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

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Integrated Vector empty HSFp-HA-HSF empty HSFp-HA-HSF empty HSFp-HA-HSF empty HSFp-HA-HSF empty HSFp-HA-HSF Heat Shock - - 5’ - - 5’ - - 5’ - - 5’ - - 5’ IP: HA-HSF WCE Strain BY4741 caf1∆ ccr4∆ gcn5∆ spt7∆ Figure17. Hsf1 shows stronger binding in some HSE-LacZ inefficient strains. Indicated strains were crosslinked at the indicated time points following heat shock and ChIP was performed on HA tagged Hsf1. DISCUSSION In response to stress, or to fluctuations in optimal growth conditions, cells halt transcription of most genes. Yet, genes involved in combating stresses are upregulated. The molecular mechanisms responsible for transcription initiation of this group of genes are not fully revealed. This work described a systematic study aimed at revealing these mechanisms in the S.cerevisiae HSP104 gene. This experimental approach (elaborating on just one promoter) is somewhat different than current prevailing strategies in the field. Common studies reported in recent years on transcription initiation in general and in response to stress in particular, addressed coregulation of many genes searching for common themes in their regulation (21, 36, 59, 103, 128, 135, 136). These approaches lead on one hand to important discoveries, but on the other hand major matters are left unresolved. Most important, data accumulated so far suggest that although common regulatory themes do exist, each promoter is induced via specific processes. Our long-term intention is therefore to dissect the processes of transcription initiation under stress of one gene in order to understand its specific and unique regulation at high resolution and in a highly detailed manner. This thesis presents the data obtained so far in the long run toward this ultimate goal. This data was obtained via three approaches I undertook: I) Promoter analysis, i.e., identification of various cis-elements which are essential for regulating promoter activity of HSP104. In order to identify the important elements and the relationships between them for HSP104 transcription we proceeded with 5’deletions of the promoter, point mutations and the establishment of heterologous reporter genes. II) Monitoring modifications of histones occurring on the promoter of HSP104 in response to heat shock. We also monitored the binding activity of the transcriptional activator Hsf1 and attempted to measure the binding of Msn2/4 on the HSP104 47
promoter. III) The identification of components of the basal transcription machinery involved specifically in transcriptionally activating HSP104. The results obtained allow us to draw a detailed working model for the molecular events occurring on the HSP104 promoter in response to heat shock (see details in the last section of the Discussion). The first aspect of our promoter analysis dealt with, as mentioned above, analyzing the promoter through 5’deletion [most aspects of promoter analysis are thoroughly described in (47)]. We sought at this stage of our study, to identify sequences which are required for the high basal activity of -334LacZ reporter. We found the putative single HSE site between (-304 and -300) to have an important role in this promoter activity. In fact, our fine-tuned mapping effort did not point at an additional particular sequence within the 34bp as responsible for the activity. We further conclude that the 34bp (particularly the HSE between - 304 and -300) may possibly be required to interact with the specific basal transcription machinery of HSP104 (this conclusion is also based on the results with the heterologous promoter, Fig. 7). We therefore attempted to analyze the interaction between the 34bp with the basal transcription machinery by deleting an internal fragment of 78bp, thereby bringing the 34bp close to the minimal promoter. The various ∆78 constructs manifested low basal activity, suggesting that perhaps the spatial organization between the 34bp and the basal transcription machinery is critical for the basal activity of the HSP104 promoter. It could also be that STREs, removed with the internal 78bp, are required for basal activity. The ∆78 constructs provided important information about the flexibility and modularity of the promoter (Fig. 8). Normally, there is complete cooperation between STRE/Msn2/4 and HSE/Hsf1 systems for optimal promoter activity. Yet, under specific conditions, such as in msn2∆msn4∆ cells, HSP104 is solely activated by the HSE/Hsf1 system. Conversely, in ras2∆ cells, the HSE/Hsf1 system is not required for the activation of HSP104 promoter activity. High activity of HSP104 in this strain is due to the hypothesized constitutive binding of Msn2/4 to the STREs present on the promoter. This flexibility and back-up capabilities of the HSE/Hsf1 and STRE/Msn2/4 systems are now further expanded and include the upstream 34bp that become Ras2 responsive in cells lacking Msn2/4 and the internal 78bp, that under various conditions, affect not only induced, but also basal promoter activity. Namely, promoter flexibility is much stronger than we previously suggested and many cis and trans-elements are backing up each other to allow promoter activity under various circumstances. Measuring 48
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 and 50: Regulation of Hsf1 As explained in
Page 51: Table 7. Comparison between HSE-Lac
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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