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

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Given that STREs are activated by many stresses and in turn induce the transcription of hundreds of genes, many of them not to a full extent, they clearly belong to the general, non-specific stress response (81, 107), part of the “just in case” expression of genes, not directly relevant to the stress inflicted. STREs serve as binding sites for two transcriptional activators, containing Cys 2 His 2 zinc fingers, known as Msn2 and Msn4 (87, 110). Since Msn2/4 are able to bind STREs, they are activators of the many STRE containing genes (e.g., CTT1, DDR2, HSP12). Their transcriptional activity is stimulated by a broad range of stresses. Indeed, the msn2∆msn4∆ double mutant shows up to ten fold reduction in the basal and induced expression of many stress related genes and similar reduction in the activity of STRE-dependent reporter genes (16, 44, 102, 115, 127). While the means by which Msn2/4 are activated remain elusive, it is well established that the Ras/cAMP/PKA pathway directly inhibits Msn2/4 translocation to the nucleus (46). Therefore, in yeast cells deleted for RAS2 or in mutants with low cAMP levels [in yeast, Ras proteins induce cAMP production and consequently PKA activity (18, 19)], Msn2/4 are constantly localized to the nucleus and the cells exhibit high and relatively constitutive expression of many stress related genes (9, 86, 116, 121, 129). Conversely, cells expressing the constitutively active mutant of Ras2 (RAS2 val19 ) are hypersensitive to stress and are defective in proper expression of stress related genes, because Msn2/4 are constantly cytoplasmic (9, 46, 86, 116, 121, 129). Another level of Msn2/4 regulation is probably protein stability. In response to numerous stresses such as heat shock or ethanol, Msn2 has been shown to be highly unstable (33). The instability of Msn2 is related to its nuclear localization as it is highly degraded in an msn5 mutant [MSN5 encodes a nuclear exportin involved in the nuclear export of many proteins (12, 32, 62)], demonstrating that constitutive nuclear localization is detrimental to Msn2 stability (15, 33, 75). Interestingly, studies have also shown that in response to heat shock, Msn2 is phosphorylated directly by Srb10 (Srb10 is a component of the mediator co-activator complex SRB/MED with intrinsic kinase activity) thereby downregulating its activity. Additional evidence demonstrating the negative effects of Srb10 on the activity of Msn2 is that srb10 mutant cells have high basal transcript levels of many Msn2 target genes (15, 22, 74). 9
Hsf1 and Msn2/4 can exclusively or cooperatively activate the yeast HSP104 gene The results described in the following paragraph describe the first phase of my analysis of HSP104 transcription initiation. As most of the data obtained in this phase were achieved during the course of my M.Sc. studies, they are described in this section of the “Introduction”. It should be appreciated however, that some experiments were completed during the course of my Ph.D. studies and formed the basis for the continuation of the work described in “Results”. These latter experiments are also described here in order to maintain the coherence of the “Introduction”. HSP104 promoter analysis As the first step towards analyzing the HSP104 promoter, we investigated the sequence of the upstream region of HSP104 as it appears in the Saccharomyces Genome Database (SGD website). We identified several putative cis-elements reaching up to 700bp upstream of the first AUG codon. These putative elements included various HSEs and STREs (Fig. 1). -713 -750 AAGGGCACTG CTAGCTCAGC CGGAACCTAA ATTGATTAGA GTTAGCGCTA -700 GAAACCGTGG ATGTTCAGGA CTAACGTACG ATCTACAATA TATCACCGAG -641 -650 CCGGGGAAAT TCGATGAGGT AGTAGAACAA GATGGCGTTA AAATTGTCAT -600 CGATTCAAAG GCGTTATTCA GCATCATTGG AAGTGAAATG GACTGGATCG -531 -550 ACGACAAGTT GGCCTCTAAG TTTGTCTTCA AGAATCCAAA CTCCAAGGGC -500 -500 ACATGCGGTT GTGGCGAGAG TTTCATGGTT TAAAAACCTT CTGCACCATT -450 TTTAGAAAAA AAGAATCTAC CTATTCACTT ATTTATTCAT TTACTTATTT -400 ATTTACATAT TTATCATACA TATTAACATT GAACCCTCCA TCGTGGTAGT -334 -305 -350 GTTTGCTGTT CCTAACTTTT CTTTCGTTGT TCTTGTAGAT ATATATTTTT -300 CCAGAATTTT CTAGAAGGGT TATTAATTAC AATCTTAAAC GTTCCATAAG -250 GGGCCGCGAT TTTTTTGTTC AATTTTCAAC AGGGGGCCCA TCTCAAAGAA -200 CTGCAAATTA TATCACAGTA AAAGGCAAAG GGGCGCAAAC TTATGCAACC -150 TGCCAGATTA TTATATAAGG CATTGTAATC TTGCCTCAAT TCCTTCATAA -100 TTCGTTCCTT TGTCACTTGT TCCTTTTTAC CCTTGAATCG AATCAGCAAT -50 AACAAAGAAA AAAGAAATCA ACTACACGTA CCATAAAATA TACAGAATAT +1 ATGAAC Legend STRE HSE STRE-like (PDS) TATA box Transcription initiation region Figure 1. Sequence corresponding to 750bp of the HSP104 promoter region. Letters in green correspond to the most 5’nucleotide in each of the deletion constructs used in our study. +1 is the first nucleotide of the coding sequence. Putative STREs are shown in red italics, STRE-like elements in pink and HSEs in blue; the putative TATA box is marked in green. The transcription initiation region is underlined in black. 10
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: In mammalian cells, Hsf1 is a monom
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 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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