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ABSTRACT: The evolutionarily conserved Mediator complex is central to the regulation of gene transcription in eukaryotes, for it serves as a physical and functional interface between upstream regulators and the Pol II transcriptional machinery. Nonetheless, its role appears to be context dependent and the detailed mechanism by which it governs the expression of most genes remains unknown. Here we investigate Mediator involvement in Heat Shock Protein (HSP) gene regulation in the yeast Saccharomyces cerevisiae. We find that in response to thermal upshift, subunits representative of each of the four Mediator modules - Head, Middle, Tail and Kinase - are rapidly, robustly and selectively recruited to the promoter regions of HSP genes. Their residence is transient, returning to near-background levels within 90 min. Heat Shock Factor 1 (Hsf1) plays a central role in recruiting Mediator as indicated by the fact that truncation of either its N- or C-terminal activation domain significantly reduces Mediator occupancy, while removal of both activation domains abolishes it. Likewise, ablation of either of two Mediator Tail subunits, Med15 or Med16, reduces Mediator recruitment to HSP promoters, while deletion of both abolishes it. Accompanying loss of Mediator, recruitment of RNA Pol II is substantially diminished. Interestingly, Mediator antagonizes Hsf1's constitutive occupancy of non-induced promoters yet facilitates enhanced Hsf1 association with activated ones. Collectively, our observations indicate that Hsf1, via its dual activation domains, recruits holo-Mediator to HSP promoters in response to acute heat stress through cooperative physical and/or functional interactions with the Tail module.
Journal of Biological Chemistry 02/2013; · 4.77 Impact Factor
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ABSTRACT: Mediator is a modular multisubunit complex that functions as a critical coregulator of RNA polymerase II (Pol II) transcription. While it is well accepted that Mediator plays important roles in the assembly and function of the preinitiation complex (PIC), less is known of its potential roles in regulating downstream steps of the transcription cycle. Here we use a combination of genetic and molecular approaches to investigate Mediator regulation of Pol II elongation in the model eukaryote, Saccharomyces cerevisiae. We find that ewe (expression without heat shock element) mutations in conserved Mediator subunits Med7, Med14, Med19, and Med21-all located within or adjacent to the middle module-severely diminish heat-shock-induced expression of the Hsf1-regulated HSP82 gene. Interestingly, these mutations do not impede Pol II recruitment to the gene's promoter but instead impair its transit through the coding region. This implies that a normal function of Mediator is to regulate a postinitiation step at HSP82. In addition, displacement of histones from promoter and coding regions, a hallmark of activated heat-shock genes, is significantly impaired in the med14 and med21 mutants. Suggestive of a more general role, ewe mutations confer hypersensitivity to the anti-elongation drug 6-azauracil (6-AU) and one of them-med21-impairs Pol II processivity on a GAL1-regulated reporter gene. Taken together, our results suggest that yeast Mediator, acting principally through its middle module, can regulate Pol II elongation at both heat-shock and non-heat-shock genes.
Genetics 02/2012; 191(1):95-106. · 4.01 Impact Factor
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ABSTRACT: The tumor suppressor p53 principally functions as a gene-specific transcription factor. p53 triggers a variety of anti-proliferative programs by activating or repressing the transcription of effector genes in response to genotoxic stress. To date, much effort has been placed on understanding p53's ability to affect transcription in the context of its DNA-binding activity. How p53 regulates transcriptional output independent of DNA binding is less well understood. Here we provide evidence that human p53 can physically interact with the large subunit of RNA polymerase II (Pol II) both in in vitro interaction assays and in whole cell extracts, and that this interaction is mediated (at least in part) through p53's core DNA-binding domain and the Ser5-phosphorylated CTD of Pol II. Ectopic expression of p53, combined with mutations in transcription elongation factors or exposure to drugs that inhibit Pol II elongation, elicit sickness or lethality in yeast cells. These phenotypes are suppressed by oncogenic point mutations within p53's core domain. The growth phenotypes raise the possibility that p53 impairs Pol II elongation. Consistent with this, a p53-dependent increase in Pol II density is seen at constitutively expressed genes without a concomitant increase in transcript accumulation. Additionally, p53-expressing yeast strains exhibit reduced transcriptional processivity at an episomal reporter gene; this inhibitory activity is abolished by a core domain point mutation. Our results suggest a novel mechanism by which p53 can regulate gene transcription, and a new biological function for its core domain that is susceptible to inactivation by oncogenic point mutations.
PLoS ONE 01/2011; 6(8):e22183. · 4.09 Impact Factor
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ABSTRACT: The chromatin structure of heat shock protein (HSP)-encoding genes undergoes dramatic alterations upon transcriptional induction, including, in extreme cases, domain-wide nucleosome disassembly. Here, we use a combination of gene knock-out, in situ mutagenesis, chromatin immunoprecipitation, and expression assays to investigate the role of histone modification complexes in regulating heat shock gene structure and expression in Saccharomyces cerevisiae. Two histone acetyltransferases, Gcn5 and Esa1, were found to stimulate HSP gene transcription. A detailed chromatin immunoprecipitation analysis of the Gcn5-containing SAGA complex (signified by Spt3) revealed its presence within the promoter of every heat shock factor 1-regulated gene examined. The occupancy of SAGA increased substantially upon heat shock, peaking at several HSP promoters within 30-45 s of temperature upshift. SAGA was also efficiently recruited to the coding regions of certain HSP genes (where its presence mirrored that of pol II), although not at others. Robust and rapid recruitment of repressive, Rpd3-containing histone deacetylase complexes was also seen and at all HSP genes examined. A detailed analysis of HSP82 revealed that both Rpd3(L) and Rpd3(S) complexes (signified by Sap30 and Rco1, respectively) were recruited to the gene promoter, yet only Rpd3(S) was recruited to its open reading frame. A consensus URS1 cis-element facilitated the recruitment of each Rpd3 complex to the HSP82 promoter, and this correlated with targeted deacetylation of promoter nucleosomes. Collectively, our observations reveal that SAGA and Rpd3 complexes are rapidly and synchronously recruited to heat shock factor 1-activated genes and suggest that their opposing activities modulate heat shock gene chromatin structure and fine-tune transcriptional output.
Journal of Biological Chemistry 09/2009; 284(47):32914-31. · 4.77 Impact Factor
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ABSTRACT: It is well accepted that for transcriptional silencing in budding yeast, the evolutionarily conserved lysine deacetylase Sir2, in concert with its partner proteins Sir3 and Sir4, establishes a chromatin structure that prevents RNA polymerase II (Pol II) transcription. However, the mechanism of repression remains controversial. Here, we show that the recruitment of Pol II, as well as that of the general initiation factors TBP and TFIIH, occurs unimpeded to the silent HMRa1 and HMLalpha1/HMLalpha2 mating promoters. This, together with the fact that Pol II is Ser5 phosphorylated, implies that SIR-mediated silencing is permissive to both preinitiation complex (PIC) assembly and transcription initiation. In contrast, the occupancy of factors critical to both mRNA capping and Pol II elongation, including Cet1, Abd1, Spt5, Paf1C, and TFIIS, is virtually abolished. In agreement with this, efficiency of silencing correlates not with a restriction in Pol II promoter occupancy but with a restriction in capping enzyme recruitment. These observations pinpoint the transition between polymerase initiation and elongation as the step targeted by Sir2 and indicate that transcriptional silencing is achieved through the differential accessibility of initiation and capping/elongation factors to chromatin. We compare Sir2-mediated transcriptional silencing to a second repression mechanism, mediated by Tup1. In contrast to Sir2, Tup1 prevents TBP, Pol II, and TFIIH recruitment to the HMLalpha1 promoter, thereby abrogating PIC formation.
Molecular and cellular biology 07/2008; 28(12):3979-94. · 6.06 Impact Factor
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ABSTRACT: Silent chromatin in budding yeast is characterized by the presence of a specialized chromatin modification complex consisting of silent information regulator (Sir) proteins, closely packed pairs of nucleosomes, and hypoacetylated and hypomethylated histones. How this specialized chromatin is established, maintained and inherited has been extensively studied. Less investigated are the determinants that constrain its linear spread along the chromatin fibre and the manner by which it represses gene transcription. Here we review the essential features of SIR-mediated heterochromatin, and discuss genomic and proteomic approaches for discerning the composition of its boundaries and for elucidating the mechanisms by which it silences transcription.
Briefings in Functional Genomics and Proteomics 01/2007; 5(4):280-8.
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ABSTRACT: We report the results of a genetic screen designed to identify transcriptional coregulators of yeast heat-shock factor (HSF). This sequence-specific activator is required to stimulate both basal and induced transcription; however, the identity of factors that collaborate with HSF in governing noninduced heat-shock gene expression is unknown. In an effort to identify these factors, we isolated spontaneous extragenic suppressors of hsp82-deltaHSE1, an allele of HSP82 that bears a 32-bp deletion of its high-affinity HSF-binding site, yet retains its two low-affinity HSF sites. Nearly 200 suppressors of the null phenotype of hsp82-deltaHSE1 were isolated and characterized, and they sorted into six expression without heat-shock element (EWE) complementation groups. Strikingly, all six groups contain alleles of genes that encode subunits of Mediator. Three of the six subunits, Med7, Med10/Nut2, and Med21/Srb7, map to Mediator's middle domain; two subunits, Med14/Rgr1 and Med16/Sin4, to its tail domain; and one subunit, Med19/Rox3, to its head domain. Mutations in genes encoding these factors enhance not only the basal transcription of hsp82-deltaHSE1, but also that of wild-type heat-shock genes. In contrast to their effect on basal transcription, the more severe ewe mutations strongly reduce activated transcription, drastically diminishing the dynamic range of heat-shock gene expression. Notably, targeted deletion of other Mediator subunits, including the negative regulators Cdk8/Srb10, Med5/Nut1, and Med15/Gal11 fail to derepress hsp82-deltaHSE1. Taken together, our data suggest that the Ewe subunits constitute a distinct functional module within Mediator that modulates both basal and induced heat-shock gene transcription.
Genetics 05/2006; 172(4):2169-84. · 4.01 Impact Factor
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ABSTRACT: We show that histone-DNA interactions are disrupted across entire yeast heat shock genes upon their transcriptional activation. At HSP82, nucleosomal disassembly spans a domain of approximately 3 kb, beginning upstream of the promoter and extending through the transcribed region. A kinetic analysis reveals that histone H4 loses contact with DNA within 45 s of thermal upshift. Nucleosomal reassembly, prompted by temperature downshift, is also rapid, detectable within 60 s. Prior to their eviction, promoter-associated histones are transiently hyperacetylated, while those in the coding region are not. An upstream activation sequence mutation that weakens the binding of heat shock factor obviates domain-wide remodeling, while deletion of the TATA box that nearly abolishes transcription is permissive to 5'-end remodeling. The Swi/Snf complex is rapidly recruited to HSP82 upon heat shock. Nonetheless, domain-wide remodeling occurs efficiently in Swi/Snf mutants despite a sixfold reduction in transcription; it is also seen in gcn5Delta, set1Delta, and paf1Delta mutants. Contrary to current models, we demonstrate that a high density of RNA polymerase (Pol) is insufficient to elicit histone displacement. This finding suggests that histone eviction is modulated by factors that are not linked to elongating Pol II. It further suggests that histone depletion plays a causal role in mediating vigorous transcription in vivo and is not merely a consequence of it.
Molecular and Cellular Biology 11/2005; 25(20):8985-99. · 5.53 Impact Factor
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ABSTRACT: Transcriptional silencing in budding yeast and fruit fly is mediated by fundamentally unrelated proteins that assemble very different chromatin structures. Surprisingly, the repressive mechanisms evolved from these very different materials have similar features, including an epigenetic mode of inheritance and a block to transcription based on interference with the assembly or function of the promoter complex rather than with the binding of gene-specific activators.
Molecular Cell 06/2005; 18(4):395-8. · 14.18 Impact Factor
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ABSTRACT: A pathological feature of Parkinson's disease is the presence of Lewy bodies within selectively vulnerable neurons. These are ubiquitinated cytoplasmic inclusions containing alpha-synuclein, an abundant protein normally associated with presynaptic terminals. Point mutations in the alpha-synuclein gene (A30P and A53T), as well as triplication of the wild-type (WT) locus, have been linked to autosomal dominant Parkinson's. How these alterations might contribute to disease progression is unclear. Using the genetically tractable yeast Saccharomyces cerevisiae as a model system, we find that both the WT and the A53T isoforms of alpha-synuclein initially localize to the plasma membrane, to which they are delivered via the classical secretory pathway. In contrast, the A30P mutant protein disperses within the cytoplasm and does not associate with the plasma membrane, and its intracellular distribution is unaffected by mutations in the secretory pathway. When their expression is elevated, WT and A53T, but not A30P, are toxic to cells. At moderate levels of expression, WT and A53T induce the cellular stress (heat-shock) response and are toxic to cells bearing mutations in the 20S proteasome. Our results reveal a link between plasma membrane targeting of alpha-synuclein and its toxicity in yeast and suggest a role for the quality control (QC) system in the cell's effort to deal with this natively unfolded protein.
Genetics 06/2005; 170(1):47-59. · 4.01 Impact Factor
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ABSTRACT: The activation domains (ADs) of transcription activators recruit a multiplicity of enzymatic activities to gene promoters. The mechanisms by which such recruitment takes place are not well understood. Using chromatin immunoprecipitation, we demonstrate dynamic alterations in the abundance of histones H2A, H3, and H4 at promoters of genes regulated by the HSF and Gal4 activators of Saccharomyces cerevisiae. Transcriptional activation of these genes, particularly those regulated by HSF, is accompanied by a significant reduction in both acetylated and unacetylated histones at promoters and may involve the transient displacement of histone octamers. To gain insight into the function of ADs, we conducted a genetic screen to identify polypeptides that could substitute for the 340-residue C-terminal activator of HSF and rescue the temperature sensitivity caused by its deletion. We found that the ts(-) phenotype of HSF(1-493) could be complemented by peptides as short as 11 amino acids. Such peptides are enriched in acidic and hydrophobic residues, and exhibit both trans-activating and chromatin-modifying activities when fused to the Gal4 DNA-binding domain. We also demonstrate that a previously identified 14-amino acid histone H3-binding module of human CTF1/NF1, which is similar to synthetic ADs, can substitute for the HSF C-terminal activator in conferring temperature resistance and can mediate the modification of promoter chromatin structure. Possible mechanisms of AD function, including one involving direct interactions with histones, are discussed.
Journal of Biological Chemistry 04/2003; 278(10):7755-64. · 4.77 Impact Factor
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ABSTRACT: In this review, we discuss recent evidence implicating chromatin structure in the etiology of cancer. In particular, we present evidence indicating that inappropriate regulation of chromatin structure inhibits normal cell differentiation pathways and stimulates uncontrolled cell proliferation, with the outcome being oncogenesis. Such inappropriate chromatin structures arise as a consequence of (i) chromosomal rearrangements that fuse gene-specific activators with global co-regulators, drastically altering activator function; (ii) hypermethylation of tumor suppressor gene promoters, resulting in their inactivation; or (iii) mistargeted nuclear compartmentalization of growth-control genes and their regulators, resulting in the up- or down-regulation of such genes. How does chromatin silence genes? Recent results from model in vivo systems argues that chromatin can repress transcription at two levels: (i) by sterically interfering with the binding of transcription factors to the promoter, thereby blocking initiation; and (ii) at a step subsequent to the binding of activators and recruitment of the preinitiation complex. J. Cell. Biochem. Suppl. 35:61–68, 2000. © 2001 Wiley-Liss, Inc.
Journal of Cellular Biochemistry 05/2001; 79(S35):61 - 68. · 2.87 Impact Factor
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ABSTRACT: We present the upstream sequences of HSP82 and HSC82, two closely related, but differentially regulated, heat-shock genes of Saccharomyces cerevisiae. Several dozen potential regulatory elements are identified within each upstream region; interestingly, only a few are conserved between the two genes. These include a consensus heat-shock element, an upstream repressor element, and a consensus TATA element. A search for motifs known actively to position nucleosomes in vitro revealed that such sequences are three- to seven-fold enriched within each promoter; a comparable enrichment is seen near the 3′ end of each transcription unit. Located ∼ 1100 bp upstream of HSC82 is an open reading frame (ORF) of 255 amino acids; ∼ 800 bp upstream of HSP82 is an ORF of 132 amino acids. The latter ORF contains several conserved ankyrin motifs and appears to be expressed under normal growth conditions. Finally, we show by clamped homogeneous electric field gel electrophoresis that the two genetic loci map to different chromosomes: HSP82 to chromosome XVI and HSC82 to chromosome XIII. The sequences have been deposited in the GenBank database under Accession Numbers U20323 and U20349.
Yeast 04/1995; 11(6):573 - 580. · 1.89 Impact Factor
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