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

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either under physiological conditions or under heat shock conditions when compared to wild type (Fig. 14). In some strains, however, such correlation was not observed. The sas3∆ strain is an extreme case of such discrepancy. When we measured β- galactosidase activity of the various constructs in sas3∆ cells very little or no activity was detected. We were therefore surprised to see that an impressive amount of HSP104 mRNA accumulated in these cells in response to heat shock. Other strains in which RNA levels were not fully correlated to levels of reporter assays are snf6∆ and srb2∆ that exhibited very low levels of HSP104 mRNA, but normal or close to normal levels of β-galactosidase induced activity. In addition, in the hos2∆ and sir2∆ strains that showed some decrease in promoter activity (Table 4) endogenous HSP104 mRNA levels were not affected (data not shown). Finally, hpr1∆ cells which showed very low or non-inducible reporter activity, expressed HSP104 mRNA at basal levels (not induced) significantly higher than those in wild type cells (Fig. 16). We therefore considered Hpr1 as a negative regulator of HSP104 rather than a positive regulator. A B time wt - Heat shock 5’ 10’ 15’ 60’ 5hrs 30 0 HSP104-LacZ(units) -HS +HS 51.2 273 time hst1∆ - 5’ 10’ 15’ 60’ 5hrs 30 0 HSP104-LacZ(units) -HS +HS 28.1 161.2 time wt - 5’ Heat shock 10’ 15’ 60’ 5hrs 30 0 time hst1∆ - Heat shock 5’ 10’ 15’ 60’ 5hrs 30 0 srb2∆ 15.7 271.7 gcn5∆ 12.8 185.4 srb2∆ srb5∆ gcn5∆ spt7∆ srb5∆ 4.7 56.3 spt7∆ 31.5 43.6 srb8∆ dst1∆ srb8∆ 24.2 56.0 dst1∆ 15.4 103.1 srb9∆ srb10∆ mft1∆ cdc73∆ srb9∆ 59.9 179.6 mft1∆ 2.0 5.8 med1∆ rpb4∆ srb10∆ 37.4 56.1 cdc73∆ 6.1 55.0 snf6∆ ACTIN sas3∆ ACTIN med1∆ 60.6 288.4 rpb4∆ 1.01 1.35 snf6∆ HSP104 7.4 175.6 sas3∆ HSP104 1.82 12.6 Figure 14. Components from the SRB/MED, SAGA and SWI/SNF complexes and RNA PolII subunit, Rpb4, are important for proper transcription of HSP104. A) S1 RNA analysis of HSP104 mRNA was performed on the indicated yeast strains following heat shock at the marked time points. Table indicates β-galactosidase activity units obtained with the -334LacZ reporter in the same strains before and after heat shock. B) Same RNAs as in A were used to measure ACTIN mRNA levels via S1 analysis as control. 41
Overall, the results nevertheless show a general correlation between reporter activity and mRNA levels. Namely, the genes deleted in 15 strains out of the 17 tested, encode proteins that are required for proper induction of the HSP104 promoter. Our results strongly suggest the involvement of the SAGA complex (represented by the spt7∆, gcn5∆ mutants), the SRB/MED coactivator complex (represented by the srb2∆, srb5∆, srb8∆, srb9∆, srb10∆, and med1∆ mutants) as well as the SWI/SNF chromatin remodeling complex (snf6∆ mutant) in mediating HSP104 transcription initiation (Table 4). In addition, we also measured promoter activity and mRNA levels of HSP104 in rpb4∆ cells and observed that HSP104 is barely, if at all, induced in this strain in response to heat shock. Rpb4 is a RNA PolII subunit which is not essential for growth under optimal growth conditions, but essential under stress and is important for the activation of PolII under heat shock conditions (23, 24, 89, 97, 98). As the components identified are part of the basal machinery we wondered whether the defective induction of HSP104 in these mutants is specific to HSP104, or whether the mutants demonstrate a general abnormality in inducing stress genes in response to heat shock. To this end we measured the mRNAs of HSP26 and SSA3, two HSP encoding genes known to be induced in response to heat shock. As shown in Figure 15, most of the mutants tested induced HSP26 and SSA3 normally, suggesting that the failure of the mutants to properly induce HSP104 is specific. We did notice however, that some mutants, like rpb4∆ and snf6∆, seem to have more general defects in inducing stress genes. Curiously, SSA3 mRNA levels were spontaneously elevated in most mutants. WT dst1∆ hst1∆ srb8∆ srb10∆ spt7∆ gcn5∆ srb2∆ HS - + - + - + - + - + - + - + - + HS srb5∆ snf6∆ rpb4∆ sas3∆ mft1∆ cdc73∆ med1∆ srb9∆ - + - + - + - + - + - + - + - + HSP26 HSP26 SSA3 SSA3 Figure 15. Most factors required for proper induction of HSP104 are specific for this gene. RNA from mutants that demonstrated impaired HSP104 induction in response to heat shock, were used to measure mRNA levels of HSP26 (sample of no heat shock and ten minutes heat shock were used) and SSA3 (sample of no heat shock and fifteen minutes heat shock were used). 42
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 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: Table 4. Strains required for posit
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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