Chromatin remodeling by GAGA factor and heat shock factor at the hypersensitive Drosophila hsp26 promoter in vitro.

ABSTRACT The heat shock response, which is accompanied by a rapid and robust upregulation of heat shock proteins (Hsps), is a highly conserved protection mechanism against protein-damaging stress. Hsp induction is mainly regulated at transcriptional level by stress-inducible heat shock factor 1 (HSF1). Upon activation, HSF1 trimerizes, binds to DNA, concentrates in the nuclear stress granules, and undergoes a marked multisite phosphorylation, which correlates with its transcriptional activity. In this study, we show that HSF1 is modified by SUMO-1 and SUMO-2 in a stress-inducible manner. Sumoylation is rapidly and transiently enhanced on lysine 298, located in the regulatory domain of HSF1, adjacent to several critical phosphorylation sites. Sumoylation analyses of HSF1 phosphorylation site mutants reveal that specifically the phosphorylation-deficient S303 mutant remains devoid of SUMO modification in vivo and the mutant mimicking phosphorylation of S303 promotes HSF1 sumoylation in vitro, indicating that S303 phosphorylation is required for K298 sumoylation. This finding is further supported by phosphopeptide mapping and analysis with S303/7 phosphospecific antibodies, which demonstrate that serine 303 is a target for strong heat-inducible phosphorylation, corresponding to the inducible HSF1 sumoylation. A transient phosphorylation-dependent colocalization of HSF1 and SUMO-1 in nuclear stress granules provides evidence for a strictly regulated subnuclear interplay between HSF1 and SUMO.

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(CT)n (GA)n repeats and heat shock elements have distinct roles in chromatin structure and transcriptional activation of the Drosophila hsp26 gene

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.2802-2814.1993 ◽

1993 ◽

Vol 13 (5) ◽

pp. 2802-2814

Author(s):

Q Lu ◽

L L Wallrath ◽

H Granok ◽

S C Elgin

Keyword(s):

Heat Shock ◽

Chromatin Structure ◽

Previous Analysis ◽

Heat Shock Factor ◽

Dnase I ◽

Inducible Expression ◽

P Element ◽

Enzyme Treatment ◽

Lacz Gene ◽

Gaga Factor

Previous analysis of the hsp26 gene of Drosophila melanogaster has shown that in addition to the TATA box and the proximal and distal heat shock elements (HSEs) (centered at -59 and -340, relative to the start site of transcription), a segment of (CT)n repeats at -135 to -85 is required for full heat shock inducibility (R.L. Glaser, G.H. Thomas, E.S. Siegfried, S.C.R. Elgin, and J.T. Lis, J. Mol. Biol. 211:751-761, 1990). This (CT)n element appears to contribute to formation of the wild-type chromatin structure of hsp26, an organized nucleosome array that leaves the HSEs in nucleosome-free, DNase I-hypersensitive (DH) sites (Q. Lu, L.L. Wallrath, B.D. Allan, R.L. Glaser, J.T. Lis, and S.C.R. Elgin, J. Mol. Biol. 225:985-998, 1992). Inspection of the sequences upstream of hsp26 has revealed an additional (CT)n element at -347 to -341, adjacent to the distal HSE. We have analyzed the contribution of this distal (CT)n element (-347 to -341), the proximal (CT)n element (-135 to -85), and the two HSEs both to the formation of the chromatin structure and to heat shock inducibility. hsp26 constructs containing site-directed mutations, deletions, substitutions, or rearrangements of these sequence elements have been fused in frame to the Escherichia coli lacZ gene and reintroduced into the D. melanogaster genome by P-element-mediated germ line transformation. Chromatin structure of the transgenes was analyzed (prior to gene activation) by DNase I or restriction enzyme treatment of isolated nuclei, and heat-inducible expression was monitored by measuring beta-galactosidase activity. The results indicate that mutations, deletions, or substitutions of either the distal or the proximal (CT)n element affect the chromatin structure and heat-inducible expression of the transgenes. These (CT)n repeats are associated with a nonhistone protein(s) in vivo and are bound by a purified Drosophila protein, the GAGA factor, in vitro. In contrast, the HSEs are required for heat-inducible expression but play only a minor role in establishing the chromatin structure of the transgenes. Previous analysis indicates that prior to heat shock, these HSEs appear to be free of protein. Our results suggest that GAGA factor, an abundant protein factor required for normal expression of many Drosophila genes, and heat shock factor, a specific transcription factor activated upon heat shock, play distinct roles in gene regulation: the GAGA factor establishes and/or maintains the DH sites prior to heat shock induction, while the activated heat shock factor recognizes and binds HSEs located within the DH sites to trigger transcription.

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The GAGA factor is required in the early Drosophila embryo not only for transcriptional regulation but also for nuclear division

Development ◽

10.1242/dev.122.4.1113 ◽

1996 ◽

Vol 122 (4) ◽

pp. 1113-1124 ◽

Cited By ~ 1

Author(s):

K.M. Bhat ◽

G. Farkas ◽

F. Karch ◽

H. Gyurkovics ◽

J. Gausz ◽

...

Keyword(s):

Gene Expression ◽

Chromatin Remodeling ◽

In Vitro Transcription ◽

Drosophila Embryo ◽

Gaga Factor ◽

Early Drosophila Embryo ◽

Hypersensitive Sites ◽

And Function ◽

Stimulatory Factor

The GAGA protein of Drosophila was first identified as a stimulatory factor in in vitro transcription assays using the engrailed and Ultrabithorax promoters. Subsequent studies have suggested that the GAGA factor promotes transcription by blocking the repressive effects of histones; moreover, it has been shown to function in chromatin remodeling, acting together with other factors in the formation of nuclease hypersensitive sites in vitro. The GAGA factor is encoded by the Trithorax-like locus and in the studies reported here we have used the maternal effect allele Trl13C to examine the functions of the protein during embryogenesis. We find that GAGA is required for the proper expression of a variety of developmental loci that contain GAGA binding sites in their upstream regulatory regions. The observed disruptions in gene expression are consistent with those expected for a factor involved in chromatin remodeling. In addition to facilitating gene expression, the GAGA factor appears to have a more global role in chromosome structure and function. This is suggested by the spectrum of nuclear cleavage cycle defects observed in Trl13C embryos. These defects include asynchrony in the cleavage cycles, failure in chromosome condensation, abnormal chromosome segregation and chromosome fragmentation. These defects are likely to be related to the association of the GAGA protein with heterochromatic satellite sequences which is observed throughout the cell cycle.

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Inhibition of the activation of heat shock factor in vivo and in vitro by flavonoids.

Molecular and Cellular Biology ◽

10.1128/mcb.12.8.3490 ◽

1992 ◽

Vol 12 (8) ◽

pp. 3490-3498 ◽

Cited By ~ 151

Author(s):

N Hosokawa ◽

K Hirayoshi ◽

H Kudo ◽

H Takechi ◽

A Aoike ◽

...

Keyword(s):

Heat Shock ◽

Transcriptional Activation ◽

Heat Shock Factor ◽

Mobility Shift ◽

Human Colon Cancer ◽

Heat Shock Element ◽

Mrna Accumulation ◽

Cell Extracts

Transcriptional activation of human heat shock protein (HSP) genes by heat shock or other stresses is regulated by the activation of a heat shock factor (HSF). Activated HSF posttranslationally acquires DNA-binding ability. We previously reported that quercetin and some other flavonoids inhibited the induction of HSPs in HeLa and COLO 320DM cells, derived from a human colon cancer, at the level of mRNA accumulation. In this study, we examined the effects of quercetin on the induction of HSP70 promoter-regulated chloramphenicol acetyltransferase (CAT) activity and on the binding of HSF to the heat shock element (HSE) by a gel mobility shift assay with extracts of COLO 320DM cells. Quercetin inhibited heat-induced CAT activity in COS-7 and COLO 320DM cells which were transfected with plasmids bearing the CAT gene under the control of the promoter region of the human HSP70 gene. Treatment with quercetin inhibited the binding of HSF to the HSE in whole-cell extracts activated in vivo by heat shock and in cytoplasmic extracts activated in vitro by elevated temperature or by urea. The binding of HSF activated in vitro by Nonidet P-40 was not suppressed by the addition of quercetin. The formation of the HSF-HSE complex was not inhibited when quercetin was added only during the binding reaction of HSF to the HSE after in vitro heat activation. Quercetin thus interacts with HSF and inhibits the induction of HSPs after heat shock through inhibition of HSF activation.

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Heat Shock Factor 1 (HSF1-pSer326) Predicts Response to Bortezomib-Containing Chemotherapy in Pediatric AML: A COG Study

Blood ◽

10.1182/blood.2020005208 ◽

2020 ◽

Author(s):

Fieke W Hoff ◽

Anneke D van Dijk ◽

Yi Hua Qiu ◽

Peter P Ruvolo ◽

Robert B Gerbing ◽

...

Keyword(s):

Heat Shock ◽

De Novo ◽

Heat Shock Factor ◽

Leukemic Cells ◽

Heat Shock Factor 1 ◽

Free Survival ◽

Heat Shock Responses ◽

Pediatric Aml ◽

Hsf1 Gene

Bortezomib (BTZ) was recently evaluated in a randomized Phase 3 clinical trial which compared standard chemotherapy (cytarabine, daunorubicin, etoposide; ADE) to standard therapy with BTZ (ADEB) for de novo pediatric acute myeloid leukemia. While the study concluded that BTZ did not improve outcome overall, we examined patient subgroups benefitting from BTZ-containing chemotherapy using proteomic analyses. The proteasome inhibitor BTZ disrupts protein homeostasis and activates cytoprotective heat shock responses. We measured total heat shock factor 1 (HSF1) and phosphorylated HSF1 (HSF1-pSer326) in leukemic cells from 483 pediatric patients using Reverse Phase Protein Arrays. HSF1-pSer326 phosphorylation was significantly lower in pediatric AML compared to CD34+ non-malignant cells. We identified a strong correlation between HSF1-pSer326 expression and BTZ sensitivity. BTZ significantly improved outcome of patients with low-HSF1-pSer326 with a 5-year event-free survival of 44% (ADE) vs. 67% for low-HSF1-pSer326 treated with ADEB (P=0.019). To determine the effect of HSF1 expression on BTZ potency in vitro, cell viability with HSF1 gene variants that mimicked phosphorylated (S326A) and non-phosphorylated (S326E) HSF1-pSer326 were examined. Those with increased HSF1 phosphorylation showed clear resistance to BTZ vs. those with wild type or reduced HSF1-phosphorylation. We hypothesize that HSF1-pSer326 expression could identify patients that benefit from BTZ-containing chemotherapy.

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(CT)n (GA)n repeats and heat shock elements have distinct roles in chromatin structure and transcriptional activation of the Drosophila hsp26 gene.

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.2802 ◽

1993 ◽

Vol 13 (5) ◽

pp. 2802-2814 ◽

Cited By ~ 152

Author(s):

Q Lu ◽

L L Wallrath ◽

H Granok ◽

S C Elgin

Keyword(s):

Heat Shock ◽

Chromatin Structure ◽

Previous Analysis ◽

Heat Shock Factor ◽

Dnase I ◽

Inducible Expression ◽

P Element ◽

Enzyme Treatment ◽

Lacz Gene ◽

Gaga Factor

Previous analysis of the hsp26 gene of Drosophila melanogaster has shown that in addition to the TATA box and the proximal and distal heat shock elements (HSEs) (centered at -59 and -340, relative to the start site of transcription), a segment of (CT)n repeats at -135 to -85 is required for full heat shock inducibility (R.L. Glaser, G.H. Thomas, E.S. Siegfried, S.C.R. Elgin, and J.T. Lis, J. Mol. Biol. 211:751-761, 1990). This (CT)n element appears to contribute to formation of the wild-type chromatin structure of hsp26, an organized nucleosome array that leaves the HSEs in nucleosome-free, DNase I-hypersensitive (DH) sites (Q. Lu, L.L. Wallrath, B.D. Allan, R.L. Glaser, J.T. Lis, and S.C.R. Elgin, J. Mol. Biol. 225:985-998, 1992). Inspection of the sequences upstream of hsp26 has revealed an additional (CT)n element at -347 to -341, adjacent to the distal HSE. We have analyzed the contribution of this distal (CT)n element (-347 to -341), the proximal (CT)n element (-135 to -85), and the two HSEs both to the formation of the chromatin structure and to heat shock inducibility. hsp26 constructs containing site-directed mutations, deletions, substitutions, or rearrangements of these sequence elements have been fused in frame to the Escherichia coli lacZ gene and reintroduced into the D. melanogaster genome by P-element-mediated germ line transformation. Chromatin structure of the transgenes was analyzed (prior to gene activation) by DNase I or restriction enzyme treatment of isolated nuclei, and heat-inducible expression was monitored by measuring beta-galactosidase activity. The results indicate that mutations, deletions, or substitutions of either the distal or the proximal (CT)n element affect the chromatin structure and heat-inducible expression of the transgenes. These (CT)n repeats are associated with a nonhistone protein(s) in vivo and are bound by a purified Drosophila protein, the GAGA factor, in vitro. In contrast, the HSEs are required for heat-inducible expression but play only a minor role in establishing the chromatin structure of the transgenes. Previous analysis indicates that prior to heat shock, these HSEs appear to be free of protein. Our results suggest that GAGA factor, an abundant protein factor required for normal expression of many Drosophila genes, and heat shock factor, a specific transcription factor activated upon heat shock, play distinct roles in gene regulation: the GAGA factor establishes and/or maintains the DH sites prior to heat shock induction, while the activated heat shock factor recognizes and binds HSEs located within the DH sites to trigger transcription.

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Cooperative Binding of Heat Shock Factor to the Yeast HSP82 Promoter In Vivo and In Vitro

Molecular and Cellular Biology ◽

10.1128/mcb.19.3.1627 ◽

1999 ◽

Vol 19 (3) ◽

pp. 1627-1639 ◽

Cited By ~ 62

Author(s):

Alexander M. Erkine ◽

Serena F. Magrogan ◽

Edward A. Sekinger ◽

David S. Gross

Keyword(s):

Heat Shock ◽

Single Point ◽

Heat Shock Factor ◽

Single Point Mutation ◽

Heat Shock Element ◽

Cooperative Interactions ◽

Before And After ◽

Order Of Magnitude

ABSTRACT Previous work has shown that heat shock factor (HSF) plays a central role in remodeling the chromatin structure of the yeastHSP82 promoter via constitutive interactions with its high-affinity binding site, heat shock element 1 (HSE1). The HSF-HSE1 interaction is also critical for stimulating both basal (noninduced) and induced transcription. By contrast, the function of the adjacent, inducibly occupied HSE2 and -3 is unknown. In this study, we examined the consequences of mutations in HSE1, HSE2, and HSE3 on HSF binding and transactivation. We provide evidence that in vivo, HSF binds to these three sites cooperatively. This cooperativity is seen both before and after heat shock, is required for full inducibility, and can be recapitulated in vitro on both linear and supercoiled templates. Quantitative in vitro footprinting reveals that occupancy of HSE2 and -3 by Saccharomyces cerevisiae HSF (ScHSF) is enhanced ∼100-fold through cooperative interactions with the HSF-HSE1 complex. HSE1 point mutants, whose basal transcription is virtually abolished, are functionally compensated by cooperative interactions with HSE2 and -3 following heat shock, resulting in robust inducibility. Using a competition binding assay, we show that the affinity of recombinant HSF for the full-length HSP82promoter is reduced nearly an order of magnitude by a single-point mutation within HSE1, paralleling the effect of these mutations on noninduced transcript levels. We propose that the remodeled chromatin phenotype previously shown for HSE1 point mutants (and lost in HSE1 deletion mutants) stems from the retention of productive, cooperative interactions between HSF and its target binding sites.

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Basal-level expression of the yeast HSP82 gene requires a heat shock regulatory element

Molecular and Cellular Biology ◽

10.1128/mcb.9.11.4789-4798.1989 ◽

1989 ◽

Vol 9 (11) ◽

pp. 4789-4798

Author(s):

D McDaniel ◽

A J Caplan ◽

M S Lee ◽

C C Adams ◽

B R Fishel ◽

...

Keyword(s):

Heat Shock ◽

Regulatory Element ◽

Heat Shock Factor ◽

Tata Box ◽

Hypersensitive Site ◽

Heat Shock Element ◽

Hsp82 Promoter ◽

Basal Level Expression

Previous studies have shown that heat shock factor is constitutively bound to heat shock elements in Saccharomyces cerevisiae. We demonstrate that mutation of the heat shock element closest to the TATA box of the yeast HSP82 promoter abolishes basal-level transcription without markedly affecting inducibility. The mutated heat shock element no longer bound putative heat shock factor, either in vitro or in vivo, but still resided within a nuclease-hypersensitive site in the chromatin. Thus, constitutive binding of heat shock factor to heat shock elements in S. cerevisiae appears to functionally direct basal-level transcription.

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Chromatin potentiation of the hsp70 promoter is linked to GAGA-factor recruitment

Biochemistry and Cell Biology ◽

10.1139/o05-060 ◽

2005 ◽

Vol 83 (4) ◽

pp. 555-565 ◽

Cited By ~ 20

Author(s):

Philippe T Georgel

Keyword(s):

Binding Site ◽

Chromatin Remodeling ◽

Binding Sites ◽

Transcription Initiation ◽

Gaga Factor ◽

Hsp70 Promoter ◽

Nucleosome Remodeling ◽

Functional Sites ◽

Factor Binding

The events leading to transcription initiation of the Drosophila melanogaster heat-shock protein (hsp)70 gene have been demonstrated to be directly connected with nucleosome remodeling factor and GAGA-dependent chromatin remodeling on its promoter region. To investigate the relative importance of the multiple GAGA-factor binding sites in the process of chromatin remodeling and their effect on DNA conformation, the position of nucleosomes over the proximal region of the promoter was mapped. No real-positioned nucleosome was detected. By matching the relative position of the GAGA-factor binding sites with the distribution of nucleosomes over the hsp70 promoter, the GAGA site 2 appeared to be the most accessible, i.e., located close to a nucleosomal edge or within the linker DNA. This result, combined with previous observations, suggest a link between increased GAGA-factor accessibility and efficiency of transcription initiation. The effect of GAGA-binding-site mutations, both individually and in combination, on DNA structure and nucleosome remodeling was assessed using free DNA and fly embryo extract chromatin templates assembled in vitro. Results indicated that both the number of functional sites and their positions within the chromatin were important determinants for nucleosome-remodeling efficiency. Ultimately, the degree of accessibility of the GAGA factor to its cognate binding site(s) appears to be proportional to chromatin-remodeling competency of the hsp70 promoter.Key words: chromatin, remodeling, nucleosome, hsp70, GAGA, Drosophila.

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