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

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Table 6. Strains which down-regulated HSE-LacZ reporter activity (not exclusive). Strain Cofatctor complex β-Gal activity (% of WT) Uninduced Induced HSP104 HSP104 β-Gal activity (% of WT) Uninduced Induced STRE-LacZ STRE-LacZ β-Gal activity (% of WT) Uninduced Induced HSE-LacZ HSE-LacZ ada2∆ ADA,SAGA 56 117 57 17 27 65 ada3∆ ADA,SAGA 42 74 8 7 2 1 gcn5∆ ADA,SAGA 23 69 99 17 13 20 spt3∆ SAGA 41 81 213 52 22 34 spt7∆ SAGA 58 16 325 37 6 8 spt8∆ SAGA 59 101 77 31 26 37 sas3∆ NuA3 3 7 0 2 7 6 hpa3∆ HAT 62 88 88 157 29 32 hos2∆ HDAC 36 64 33 55 35 26 hos3∆ HDAC 64 65 77 47 23 30 sir2∆ HDAC 38 71 29 77 38 46 hst2∆ HDAC 59 85 109 116 54 41 tfg3∆ Multiple 62 69 259 91 4 23 swi3∆ SWI/SNF 46 96 143 95 2 14 snf6∆ SWI/SNF 13 50 0 0 10 23 not4∆ CCR4-NOT 111 109 1161 118 35 39 caf1∆ CCR4-NOT 227 60 237 87 15 13 caf4∆ CCR4-NOT 51 71 47 60 35 31 caf16∆ CCR4-NOT 61 65 56 83 39 38 dbf2∆ CCR4-NOT 62 51 342 415 32 23 dhh1∆ CCR4-NOT 319 67 873 132 17 7 srb9∆ SRB/MED,CCR4- NOT 150 59 723 104 52 38 srb10∆ SRB/MED,CCR4- NOT 72 15 469 41 13 6 srb2∆ SRB/MED 33 110 154 69 32 94 srb5∆ SRB/MED 10 23 78 23 12 29 srb8∆ SRB/MED 60 28 614 90 69 28 med1∆ SRB/MED 154 107 715 229 42 38 med9∆ SRB/MED 179 93 1550 484 29 32 rox3∆ SRB/MED 86 41 1605 251 4 16 cdc73∆ Paf1 complex 11 20 11 17 4 12 rtf1∆ Paf1 complex 49 52 100 51 34 28 leo1∆ Paf1 complex 21 29 37 51 34 28 ccr4∆ CCR4-NOT,Paf1 157 43 399 107 11 5 hpr1∆ Paf1,THO/TREX 0.1 1 0 1.5 0 0.1 mft1∆ THO/TREX 4 3 5 0.1 0.4 0.5 tex1∆ THO/TREX 34 38 44 61 32 33 spt4∆ SPT 10 2 62 8 0.5 0.3 rpb9∆ RNA PolII core 48 62 169 41 34 30 rpb4∆ RNA PolII core 2 0.4 17 0.6 1 0.4 45
Table 7. Comparison between HSE-LacZ and SV40-LacZ in various mutant strains. Strains Cofactor complex β-Gal activity (% of WT) β-Gal activity (% of WT) Uninduced Induced Uninduced Induced HSE-LacZ HSE-LacZ SV40-LacZ SV40-LacZ ada2∆ ADA,SAGA 32 89 125 149 ada3∆ ADA,SAGA 0.5 0.4 111 111 gcn5∆ ADA,SAGA 25 40 137 126 spt3∆ SAGA 32 57 350 194 spt7∆ SAGA 6 10 169 92 spt8∆ SAGA 32 54 313 168 sas3∆ NuA3 8 6 10 9 hpa3∆ HAT 40 45 101 103 hos2∆ HDAC 35 28 67 72 hos3∆ HDAC 40 43 132 101 sir2∆ HDAC 43 47 165 102 hst2∆ HDAC 64 50 102 90 To begin and address the mechanism of action of the mutations responsible for reduced Hsf1 activity, we tested in some of the strains the ability of Hsf1 to bind in vivo to the HSP104 promoter. Since we showed that Hsf1 constitutively binds HSEs on the HSP104 promoter regardless of heat shock conditions, we predicted reduced DNA binding activity of Hsf1 in strains in which HSE-LacZ activity was low. Quite surprisingly, we actually observed an inverse correlation between the transcriptional activity of Hsf1 and its DNA binding activity. As is shown in Figure 17, gcn5∆, spt7∆, caf1∆, and ccr4∆ cells which demonstrated very poor reporter activity of HSE- LacZ (Table 6), displayed stronger binding of Hsf1 on the HSP104 promoter compared to wild type. Perhaps in these strains stronger Hsf1 DNA binding activity is inhibitory. Transcription repression activity of Hsf1 was previously suggested (131). It should be noted that the studies on the mechanism of action of the mutants is only at its beginning and a large scale study is required for revealing the role of each factor on Hsf1 activity. Nevertheless, the finding of elements of the basal transcription machinery that are highly specific to Hsf1 is novel and unexpected. 46
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 and 46: Table 4. Strains required for posit
Page 47 and 48: Overall, the results nevertheless s
Page 49: Regulation of Hsf1 As explained in
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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