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

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HSP104 mRNA in different mutants show, indeed, the robustness of HSP104 transcription that is rarely affected by the absence of activators and other transcription factors. Through monitoring the acetylation states of histones, we suggest that induction of HSP104 transcription requires that acetylated H3 and H4 molecules be absent. We observed that under optimal growth conditions all H4 histones on the HSP104 promoter are acetylated as well as major fractions of H3 histones. Under these conditions, only in ras2∆ cells, concentrations of acetylated H3 and H4 are lower and the promoter is even occupied with non-acetylated H4. As in this strain the HSP104 promoter is constitutively active, there is clear correlation between promoter activity and non-acetylated histones. Current dogma of gene activation claims that acetylation of histones followed by the dissociation of nucleosomes from promoters is required for promoter activation (28, 49, 50). The case for HSP104 is different. There is no global dissociation of histones from the promoter, but rather a specific dissociation of acetylated H3 molecules from nucleosomal structures and deacetylation of H4 molecules. Non-acetylated H3 proteins remain associated with nucleosomes even in response to heat shock and so do histone H4 molecules that are now not acetylated. Although not very common, the disassembly and deacetylation patterns we observe for HSP104 in response to heat shock should not be that surprising. As described in Deckert et al (31) histone deacetylation can also lead to transcription initiation of several stress-responsive genes. Also, a recent study also demonstrated that some promoters, namely GAL induced promoters, are activated by histone deacetylation (31). However, our conclusion differs somewhat from that of Deckert and Struhl (31). These investigators also monitored a dramatic decrease in acetylated histones on the HSP104 promoter in response to heat shock [18 fold decrease of acetylated H4 and 10 fold decrease in acetylated H3 (31)]. Yet, as there was some decrease in the level of unacetylated H4 occupancy (30%) they concluded that there is altogether chromatin remodeling or disassembly of the nucleosomes. We suggest that the changes on the chromatin should not be taken as all-or-non activity (disassembly, or deacetylation). There seems to be a series of different events which we partly revealed; i.e., histones H4 are deacetylated, and acetylated H3 molecules are disassembled from nucleosomes. Also, these changes are transient and reflect the dynamic nature of the changes occurring on the promoter during continuous exposure 49
to stress. This change in nucleosome composition may also be part of the shift of the cell’s response from acute stage to adaptive stage. Perhaps fifteen minutes after heat shock, nucleosomes are reassembled in a different combination of modified histones and transcription continues through a different mechanism than that of the first fifteen minutes following heat shock. In spite of mass research it is still not possible to describe in fine details events leading to transcription activation. To a certain extent, it is possible for some promoters. For instance, the transcription of the HO gene, transcribed during the G1 phase of the cell cycle includes the recruitment (by a transcriptional activator) of the chromatin remodeling complex SWI/SNF which is followed by the recruitment of Gcn5 that acetylates histones on the nucleosomes present on the promoter. Acetylation via Gcn5 then induces the recruitment of another transcriptional activator to the HO promoter (29). Another study showed that within a group of HSP genes, thought to be co-regulated, there are differences in the steps leading to their transcription initiation (36). For instance, HSP12 which showed the highest level of nucleosome displacement also showed highest level of histone H3 acetylation (36). SSA4 which showed the lowest nucleosome displacement also demonstrated the lowest acetylation state. Finally HSP82 also showed an increase in nucleosome displacement which correlated with an increase with histone acetylation (36) and yet all three genes are activated in response to heat shock in an Hsf1 dependent manner. For the first time, we show that transcriptional activation of a single promoter involves a combination of mechanisms for remodeling nucleosomes; histone displacement (partial nucleosome disassembly) and histone deacetylation. What factor(s) is responsible for inducing the changes observed on the HSP104 promoter? Results from our small genetic screen indicate a role for SWI/SNF in regulating promoter activity and transcription of HSP104. The main role for SWI/SNF is to promote nucleosome disassembly raising the question whether SWI/SNF is responsible for disassembling acetylated histone H3 from the HSP104 promoter in response to heat shock. ChIP assays may answer this question by monitoring acetylation state of histone H3 in response to heat shock in mutant SWI/SNF strains (snf6∆, for example). Another open question is the identity of the histone deacetylases (HDACs) involved in deacetylating histone H4 on the promoter of HSP104 in response to heat shock. It is clear that these enzymes display some redundancy in the cell (34, 73). In order to target which family of histone deacetylase 50
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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 and 52: Table 7. Comparison between HSE-Lac
Page 53: promoter. III) The identification o
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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