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Dynamic Chromatin Alterations Triggered by Natural and Synthetic Activation Domains*

  • Alexander M. Erkine
    Correspondence
    To whom correspondence should be addressed: Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA 71130-3932. Tel.: 318-675-8204; Fax: 318-675-5180;
    Affiliations
    From the Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130
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  • David S. Gross
    Affiliations
    From the Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130
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  • Author Footnotes
    * This work was supported by National Institute of General Medical Sciences Grant GM45842 and the Center for Excellence in Cancer Research at Louisiana State University Health Sciences Center, Shreveport, LA (to D. S. G.) and by National Science Foundation Grant MCB-0215758 (to A. M. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:December 23, 2002DOI:https://doi.org/10.1074/jbc.M211703200
      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 ofSaccharomyces 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 thets− 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 bothtrans-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.
      AD
      activation domain
      HSF
      heat shock factor
      NTA
      N-terminal activator
      CTA
      C-terminal activator
      PIC
      preinitiation complex
      ChIP
      chromatin immunoprecipitation
      YPD
      yeast extract/peptone/dextrose
      WT
      wild type
      ORF
      open reading frame
      Despite major advances in characterizing components of the eukaryotic transcription machinery, the mechanisms by which these components are recruited to gene promoters are poorly understood. Activation domains (ADs)1 of gene-specific transcription factors are critical for these recruitment steps. ADs are believed to work by directly contacting coactivators, components of the mediator complex and basal transcriptional machinery, and polymerase II holoenzyme itself (reviewed in Refs.
      • Ptashne M.
      • Gann A.
      ,
      • Struhl K.
      ,
      • Kingston R.E.
      • Narlikar G.J.
      ,
      • Strahl B.D.
      • Allis C.D.
      ,
      • Brown C.E.
      • Lechner T.
      • Howe L.
      • Workman J.L.
      ,
      • Naar A.M.
      • Lemon B.D.
      • Tjian R.
      )). These interactions have been previously shown to occur in vitro or in whole cell lysates. In certain cases, the biochemical data have been buttressed by mutational analyses showing a correlation between the change in the affinity of these contacts and transcriptional activity in vivo (
      • Koh S.S.
      • Ansari A.Z.
      • Ptashne M.
      • Young R.A.
      ,
      • Neely K.E.
      • Hassan A.H.
      • Wallberg A.E.
      • Steger D.J.
      • Cairns B.R.
      • Wright A.P.
      • Workman J.L.
      ,
      • Natarajan K.
      • Jackson B.M.
      • Zhou H.
      • Winston F.
      • Hinnebusch A.G.
      ,
      • Utley R.T.
      • Ikeda K.
      • Grant P.A.
      • Cote J.
      • Steger D.J.
      • Eberharter A.
      • John S.
      • Workman J.L.
      ,
      • Neely K.E.
      • Hassan A.H.
      • Brown C.E.
      • Howe L.
      • Workman J.L.
      ,
      • Brown C.E.
      • Howe L.
      • Sousa K.
      • Alley S.C.
      • Carrozza M.J.
      • Tan S.
      • Workman J.L.
      ). Recent studies indicate that recruitment of coactivators is often sequential and gene-specific (
      • Cosma M.P.
      • Tanaka T.
      • Nasmyth K.
      ,
      • Krebs J.E.
      • Fry C.J.
      • Samuels M.L.
      • Peterson C.L.
      ,
      • Agalioti T.
      • Lomvardas S.
      • Parekh B.
      • Yie J.
      • Maniatis T.
      • Thanos D.
      ). Despite these advances, there is little clarity of how multiple factors are recruited to a given promoter or what determines their order of recruitment.
      An important quality of ADs is that they can substitute for one another not only between transcription factors of a given species, but also between transcription factors from such diverse eukaryotes as human, fungi, and plants. This suggests some uniformity in the mechanism(s) by which ADs work and implies that ADs share sequence or structural homology. In fact, although most ADs fall into one of three categories, acidic, glutamine-rich, or proline-rich, there is no consensus sequence or clear homology, even within a particular class. Moreover, ADs often lack any discernable secondary structure (
      • Ptashne M.
      • Gann A.
      ,
      • Triezenberg S.J.
      ,
      • Uesugi M.
      • Nyanguile O.
      • Lu H.
      • Levine A.J.
      • Verdine G.L.
      ,
      • Cho H.S.
      • Liu C.W.
      • Damberger F.F.
      • Pelton J.G.
      • Nelson H.C.
      • Wemmer D.E.
      ). The conservation of mechanism in the absence of any sequence or structural homology is paradoxical and reflects a gap in our understanding of transcriptional regulatory mechanisms.
      Recruitment of ATP-dependent nucleosome remodeling and histone modifying enzymes to gene promoters is especially important since chromatin remodeling is a fundamental prerequisite to transcriptional activation. It has been shown that yeast Swi/Snf and histone acetyltransferase-containing complexes interact in vitro with a variety of transcription activators, including Gal4, Gcn4, Swi5, and Gal4-VP16 (
      • Koh S.S.
      • Ansari A.Z.
      • Ptashne M.
      • Young R.A.
      ,
      • Neely K.E.
      • Hassan A.H.
      • Wallberg A.E.
      • Steger D.J.
      • Cairns B.R.
      • Wright A.P.
      • Workman J.L.
      ,
      • Natarajan K.
      • Jackson B.M.
      • Zhou H.
      • Winston F.
      • Hinnebusch A.G.
      ,
      • Utley R.T.
      • Ikeda K.
      • Grant P.A.
      • Cote J.
      • Steger D.J.
      • Eberharter A.
      • John S.
      • Workman J.L.
      ). Recruitment of the SAGA complex by ADs of Gal4 and Gal4-VP16 has also been demonstrated in vivo(
      • Bhaumik S.R.
      • Green M.R.
      ,
      • Larschan E.
      • Winston F.
      ). Recent investigations employing photo-cross-linking label transfer methodology identified the Tra1 subunit of SAGA and three subunits of the Swi/Snf complex (Snf5, Swi1, and Swi2/Snf2) as primary targets of a number of ADs (
      • Neely K.E.
      • Hassan A.H.
      • Brown C.E.
      • Howe L.
      • Workman J.L.
      ,
      • Brown C.E.
      • Howe L.
      • Sousa K.
      • Alley S.C.
      • Carrozza M.J.
      • Tan S.
      • Workman J.L.
      ). In these studies a strong correlation was observed between the level of transcription and strength of AD-target interactions for mutated ADs. Yet the multiplicity of targets capable of interacting with the same AD suggests the existence of a hierarchy of recruitment steps in vivo, and how such a hierarchy might be established is unknown.
      Heat shock factor (HSF) is a key transcriptional activator of stress-responsive genes in yeast. It is also responsible for establishing the constitutive DNase I hypersensitive chromatin structures within the promoter regions of at least two genes,HSP82 and HSC82 (
      • Gross D.S.
      • Adams C.C.
      • Lee S.
      • Stentz B.
      ,
      • Erkine A.M.
      • Adams C.C.
      • Diken T.
      • Gross D.S.
      ). As such, it is likely that HSF recruits nucleosome-remodeling and histone-modifying activities to target promoters using its ADs. Yeast HSF is comprised of a highly conserved core, consisting of DNA-binding and trimerization domains, and two activation domains, one located at the N terminus and the other at the C terminus (Fig. 1). The N-terminal activator (NTA) has been shown to be critical for the transient heat shock response of the cell, while the C-terminal activator (CTA) is important for sustained stimuli, and its deletion results in lethality at elevated temperatures (
      • Sorger P.K.
      ,
      • Nieto-Sotelo J.
      • Wiederrecht G.
      • Okuda A.
      • Parker C.S.
      ). As HSF has been shown to bypass the requirement for a number of key PIC components, including TFIIA, TAF17, Srb4, Srb6, Kin28, and the C-terminal domain of the large subunit of polymerase II (
      • Apone L.M.
      • Virbasius C.A.
      • Holstege F.C.
      • Wang J.
      • Young R.A.
      • Green M.R.
      ,
      • Lee D.
      • Lis J.T.
      ,
      • Lee D.K.
      • Kim S.
      • Lis J.T.
      ,
      • McNeil J.B.
      • Agah H.
      • Bentley D.
      ,
      • Moqtaderi Z.
      • Keaveney M.
      • Struhl K.
      ,
      • Chou S.
      • Chatterjee S.
      • Lee M.
      • Struhl K.
      ), the targets of its ADs may extend beyond the typical repertoire of most yeast activators (
      • Lee D.K.
      • Kim S.
      • Lis J.T.
      ).
      Figure thumbnail gr1
      Figure 1Map of yeast HSF functional domains (compilation from Refs.
      • Sorger P.K.
      ,
      • Nieto-Sotelo J.
      • Wiederrecht G.
      • Okuda A.
      • Parker C.S.
      ,
      • Flick K.E.
      • Gonzalez Jr., L.
      • Harrison C.J.
      • Nelson H.C.
      ,
      • Hoj A.
      • Jakobsen B.K.
      ,
      • Harrison C.J.
      • Bohm A.A.
      • Nelson H.C.
      ,
      • Sorger P.K.
      • Nelson H.C.
      ). CE2, implicated in repression of CTA.
      In this work, we use chromatin immunoprecipitation (ChIP) to provide evidence for dynamic alterations of chromatin structure at heat shock promoters upon induction. Similar changes in histone content are observed at two promoters regulated by the Gal4 activator,GAL1 and GAL7. For both sets of genes, chromatin remodeling depends on the presence of an AD in the corresponding gene-specific activator. Using a genetic screen, we found that the temperature sensitivity associated with deletion of the HSF CTA can be alleviated by substituting short synthetic peptides enriched in hydrophobic and acidic residues for the large, native C-terminal domain. These peptides also function as ADs when tethered to the heterologous Gal4 DNA-binding domain. At least some of them can trigger histone modifications at Gal4-regulated promoters. The nature of these peptides suggests that their interacting targets likely are hydrophobic and basic, pointing toward histones as possible targets. Consistent with this notion, we found that the ts phenotype resulting from a CTA deletion can be rescued by fusing short histone-binding modules, derived from either human CTF1/NF1 or the mouse liver-specific activator HNF3, to HSF(1–493).

      Acknowledgments

      We thank Michael Hampsey, Brett Keiper, Neal Mathias, Lucy Robinson, Sergey Slepenkov, and Kelly Tatchell for helpful discussions, and Yves Dusserre, Philip James, Nicolas Mermod, Peter Sorger, Robert Tjian, and Kenneth Zaret for gifts of strains and plasmids.

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