Cell Cycle-Dependent Binding of Yeast Heat Shock Factor to Nucleosomes

ABSTRACT In the nucleus, transcription factors must contend with the presence of chromatin in order to gain access to their cognate regulatory sequences. As most nuclear DNA is assembled into nucleosomes, activators must either invade a stable, preassembled nucleosome or preempt the formation of nucleosomes on newly replicated DNA, which is transiently free of histones. We have investigated the mechanism by which heat shock factor (HSF) binds to target nucleosomal heat shock elements (HSEs), using as our model a dinucleosomal heat shock promoter (hsp82-ΔHSE1). We find that activated HSF cannot bind a stable, sequence-positioned nucleosome in G1-arrested cells. It can do so readily, however, following release from G1 arrest or after the imposition of either an early S- or late G2-phase arrest. Surprisingly, despite the S-phase requirement, HSF nucleosomal binding activity is restored in the absence of hsp82 replication. These results contrast with the prevailing paradigm for activator-nucleosome interactions and implicate a nonreplicative, S-phase-specific event as a prerequisite for HSF binding to nucleosomal sites in vivo.

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Phosphorylation of Serine 303 Is a Prerequisite for the Stress-Inducible SUMO Modification of Heat Shock Factor 1

Molecular and Cellular Biology ◽

10.1128/mcb.23.8.2953-2968.2003 ◽

2003 ◽

Vol 23 (8) ◽

pp. 2953-2968 ◽

Cited By ~ 205

Author(s):

Ville Hietakangas ◽

Johanna K. Ahlskog ◽

Annika M. Jakobsson ◽

Maria Hellesuo ◽

Niko M. Sahlberg ◽

...

Keyword(s):

Heat Shock ◽

Phosphorylation Site ◽

Stress Granules ◽

Heat Shock Factor ◽

Transcriptional Level ◽

Heat Shock Factor 1 ◽

Protection Mechanism ◽

Sumo Modification

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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Heat shock factor can activate transcription while bound to nucleosomal DNA in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.189-199.1994 ◽

1994 ◽

Vol 14 (1) ◽

pp. 189-199

Author(s):

D S Pederson ◽

T Fidrych

Keyword(s):

Saccharomyces Cerevisiae ◽

Heat Shock ◽

Transcription Initiation ◽

Heat Shock Factor ◽

Micrococcal Nuclease ◽

Nucleosome Assembly ◽

Heat Shock Element ◽

Yeast Saccharomyces Cerevisiae ◽

Nucleosomal Dna

After each round of replication, new transcription initiation complexes must assemble on promoter DNA. This process may compete with packaging of the same promoter sequences into nucleosomes. To elucidate interactions between regulatory transcription factors and nucleosomes on newly replicated DNA, we asked whether heat shock factor (HSF) could be made to bind to nucleosomal DNA in vivo. A heat shock element (HSE) was embedded at either of two different sites within a DNA segment that directs the formation of a stable, positioned nucleosome. The resulting DNA segments were coupled to a reporter gene and transfected into the yeast Saccharomyces cerevisiae. Transcription from these two plasmid constructions after induction by heat shock was similar in amount to that from a control plasmid in which HSF binds to nucleosome-free DNA. High-resolution genomic footprint mapping of DNase I and micrococcal nuclease cleavage sites indicated that the HSE in these two plasmids was, nevertheless, packaged in a nucleosome. The inclusion of HSE sequences within (but relatively close to the edge of) the nucleosome did not alter the position of the nucleosome which formed with the parental DNA fragment. Genomic footprint analyses also suggested that the HSE-containing nucleosome was unchanged by the induction of transcription. Quantitative comparisons with control plasmids ruled out the possibility that HSF was bound only to a small fraction of molecules that might have escaped nucleosome assembly. Analysis of the helical orientation of HSE DNA in the nucleosome indicated that HSF contacted DNA residues that faced outward from the histone octamer. We discuss the significance of these results with regard to the role of nucleosomes in inhibiting transcription and the normal occurrence of nucleosome-free regions in promoters.

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Characterization of constitutive HSF2 DNA-binding activity in mouse embryonal carcinoma cells

Molecular and Cellular Biology ◽

10.1128/mcb.14.8.5309-5317.1994 ◽

1994 ◽

Vol 14 (8) ◽

pp. 5309-5317

Author(s):

S P Murphy ◽

J J Gorzowski ◽

K D Sarge ◽

B Phillips

Keyword(s):

Transcription Factors ◽

Heat Shock ◽

Dna Binding ◽

Mammalian Cells ◽

Embryonal Carcinoma ◽

Embryonic Stem ◽

Binding Activity ◽

Hsp70 Promoter ◽

Ec Cells

Two distinct murine heat shock transcription factors, HSF1 and HSF2, have been identified. HSF1 mediates the transcriptional activation of heat shock genes in response to environmental stress, while the function of HSF2 is not understood. Both factors can bind to heat shock elements (HSEs) but are maintained in a non-DNA-binding state under normal growth conditions. Mouse embryonal carcinoma (EC) cells are the only mammalian cells known to exhibit HSE-binding activity, as determined by gel shift assays, even when maintained at normal physiological temperatures. We demonstrate here that the constitutive HSE-binding activity present in F9 and PCC4.aza.R1 EC cells, as well as a similar activity found to be present in mouse embryonic stem cells, is composed predominantly of HSF2. HSF2 in F9 EC cells is trimerized and is present at higher levels than in a variety of nonembryonal cell lines, suggesting a correlation of these properties with constitutive HSE-binding activity. Surprisingly, transcription run-on assays suggest that HSF2 in unstressed EC cells does not stimulate transcription of two putative target genes, hsp70 and hsp86. Genomic footprinting analysis indicates that HSF2 is not bound in vivo to the HSE of the hsp70 promoter in unstressed F9 EC cells, although HSF2 is present in the nucleus and the promoter is accessible to other transcription factors and to HSF1 following heat shock. Thus trimerization and nuclear localization of HSF2 do not appear to be sufficient for in vivo binding of HSF2 to the HSE of the hsp70 promoter in unstressed F9 EC cells.

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The Skn7 Response Regulator ofSaccharomyces cerevisiaeInteracts with Hsf1 In Vivo and Is Required for the Induction of Heat Shock Genes by Oxidative Stress

Molecular Biology of the Cell ◽

10.1091/mbc.11.7.2335 ◽

2000 ◽

Vol 11 (7) ◽

pp. 2335-2347 ◽

Cited By ~ 108

Author(s):

Desmond C. Raitt ◽

Anthony L. Johnson ◽

Alexander M. Erkine ◽

Kozo Makino ◽

Brian Morgan ◽

...

Keyword(s):

Oxidative Stress ◽

Heat Shock ◽

Response Regulator ◽

Coiled Coil ◽

Normal Growth ◽

Temperature Sensitive ◽

Regulatory Sequences ◽

Heat Shock Genes

The Skn7 response regulator has previously been shown to play a role in the induction of stress-responsive genes in yeast, e.g., in the induction of the thioredoxin gene in response to hydrogen peroxide. The yeast Heat Shock Factor, Hsf1, is central to the induction of another set of stress-inducible genes, namely the heat shock genes. These two regulatory trans-activators, Hsf1 and Skn7, share certain structural homologies, particularly in their DNA-binding domains and the presence of adjacent regions of coiled-coil structure, which are known to mediate protein–protein interactions. Here, we provide evidence that Hsf1 and Skn7 interact in vitro and in vivo and we show that Skn7 can bind to the same regulatory sequences as Hsf1, namely heat shock elements. Furthermore, we demonstrate that a strain deleted for the SKN7 gene and containing a temperature-sensitive mutation in Hsf1 is hypersensitive to oxidative stress. Our data suggest that Skn7 and Hsf1 cooperate to achieve maximal induction of heat shock genes in response specifically to oxidative stress. We further show that, like Hsf1, Skn7 can interact with itself and is localized to the nucleus under normal growth conditions as well as during oxidative stress.

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Constitutive binding of yeast heat shock factor to DNA in vivo

Molecular and Cellular Biology ◽

10.1128/mcb.8.11.5040-5042.1988 ◽

1988 ◽

Vol 8 (11) ◽

pp. 5040-5042

Author(s):

B K Jakobsen ◽

H R Pelham

Keyword(s):

Heat Shock ◽

Transcriptional Activation ◽

Binding Sites ◽

Heat Shock Factor ◽

Promoter Element ◽

Synthetic Promoter

We measured the binding of yeast heat shock factor (HSF) to DNA in vivo by using an interference assay in which HSF excludes GAL4 from a synthetic promoter element containing overlapping binding sites for each protein. The results show that HSF binds to DNA in unstressed cells and that binding is not sufficient for transcriptional activation.

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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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Intramolecular Repression of Mouse Heat Shock Factor 1

Molecular and Cellular Biology ◽

10.1128/mcb.18.2.906 ◽

1998 ◽

Vol 18 (2) ◽

pp. 906-918 ◽

Cited By ~ 56

Author(s):

Thomas Farkas ◽

Yulia A. Kutskova ◽

Vincenzo Zimarino

Keyword(s):

Heat Shock ◽

Transcriptional Activation ◽

Mammalian Cells ◽

Coiled Coil ◽

Heat Shock Factor ◽

Heat Shock Factor 1 ◽

Reticulocyte Lysate ◽

Heptad Repeats ◽

Intramolecular Mechanism

ABSTRACT The pathway leading to transcriptional activation of heat shock genes involves a step of heat shock factor 1 (HSF1) trimerization required for high-affinity binding of this activator protein to heat shock elements (HSEs) in the promoters. Previous studies have shown that in vivo the trimerization is negatively regulated at physiological temperatures by a mechanism that requires multiple hydrophobic heptad repeats (HRs) which may form a coiled coil in the monomer. To investigate the minimal requirements for negative regulation, in this work we have examined mouse HSF1 translated in rabbit reticulocyte lysate or extracted from Escherichia coli after limited expression. We show that under these conditions HSF1 behaves as a monomer which can be induced by increases in temperature to form active HSE-binding trimers and that mutations of either HR region cause activation in both systems. Furthermore, temperature elevations and acidic buffers activate purified HSF1, and mild proteolysis excises fragments which form HSE-binding oligomers. These results suggest that oligomerization can be repressed in the monomer, as previously proposed, and that repression can be relieved in the apparent absence of regulatory proteins. An intramolecular mechanism may be central for the regulation of this transcription factor in mammalian cells, although not necessarily sufficient.

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Presence of a Poly(A) Binding Protein and Two Proteins with Cell Cycle-Dependent Phosphorylation in Crithidia fasciculata mRNA Cycling Sequence Binding Protein II

Eukaryotic Cell ◽

10.1128/ec.3.5.1185-1197.2004 ◽

2004 ◽

Vol 3 (5) ◽

pp. 1185-1197 ◽

Cited By ~ 22

Author(s):

Bidyottam Mittra ◽

Dan S. Ray

Keyword(s):

Cell Cycle ◽

Binding Protein ◽

High Specificity ◽

Binding Activity ◽

Mrna Levels ◽

Crithidia Fasciculata ◽

Target Mrna ◽

Cycle Dependent

ABSTRACT Crithidia fasciculata cycling sequence binding proteins (CSBP) have been shown to bind with high specificity to sequence elements present in several mRNAs that accumulate periodically during the cell cycle. The first described CSBP has subunits of 35.6 (CSBPA) and 42 kDa (CSBPB). A second distinct binding protein termed CSBP II has been purified from CSBPA null mutant cells, lacking both CSBPA and CSBPB proteins, and contains three major polypeptides with predicted molecular masses of 63, 44.5, and 33 kDa. Polypeptides of identical size were radiolabeled in UV cross-linking assays performed with purified CSBP II and 32P-labeled RNA probes containing six copies of the cycling sequence. The CSBP II binding activity was found to cycle in parallel with target mRNA levels during progression through the cell cycle. We have cloned genes encoding these three CSBP II proteins, termed RBP63, RBP45, and RBP33, and characterized their binding properties. The RBP63 protein is a member of the poly(A) binding protein family. Homologs of RBP45 and RBP33 proteins were found only among the kinetoplastids. Both RBP45 and RBP33 proteins and their homologs have a conserved carboxy-terminal half that contains a PSP1-like domain. All three CSBP II proteins show specificity for binding the wild-type cycling sequence in vitro. RBP45 and RBP33 are phosphoproteins, and RBP45 has been found to bind in vivo specifically to target mRNA containing cycling sequences. The levels of phosphorylation of both RBP45 and RBP33 were found to cycle during the cell cycle.

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Identification of HiNF-P, a Key Activator of Cell Cycle-Controlled Histone H4 Genes at the Onset of S Phase

Molecular and Cellular Biology ◽

10.1128/mcb.23.22.8110-8123.2003 ◽

2003 ◽

Vol 23 (22) ◽

pp. 8110-8123 ◽

Cited By ~ 42

Author(s):

Partha Mitra ◽

Rong-Lin Xie ◽

Ricardo Medina ◽

Hayk Hovhannisyan ◽

S. Kaleem Zaidi ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Cyclin E ◽

S Phase ◽

Phase Cell ◽

Regulatory Sequences ◽

Histone H4 ◽

Restriction Point ◽

Histone H4 Gene

ABSTRACT At the G1/S phase cell cycle transition, multiple histone genes are expressed to ensure that newly synthesized DNA is immediately packaged as chromatin. Here we have purified and functionally characterized the critical transcription factor HiNF-P, which is required for E2F-independent activation of the histone H4 multigene family. Using chromatin immunoprecipitation analysis and ligation-mediated PCR-assisted genomic sequencing, we show that HiNF-P interacts with conserved H4 cell cycle regulatory sequences in vivo. Antisense inhibition of HiNF-P reduces endogenous histone H4 gene expression. Furthermore, we find that HiNF-P utilizes NPAT/p220, a substrate of the cyclin E/cyclin-dependent kinase 2 (CDK2) kinase complex, as a key coactivator to enhance histone H4 gene transcription. The biological role of HiNF-P is reflected by impeded cell cycle progression into S phase upon antisense-mediated reduction of HiNF-P levels. Our results establish that HiNF-P is the ultimate link in a linear signaling pathway that is initiated with the growth factor-dependent induction of cyclin E/CDK2 kinase activity at the restriction point and culminates in the activation of histone H4 genes through HiNF-P at the G1/S phase transition.

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