, (but with very low affinity) (135). We still do not know whether the reason is technical or conceptual (reflecting no direct association of Msn2/4 with DNA). SAGA, SRB/MED and SWI/SNF are important for HSP104 promoter activity The experiments described above analyzed the HSP104 promoter and provided an insight into the events occurring on nucleosomes located on the HSP104 promoter. The experiments further showed that these events are controlled, at least in part, by the activators Msn2/4. Having identified the major activators of the promoter and some of their effects on chromatin organization we sought to identify the basal transcription factors required for heat shock induced HSP104 transcription. To uncover these factors, we tested HSP104 promoter activity in various mutants from the Saccharomyces Genome Deletion Project. Mutants included those lacking a gene encoding a basal transcription factor. Into each of these mutants we introduced (separately) three reporter genes: i) -334LacZ, ii) STRE-LacZ and iii) HSE-LacZ. Activity of the -334LacZ manifests the activity of the full length promoter. Activity of STRE-LacZ [-260LacZ in (47)] manifests the function of the promoter activity dependent on Msn2/4. The HSE-LacZ reporter contains the HSE cluster of HSP104 in four repeats fused to the CYC1 minimal promoter. This reporter reflects the activity of Hsf1. 69 strains (Table 3) were tested with the three different plasmids. 36
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 and 36: nucleosome disassembly involving sp
Page 37 and 38: A) Strain W.T. msn2∆msn4∆ ras2
Page 39: Hsf1 constitutively binds the HSP10
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

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

Create successful ePaper yourself

Delete template?

Save as template?