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1
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Sicoli G, Vezin H, Ledolter K, Kress T, Kurzbach D. Conformational tuning of a DNA-bound transcription factor. Nucleic Acids Res 2019; 47:5429-5435. [PMID: 31020309 PMCID: PMC6547406 DOI: 10.1093/nar/gkz291] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2018] [Revised: 04/05/2019] [Accepted: 04/10/2019] [Indexed: 11/14/2022] Open
Abstract
Transcription factors are involved in many cellular processes that take place remote from their cognate DNA sequences. The efficiencies of these activities are thus in principle counteracted by high binding affinities of the factors to their cognate DNAs. Models such as facilitated diffusion or dissociation address this apparent contradiction. We show that the MYC associated transcription factor X (MAX) undergoes nanoscale conformational fluctuations in the DNA-bound state, which is consistent with facilitated dissociation from or diffusion along DNA strands by transiently reducing binding energies. An integrative approach involving EPR, NMR, crystallographic and molecular dynamics analyses demonstrates that the N-terminal domain of MAX constantly opens and closes around a bound DNA ligand thereby dynamically tuning the binding epitope and the mode of interaction.
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Affiliation(s)
- Giuseppe Sicoli
- Univ. Lille, CNRS, UMR 8516 - LASIR - Laboratoire de Spectrochimie Infrarouge et Raman, F-59000 Lille, France
| | - Hervé Vezin
- Univ. Lille, CNRS, UMR 8516 - LASIR - Laboratoire de Spectrochimie Infrarouge et Raman, F-59000 Lille, France
| | - Karin Ledolter
- University Vienna, Department for Structural and Computational Biology, Max F. Perutz Laboratories, Campus Vienna BioCenter 5, 1030 Vienna, Austria
| | - Thomas Kress
- Laboratoire des biomolécules, LBM, Département de chimie, École normale supérieure, PSL University, Sorbonne Université, CNRS, 75005 Paris, France.,University of Vienna, Faculty of Chemistry, Institute of Biological Chemistry, Währinger Str. 38, 1090 Vienna, Austria
| | - Dennis Kurzbach
- University of Vienna, Faculty of Chemistry, Institute of Biological Chemistry, Währinger Str. 38, 1090 Vienna, Austria
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2
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Somlyay M, Ledolter K, Kitzler M, Sandford G, Cobb SL, Konrat R. 19 F NMR Spectroscopy Tagging and Paramagnetic Relaxation Enhancement-Based Conformation Analysis of Intrinsically Disordered Protein Complexes. Chembiochem 2019; 21:696-701. [PMID: 31529763 DOI: 10.1002/cbic.201900453] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2019] [Indexed: 11/06/2022]
Abstract
The combination of 19 F NMR spectroscopy tagging and paramagnetic relaxation enhancement (PRE) NMR spectroscopy experiments was evaluated as a versatile method to probe protein-protein interactions and conformational changes of intrinsically disordered proteins upon complex formation. The feasibility of the approach is illustrated with an application to the Myc-Max protein complex; this is an oncogenic transcription factor that binds enhancer box DNA fragments. The single cysteine residue of Myc was tagged with highly fluorinated [19 F]3,5-bis(trifluoromethyl)benzyl bromide. Structural dynamics of the protein complex were monitored through intermolecular PREs between 19 F-Myc and paramagnetic (1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)methanethiosulfonate (MTSL)-tagged) Max. The 19 F R2 relaxation rates obtained with three differently MTSL-tagged Max mutants revealed novel insights into the differential structural dynamics of Myc-Max bound to DNA and the tumour suppressor breast cancer antigen 1. Given its ease of implementation, fruitful applications of this strategy to structural biology and inhibitor screening can be envisaged.
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Affiliation(s)
- Máté Somlyay
- Department of Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, Campus Vienna Biocenter 5, 1030, Vienna, Austria
| | - Karin Ledolter
- Department of Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, Campus Vienna Biocenter 5, 1030, Vienna, Austria
| | - Manuel Kitzler
- Department of Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, Campus Vienna Biocenter 5, 1030, Vienna, Austria
| | - Graham Sandford
- Department of Chemistry, Durham University, Stockton Road, DH1 3LE, Durham, UK
| | - Steven L Cobb
- Department of Chemistry, Durham University, Stockton Road, DH1 3LE, Durham, UK
| | - Robert Konrat
- Department of Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, Campus Vienna Biocenter 5, 1030, Vienna, Austria
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3
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Zhu Q, Liu N, Orkin SH, Yuan GC. CUT&RUNTools: a flexible pipeline for CUT&RUN processing and footprint analysis. Genome Biol 2019; 20:192. [PMID: 31500663 PMCID: PMC6734249 DOI: 10.1186/s13059-019-1802-4] [Citation(s) in RCA: 91] [Impact Index Per Article: 15.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2019] [Accepted: 08/26/2019] [Indexed: 01/20/2023] Open
Abstract
We introduce CUT&RUNTools as a flexible, general pipeline for facilitating the identification of chromatin-associated protein binding and genomic footprinting analysis from antibody-targeted CUT&RUN primary cleavage data. CUT&RUNTools extracts endonuclease cut site information from sequences of short-read fragments and produces single-locus binding estimates, aggregate motif footprints, and informative visualizations to support the high-resolution mapping capability of CUT&RUN. CUT&RUNTools is available at https://bitbucket.org/qzhudfci/cutruntools/ .
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Affiliation(s)
- Qian Zhu
- Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute and Harvard Chan School of Public Health, Boston, MA, USA
| | - Nan Liu
- Cancer and Blood Disorders Center, Dana-Farber Cancer Institute and Boston Children's Hospital, Harvard Medical School, Boston, MA, USA
| | - Stuart H Orkin
- Cancer and Blood Disorders Center, Dana-Farber Cancer Institute and Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.
- Howard Hughes Medical Institute, Boston, MA, USA.
| | - Guo-Cheng Yuan
- Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute and Harvard Chan School of Public Health, Boston, MA, USA.
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4
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Scovell WM. High mobility group protein 1: A collaborator in nucleosome dynamics and estrogen-responsive gene expression. World J Biol Chem 2016; 7:206-222. [PMID: 27247709 PMCID: PMC4877529 DOI: 10.4331/wjbc.v7.i2.206] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/03/2015] [Revised: 02/19/2016] [Accepted: 03/14/2016] [Indexed: 02/05/2023] Open
Abstract
High mobility group protein 1 (HMGB1) is a multifunctional protein that interacts with DNA and chromatin to influence the regulation of transcription, DNA replication and repair and recombination. We show that HMGB1 alters the structure and stability of the canonical nucleosome (N) in a nonenzymatic, adenosine triphosphate-independent manner. As a result, the canonical nucleosome is converted to two stable, physically distinct nucleosome conformers. Although estrogen receptor (ER) does not bind to its consensus estrogen response element within a nucleosome, HMGB1 restructures the nucleosome to facilitate strong ER binding. The isolated HMGB1-restructured nucleosomes (N’ and N’’) remain stable and exhibit a number of characteristics that are distinctly different from the canonical nucleosome. These findings complement previous studies that showed (1) HMGB1 stimulates in vivo transcriptional activation at estrogen response elements and (2) knock down of HMGB1 expression by siRNA precipitously reduced transcriptional activation. The findings indicate that a major facet of the mechanism of HMGB1 action involves a restructuring of aspects of the nucleosome that appear to relax structural constraints within the nucleosome. The findings are extended to reveal the differences between ER and the other steroid hormone receptors. A working proposal outlines mechanisms that highlight the multiple facets that HMGB1 may utilize in restructuring the nucleosome.
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5
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Pioneer transcription factors target partial DNA motifs on nucleosomes to initiate reprogramming. Cell 2015; 161:555-568. [PMID: 25892221 DOI: 10.1016/j.cell.2015.03.017] [Citation(s) in RCA: 573] [Impact Index Per Article: 57.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2014] [Revised: 12/24/2014] [Accepted: 02/15/2015] [Indexed: 12/23/2022]
Abstract
Pioneer transcription factors (TFs) access silent chromatin and initiate cell-fate changes, using diverse types of DNA binding domains (DBDs). FoxA, the paradigm pioneer TF, has a winged helix DBD that resembles linker histone and thereby binds its target sites on nucleosomes and in compacted chromatin. Herein, we compare the nucleosome and chromatin targeting activities of Oct4 (POU DBD), Sox2 (HMG box DBD), Klf4 (zinc finger DBD), and c-Myc (bHLH DBD), which together reprogram somatic cells to pluripotency. Purified Oct4, Sox2, and Klf4 proteins can bind nucleosomes in vitro, and in vivo they preferentially target silent sites enriched for nucleosomes. Pioneer activity relates simply to the ability of a given DBD to target partial motifs displayed on the nucleosome surface. Such partial motif recognition can occur by coordinate binding between factors. Our findings provide insight into how pioneer factors can target naive chromatin sites.
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6
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He Q, Johnston J, Zeitlinger J. ChIP-nexus enables improved detection of in vivo transcription factor binding footprints. Nat Biotechnol 2015; 33:395-401. [PMID: 25751057 PMCID: PMC4390430 DOI: 10.1038/nbt.3121] [Citation(s) in RCA: 173] [Impact Index Per Article: 17.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2014] [Accepted: 12/08/2014] [Indexed: 11/13/2022]
Abstract
Understanding how eukaryotic enhancers are bound and regulated by specific combinations of transcription factors is still a major challenge. To better map transcription factor binding genome-wide at nucleotide resolution in vivo, we have developed a robust ChIP-exo protocol called ChIP experiments with nucleotide resolution through exonuclease, unique barcode and single ligation (ChIP-nexus), which utilizes an efficient DNA self-circularization step during library preparation. Application of ChIP-nexus to four proteins—human TBP and Drosophila NFkB, Twist and Max— demonstrates that it outperforms existing ChIP protocols in resolution and specificity, pinpoints relevant binding sites within enhancers containing multiple binding motifs and allows the analysis of in vivo binding specificities. Notably, we show that Max frequently interacts with DNA sequences next to its motif, and that this binding pattern correlates with local DNA sequence features such as DNA shape. ChIP-nexus will be broadly applicable to studying in vivo transcription factor binding specificity and its relationship to cis-regulatory changes in humans and model organisms.
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Affiliation(s)
- Qiye He
- Stowers Institute for Medical Research, Kansas City, Missouri, USA
| | - Jeff Johnston
- Stowers Institute for Medical Research, Kansas City, Missouri, USA
| | - Julia Zeitlinger
- 1] Stowers Institute for Medical Research, Kansas City, Missouri, USA. [2] Department of Pathology, Kansas University Medical Center, Kansas City, Kansas, USA
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7
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Shachaf CM, Gentles AJ, Elchuri S, Sahoo D, Soen Y, Sharpe O, Perez OD, Chang M, Mitchel D, Robinson WH, Dill D, Nolan GP, Plevritis SK, Felsher DW. Genomic and proteomic analysis reveals a threshold level of MYC required for tumor maintenance. Cancer Res 2008; 68:5132-42. [PMID: 18593912 PMCID: PMC4191850 DOI: 10.1158/0008-5472.can-07-6192] [Citation(s) in RCA: 79] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
MYC overexpression has been implicated in the pathogenesis of most types of human cancers. MYC is likely to contribute to tumorigenesis by its effects on global gene expression. Previously, we have shown that the loss of MYC overexpression is sufficient to reverse tumorigenesis. Here, we show that there is a precise threshold level of MYC expression required for maintaining the tumor phenotype, whereupon there is a switch from a gene expression program of proliferation to a state of proliferative arrest and apoptosis. Oligonucleotide microarray analysis and quantitative PCR were used to identify changes in expression in 3,921 genes, of which 2,348 were down-regulated and 1,573 were up-regulated. Critical changes in gene expression occurred at or near the MYC threshold, including genes implicated in the regulation of the G(1)-S and G(2)-M cell cycle checkpoints and death receptor/apoptosis signaling. Using two-dimensional protein analysis followed by mass spectrometry, phospho-flow fluorescence-activated cell sorting, and antibody arrays, we also identified changes at the protein level that contributed to MYC-dependent tumor regression. Proteins involved in mRNA translation decreased below threshold levels of MYC. Thus, at the MYC threshold, there is a loss of its ability to maintain tumorigenesis, with associated shifts in gene and protein expression that reestablish cell cycle checkpoints, halt protein translation, and promote apoptosis.
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Affiliation(s)
- Catherine M. Shachaf
- Division of Medical Oncology, Departments of Medicine and Pathology, Stanford University School of Medicine, Stanford University, Stanford, California
- Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford University, Stanford, California
- The Baxter Laboratory for Genetic Pharmacology, Stanford University School of Medicine, Stanford University, Stanford, California
| | - Andrew J. Gentles
- Department of Radiology, Stanford University School of Medicine, Stanford University, Stanford, California
| | - Sailaja Elchuri
- Division of Medical Oncology, Departments of Medicine and Pathology, Stanford University School of Medicine, Stanford University, Stanford, California
| | - Debashis Sahoo
- Department of Computer Science, Stanford University School of Medicine, Stanford University, Stanford, California
| | - Yoav Soen
- Department of Biochemistry, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford University, Stanford, California
| | - Orr Sharpe
- Division of Immunology and Rheumatology, Department of Medicine, Stanford University School of Medicine, Stanford University, Stanford, California
| | - Omar D. Perez
- Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford University, Stanford, California
- The Baxter Laboratory for Genetic Pharmacology, Stanford University School of Medicine, Stanford University, Stanford, California
| | - Maria Chang
- Division of Medical Oncology, Departments of Medicine and Pathology, Stanford University School of Medicine, Stanford University, Stanford, California
| | - Dennis Mitchel
- Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford University, Stanford, California
- The Baxter Laboratory for Genetic Pharmacology, Stanford University School of Medicine, Stanford University, Stanford, California
| | - William H. Robinson
- Division of Immunology and Rheumatology, Department of Medicine, Stanford University School of Medicine, Stanford University, Stanford, California
| | - David Dill
- Department of Computer Science, Stanford University School of Medicine, Stanford University, Stanford, California
| | - Garry P. Nolan
- Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford University, Stanford, California
- The Baxter Laboratory for Genetic Pharmacology, Stanford University School of Medicine, Stanford University, Stanford, California
| | - Sylvia K. Plevritis
- Department of Radiology, Stanford University School of Medicine, Stanford University, Stanford, California
| | - Dean W. Felsher
- Division of Medical Oncology, Departments of Medicine and Pathology, Stanford University School of Medicine, Stanford University, Stanford, California
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8
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Farina A, Faiola F, Martinez E. Reconstitution of an E box-binding Myc:Max complex with recombinant full-length proteins expressed in Escherichia coli. Protein Expr Purif 2004; 34:215-22. [PMID: 15003254 PMCID: PMC4004042 DOI: 10.1016/j.pep.2003.11.021] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2003] [Revised: 11/12/2003] [Indexed: 11/26/2022]
Abstract
The c-Myc oncoprotein (Myc) is a DNA sequence-specific transcription factor that regulates transcription of a wide variety of genes involved in the control of cell growth, proliferation, differentiation, and apoptosis and its deregulated expression is implicated in many types of human cancer. Myc has an N-terminal transcription activation domain (TAD) that interacts with various coactivators and a C-terminal basic-helix-loop-helix-leucine zipper (bHLHZip) domain required for E box-specific DNA-binding and heterodimerization with its obligatory bHLHZip protein partner Max. The analysis of the mechanisms by which the Myc:Max complex regulates transcription at the molecular level in vitro has been hampered by the difficulty in obtaining highly pure recombinant Myc:Max heterodimers that contain full-length Myc with its complete TAD domain and that have sequence-specific DNA-binding activity. Here, we describe a simple method to reconstitute recombinant Myc:Max complexes from highly purified full-length proteins expressed in Escherichia coli that are soluble and highly active in E box-specific DNA-binding in vitro. The reconstituted Myc:Max complexes are stable and lack Max:Max homodimers. This procedure should facilitate the characterization of the DNA-binding and transcription activation functions of full-length Myc:Max complexes in vitro and in particular the role of Myc TAD-interacting cofactors and Myc:Max post-translational modifications.
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9
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White CL, Luger K. Defined structural changes occur in a nucleosome upon Amt1 transcription factor binding. J Mol Biol 2004; 342:1391-402. [PMID: 15364568 DOI: 10.1016/j.jmb.2004.07.080] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2004] [Revised: 07/17/2004] [Accepted: 07/20/2004] [Indexed: 11/19/2022]
Abstract
Here, we study the binding of the transcription factor Amt1 to its recognition site near the dyad of a highly positioned nucleosome. We find that the DNA binding domain of Amt1 binds to nucleosomes with only threefold reduced affinity compared to free DNA. We show by fluorescence resonance energy transfer that factor binding at the nucleosomal dyad is accompanied by the partial dissociation of the DNA ends from the histone octamer surface; however, no dissociation or subtle rearrangements of histone subunits is observed. A poly(dA.dT) DNA sequence element adjacent to the transcription factor binding site appears to facilitate factor binding, but is not essential. The methods that we describe here characterize for the first time the subtle structural changes that occur upon transcription factor binding to nucleosomes, and demonstrate the ability of the nucleosome to structurally adapt in response to outside influences.
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Affiliation(s)
- Cindy L White
- Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO-80523-1870, USA
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10
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Ruh MF, Chrivia JC, Cox LK, Ruh TS. The interaction of the estrogen receptor with mononucleosomes. Mol Cell Endocrinol 2004; 214:71-9. [PMID: 15062546 DOI: 10.1016/j.mce.2003.11.023] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/30/2003] [Accepted: 11/12/2003] [Indexed: 11/28/2022]
Abstract
To directly activate specific gene expression, the estrogen receptor (ER) must bind to estrogen receptor response elements (EREs) in the context of nucleosomes. In order to investigate the interaction of the ER with mononucleosomes, we developed a mononucleosome gel shift assay. A 164 bp high specific activity [(32)P]probe DNA (32 bp consensus ERE with flanking regions separated by 23 nucleotides from an artificial nucleosome positioning sequence) was prepared. Nuclear extracts from MCF-7 cells or recombinant human ERalpha were incubated with the labeled ERE +/- excess ERE. A retarded band was seen which was completely obliterated with excess ERE, confirming the specificity of binding. This probe was then used to make reconstituted mononucleosomes by sequential dilution of a high salt histone preparation. The nucleosomes were purified by sucrose density gradients and footprinting analysis was performed to demonstrate that the mononucleosomes were rotationally phased as seen by a periodic digestion pattern (10 bp) of the nucleosomes versus ERE. Nucleosomes were incubated with nuclear extracts containing ER or recombinant ERalpha. Dose dependence in the shift of the mononucleosomes with increasing concentrations of ER was observed. Specificity was demonstrated in experiments with excess ERE and anti-ER antibody. Footprinting analysis was also performed. We also determined that addition of high mobility group protein-2 (HMGB-2, a protein closely related to HMGB-1) with the ER increased the interaction of ER with mononucleosomes. These studies will allow us to address the interactions of ER with core histones containing a multiplicity of variants and modifications in nucleosomal structure.
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Affiliation(s)
- Mary F Ruh
- Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, 1402 S Grand Boulevard, St Louis, MO 63104, USA.
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11
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Abstract
The Drosophila nucleosome remodeling factor (NURF) is an imitation switch (ISWI)-containing chromatin remodeling complex that can catalyze nucleosome repositioning at promoter regions to regulate access by the transcription machinery. Mononucleosomes reconstituted in vitro by salt dialysis adopt an ensemble of translational positions on DNA templates. NURF induces bi-directional 'sliding' of these nucleosomes to a subset of preferred positions. Here we show that mononucleosome sliding catalyzed by NURF bears similarity to nucleosome movement induced by elevated temperature. Moreover, we demonstrate that the GAL4 DNA-binding domain can extend NURF-induced nucleosome movement on a GAL4-E4 promoter, expanding the stretch of histone-free DNA at GAL4 recognition sites. The direction of NURF-induced nucleosome movement can be significantly modulated by asymmetric placement of tandem GAL4 sites relative to the nucleosome core particle. As such, sequence-specific, transcription factor-directed nucleosome sliding is likely to have substantial influence on promoter activation.
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Affiliation(s)
| | - Ali Hamiche
- Laboratory of Molecular Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Building 37, Room 6068, Bethesda, MD 20892-4255, USA
Present address: LBME–IBCG–CNRS, 118 Route de Narbonne, 31062 Toulouse, France Corresponding author e-mail:
| | - Carl Wu
- Laboratory of Molecular Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Building 37, Room 6068, Bethesda, MD 20892-4255, USA
Present address: LBME–IBCG–CNRS, 118 Route de Narbonne, 31062 Toulouse, France Corresponding author e-mail:
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12
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Rossetti L, Cacchione S, De Menna A, Chapman L, Rhodes D, Savino M. Specific interactions of the telomeric protein Rap1p with nucleosomal binding sites. J Mol Biol 2001; 306:903-13. [PMID: 11237607 DOI: 10.1006/jmbi.2001.4458] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The telomeres of Saccharomyces cerevisiae are structurally and functionally well characterized. Their telomeric DNA is packaged by the protein Rap1p (repressor activator protein 1). Rap1p is a multifunctional, sequence-specific, DNA-binding protein which, besides participating in the regulation of telomeres structure and length, is also involved in transcriptional regulation of genes essential for cell growth and in silencing. Whereas the long tracts of telomeric DNA repeats of higher eukaryotes are mostly organized in closely spaced canonical nucleosomal arrays, it has been proposed that the 300 base-pairs of S. cerevisiae telomeric DNA are organized in a large non-nucleosomal structure that has been called the telosome. Recently, nucleosomes have been found also in Tetrahymena thermophila telomeres, suggesting that, in general, telomere structural differences between lower and higher eukaryotes could be quantitative, rather than qualitative. Using an in vitro model system, we have addressed the question of whether Rap1p can form a stable ternary complex with nucleosomes containing telomeric binding sites, or competes with nucleosome core formation. The approach we have taken is to place a single Rap1p-binding site at different positions within a nucleosome core and then test the binding of Rap1p and its DNA-binding domain (Rap1p-DBD). We show here that both proteins are able to specifically recognize their nucleosomal binding site, but that binding is dependent on the location of the site within the nucleosome core structure. These results show that a ternary complex between a nucleosome and Rap1p is stable and could be a possible intermediate between telomeric nucleosomes and telosomes in the dynamics of S. cerevisiae telomere organization.
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Affiliation(s)
- L Rossetti
- Dipartimento di Genetica e Biologia Molecolare, Fondazione Istituto Pasteur -Fondazione Cenci Bolognetti, Università di Roma La Sapienza, Piazzale A Moro 5,00185, Roma, Italy
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13
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Dang CV, Resar LM, Emison E, Kim S, Li Q, Prescott JE, Wonsey D, Zeller K. Function of the c-Myc oncogenic transcription factor. Exp Cell Res 1999; 253:63-77. [PMID: 10579912 DOI: 10.1006/excr.1999.4686] [Citation(s) in RCA: 284] [Impact Index Per Article: 10.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The c-myc gene and the expression of the c-Myc protein are frequently altered in human cancers. The c-myc gene encodes the transcription factor c-Myc, which heterodimerizes with a partner protein, termed Max, to regulate gene expression. Max also heterodimerizes with the Mad family of proteins to repress transcription, antagonize c-Myc, and promote cellular differentiation. The constitutive activation of c-myc expression is key to the genesis of many cancers, and hence the understanding of c-Myc function depends on our understanding of its target genes. In this review, we attempt to place the putative target genes of c-Myc in the context of c-Myc-mediated phenotypes. From this perspective, c-Myc emerges as an oncogenic transcription factor that integrates the cell cycle machinery with cell adhesion, cellular metabolism, and the apoptotic pathways.
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Affiliation(s)
- C V Dang
- Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205, USA.
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14
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Lüscher B, Larsson LG. The basic region/helix-loop-helix/leucine zipper domain of Myc proto-oncoproteins: function and regulation. Oncogene 1999; 18:2955-66. [PMID: 10378692 DOI: 10.1038/sj.onc.1202750] [Citation(s) in RCA: 157] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
A large body of evidence has been accumulated that demonstrates dominant effects of Myc proto-oncoproteins on different aspects of cellular growth. Myc is one of the few proteins that is sufficient to drive resting cells into the cell cycle and promote DNA synthesis. In line with this finding is that the constitutive expression of Myc in cells blocks their differentiation. These growth stimulating properties are most likely responsible for Myc's ability to initiate and promote tumor formation. Interestingly Myc can also sensitize cells to apoptosis, suggesting that this protein is part of a life-and-death switch. Molecularly Myc functions as a transcriptional regulator that needs to heterodimerize with Max to exert the biological activities described above and to regulate gene transcription. Myc and Max are just two members of a growing family of proteins referred to as the Myc/Max/Mad network. A hallmark of these proteins is that they possess a C-terminal basic region/helix-loop-helix/leucine zipper domain (bHLHZip). The bHLHZip domain specifies dimerization within the network and determines sequence specific DNA binding. Importantly this domain together with the N-terminal transactivation domain is essential for Myc biology. Here we have summarized the structural, functional, and regulatory aspects of the bHLHZip domain of Myc proteins.
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Affiliation(s)
- B Lüscher
- Institut für Molekularbiologie, Medizinische Hochschule Hannover, Germany.
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15
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Motta MC, Caretti G, Badaracco GF, Mantovani R. Interactions of the CCAAT-binding trimer NF-Y with nucleosomes. J Biol Chem 1999; 274:1326-33. [PMID: 9880503 DOI: 10.1074/jbc.274.3.1326] [Citation(s) in RCA: 24] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
NF-Y is a sequence-specific evolutionary conserved activator binding to CCAAT boxes with high affinity and specificity. It is a trimer formed by NF-YA and two putative histone-like subunits, NF-YB and NF-YC, showing similarity to histones H2B and H2A, respectively. We investigated the relationships between NF-Y and chromatin using an Artemia franciscana chromatin assembly system with plasmids containing the Major HistoCompatibility complex class II Ea promoter. The NF-Y trimer, but not single subunits, protects the Y box in the presence of reconstituted chromatin, and it can bind the target sequence during and after assembly. Using reconstitution assays with purified chicken histones, we show that NF-Y associates with preformed nucleosomes. Translational analysis of various Ea fragments of identical length in which the CCAAT box is at different positions indicated that the lateral fragment was slightly more prone to NF-Y binding. In competition experiments, NF-Y is able to prevent formation of nucleosomes significantly. These data support the idea that NF-Y is a gene-specific activator with a built-in capacity to interface with chromatin structures.
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Affiliation(s)
- M C Motta
- Dipartimento di Genetica e Biologia dei Microrganismi, Università di Milano, Via Celoria 26, 20133 Milano, Italy
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Blomquist P, Belikov S, Wrange O. Increased nuclear factor 1 binding to its nucleosomal site mediated by sequence-dependent DNA structure. Nucleic Acids Res 1999; 27:517-25. [PMID: 9862974 PMCID: PMC148209 DOI: 10.1093/nar/27.2.517] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
The organization of DNA into chromatin is important in the regulation of transcription, by influencing the access of transcription factors to their DNA binding sites. Nuclear factor 1 (NF-1) is a transcription factor which binds to DNA constitutively and which interacts with its cognate DNA site with high affinity. However, this affinity is drastically reduced, approximately 100- to 300-fold, when the binding site is organized into a nucleosome. Here we demonstrate that the introduction of stretches of adenines of length 5 nt (A-tracts) on both sides of the NF-1 binding site has a distinct effect on NF-1 binding to a nucleosomal, but not to a free, NF-1 binding site. The position of the A-tracts, relative to the rotational phase of a synthetic DNA bending sequence, the TG-motif, decides whether the NF-1 affinity increases or decreases. The NF-1 binding affinity is seven times stronger when the flanking A-tracts are positioned out-of-phase with the TG-motif than it is when the A-tracts are positioned in-phase with the TG-motif. We demonstrate that this effect correlates with differences in DNA curvature and apparent histone octamer affinity. We conclude that DNA curvature influences the local histone-DNA contacts and hence the accessibility of the NF-1 site in a nucleosome context.
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Affiliation(s)
- P Blomquist
- Laboratory of Molecular Genetics, Department of Cell and Molecular Biology, Medical Nobel Institute,Karolinska Institute, S-171 77 Stockholm, Sweden
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17
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Imbalzano AN. SWI/SNF complexes and facilitation of TATA binding protein:nucleosome interactions. Methods 1998; 15:303-14. [PMID: 9740718 DOI: 10.1006/meth.1998.0634] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
It has become increasingly apparent that eukaryotic cells possess machinery that modifies chromatin structure and that this machinery contributes to the regulation of gene expression. Identification of factors that alter chromatin structure has made possible biochemical analyses that have begun to define what structural changes each factor can cause as well as what consequences these changes have on transcription factor function. Here, a protocol that has facilitated study of energy-dependent chromatin remodeling complexes containing SWI/SNF proteins is described. Rotationally phased mononucleosome particles were assembled in vitro and used to demonstrate that human SWI/SNF complexes and the yeast RNA polymerase II holoenzyme, which contains yeast SWI/SNF proteins, can directly alter nucleosome structure in an ATP-dependent manner. A functional consequence of this nucleosome disruption is that the pol II general transcription factor, TATA binding protein (TBP), which cannot bind to unaltered nucleosomal DNA, can bind to its site on the altered nucleosome. This experimental system has been invaluable for characterization of both nucleosome alteration and facilitated transcription factor binding mediated by SWI/SNF complexes. These procedures should also be useful to examine other factors that interact with or structurally affect nucleosome particles.
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Affiliation(s)
- A N Imbalzano
- Department of Cell Biology, University of Massachusetts Medical Center, Worcester 01655, USA
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18
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Boyes J, Omichinski J, Clark D, Pikaart M, Felsenfeld G. Perturbation of nucleosome structure by the erythroid transcription factor GATA-1. J Mol Biol 1998; 279:529-44. [PMID: 9641976 DOI: 10.1006/jmbi.1998.1783] [Citation(s) in RCA: 52] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The ability of transcription factors to gain access to their sites in chromatin requires the disruption or displacement of nucleosomes covering the promoter, signalled by the generation of a nuclease hypersensitive site. We characterise here the alterations in nucleosome structure caused by binding of the erythroid factor GATA-1 to a nucleosome carrying GATA-1 sites. DNase I and micrococcal nuclease probes show that GATA-1 binding causes extensive, cooperative breakage of the histone/DNA contacts to generate a complex very similar to that formed by the factor with free DNA. The only region which differs is confined to about 50 bp surrounding the nucleosome dyad axis which appears to be the domain of residual contact between the DNA and histone octamer. Despite considerable breakage of the histone/DNA contacts, the complex is completely stable in solution, and disruption of the nucleosome is entirely reversible: it is regenerated quantitatively upon removal of the transcription factor. Moreover, the histone 2A/2B component of the octamer does not exchange to external competitor. We suggest that formation of this complex may be a step in the generation of a fully hypersensitive site in vivo over regulatory elements containing GATA family binding sites.
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Affiliation(s)
- J Boyes
- Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA
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19
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Hassig CA, Schreiber SL. Nuclear histone acetylases and deacetylases and transcriptional regulation: HATs off to HDACs. Curr Opin Chem Biol 1997; 1:300-8. [PMID: 9667866 DOI: 10.1016/s1367-5931(97)80066-x] [Citation(s) in RCA: 294] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Reversible acetylation of lysines on the amino-terminal tails of nucleosomal histones is correlated with changes in chromatin structure and transcription. The recent characterization of enzymes directly responsible for regulating histone acetylation and deacetylation and the cloning of their encoding cDNAs have provided insights into the possible functional and regulatory mechanisms of these classes of molecules. Nuclear histone acetylases have been shown to be transcriptional coactivators and coactivator-associated proteins, while histone deacetylases have been identified as components of nuclear co-repressor complexes. These findings confirm previous studies linking histone acetylation and transcriptional regulation.
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Affiliation(s)
- C A Hassig
- Howard Hughes Medical Institute, Harvard University Department of Chemistry, 12 Oxford Street, Cambridge, MA 02138, USA. hassig@slsiris harvard.edu
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20
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Abstract
Transcriptional activation is mediated by the facilitated binding of the basal transcription complex to the transcription start site of a promoter. The activation procedure involves protein-protein interactions between specific transcription factors and members of the basal transcription complex. However, since eukaryotic DNA is packaged with histones into nucleosomes the accessibility of the transcription factors is limited. In order to activate transcription, some of the specific transcription factors must have the capacity to bind to their binding sites when organized into nucleosomes. As a next step, the chromatin structure of the promoter needs to be decondensed in order to facilitate the binding of the basal transcription machinery. Recent data have addressed these issues and both binding of transcription factors to their chromatin binding site as well as transcription factor-induced chromatin remodelling have been demonstrated. In addition, factors that are candidates to mediate the chromatin remodelling have recently been identified and characterized. The ability of a transcription factor to recognize its cognate element in a nucleosome is an inheret property that differs among different transcription factors. The implications of the rotational and translational positioning of the DNA within a nucleosome on the accessibility of a transcription factor is described in this review. In addition, nucleosome rearrangement and juxtaposing in the context of transcriptional activation is also discussed.
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Affiliation(s)
- Q Li
- Department of Cell and Molecular Biology, Nobel Medical Institute, Karolinska Institute, Stockholm, Sweden
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21
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Choi JK, Shen CP, Radomska HS, Eckhardt LA, Kadesch T. E47 activates the Ig-heavy chain and TdT loci in non-B cells. EMBO J 1996; 15:5014-21. [PMID: 8890174 PMCID: PMC452239] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023] Open
Abstract
The E2A proteins, E12 and E47, are basic helix-loop-helix (bHLH) proteins essential for the B-cell lineage. Initially identified as immunoglobulin enhancer-binding proteins, they have also been shown to activate immunoglobulin enhancer-based reporters in transient transfection assays. Here, we examine the relationship between E2A DNA binding activity and activation of the endogenous, chromosomal immunoglobulin heavy chain (IgH) locus. Using sterile I(mu) transcription as an indicator of IgH enhancer activity, we see a direct correlation between E2A DNA binding activity and I(mu) transcription in stable BxT hybrids. We also observe a 1000-fold stimulation of endogenous I(mu) transcription in fibroblasts that express high levels of E47 and less stimulation in cells that express E12. By contrast, none of the other IgH enhancer-binding proteins tested (E2-2, Pu.1, Oct-2, OCA-B, TFE3 and USF) were able to activate I(mu) transcription. E47 overexpression also resulted in transcriptional activation of the endogenous gene encoding TdT, indicating that it, too, is a target of E2A proteins early in the B-cell lineage. Our results indicate that E2A proteins have the distinctive property of activating silent, chromatin-embedded B-cell-specific genes, underscoring their crucial role in B-cell development.
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Affiliation(s)
- J K Choi
- Department of Genetics and Pathology, University of Pennsylvania School of Medicine, Philadelphia 19104-6145, USA
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22
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Niu X, Adams CC, Workman JL, Guiltinan MJ. Binding of the wheat basic leucine zipper protein EmBP-1 to nucleosomal binding sites is modulated by nucleosome positioning. THE PLANT CELL 1996; 8:1569-1587. [PMID: 8837510 PMCID: PMC161299 DOI: 10.1105/tpc.8.9.1569] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/22/2023]
Abstract
To investigate interactions of the basic leucine zipper transcription factor EmBP-1 with its recognition sites in nucleosomal DNA, we reconstituted an abscisic acid response element and a high-affinity binding site for EmBP-1 into human and wheat nucleosome cores in vitro. DNA binding studies demonstrated that nucleosomal elements can be bound by EmBP-1 at reduced affinities relative to naked DNA. EmBP-1 affinity was lowest when the recognition sites were positioned near the center of the nucleosome. Binding was achieved with a truncated DNA binding domain; however, binding of full-length EmBP-1 caused additional strong DNase I hypersensitivity flanking the binding sites. Similar results were observed with nucleosomes reconstituted with either human or wheat histones, demonstrating a conserved mechanism of transcription factor-nucleosome interactions. We conclude that positioning of recognition sequences on a nucleosome may play an important role in regulating interactions of EmBP-1 with its target sites in plant cells.
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Affiliation(s)
- X Niu
- Department of Horticulture, Pennsylvania State University, University Park 16802, USA
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Imbalzano AN, Schnitzler GR, Kingston RE. Nucleosome disruption by human SWI/SNF is maintained in the absence of continued ATP hydrolysis. J Biol Chem 1996; 271:20726-33. [PMID: 8702824 DOI: 10.1074/jbc.271.34.20726] [Citation(s) in RCA: 85] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023] Open
Abstract
We have examined the requirement for ATP in human (h) SWI/SNF-mediated alteration of nucleosome structure and facilitation of transcription factor binding to nucleosomal DNA. hSWI/SNF-mediated nucleosome alteration requires hydrolysis of ATP or dATP. The alteration is stable upon removal of ATP from the reaction or upon inhibition of activity by excess ATPgammaS, indicating that continued ATP hydrolysis is not required to maintain the altered nucleosome structure. This stable alteration is sufficient to facilitate binding of a transcriptional activator protein; concurrent ATP hydrolysis was not required to facilitate binding. These data suggest sequential steps that can occur in the process by which transcription factors gain access to nucleosomal DNA.
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Affiliation(s)
- A N Imbalzano
- Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA
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Vettese-Dadey M, Adams CC, Côté J, Walter P, Workman JL. [7] Experimental analysis of transcription factor-nucleosome interactions. MICROBIAL GENE TECHNIQUES 1995. [DOI: 10.1016/s1067-2389(06)80010-5] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
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Imbalzano AN, Kwon H, Green MR, Kingston RE. Facilitated binding of TATA-binding protein to nucleosomal DNA. Nature 1994; 370:481-5. [PMID: 8047170 DOI: 10.1038/370481a0] [Citation(s) in RCA: 488] [Impact Index Per Article: 15.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
BINDING of the TATA-binding protein (TBP) to the TATA box is required for transcription from many eukaryotic promoters in gene expression. Regulation of this binding is therefore likely to be an important determinant of promoter activity. Incorporation of the TATA sequence into nucleosomes dramatically reduces transcription initiation, presumably because of stereochemical constraints on binding of general transcription factors. Biochemical and genetic studies imply that cellular factors such as yeast SWI/SNF are required for activator function and might alter chromatin structure. One step that could be regulated during the activation process is TBP binding in chromatin 12, 13. We show here that binding of TBP to the TATA sequence is severely inhibited by incorporation of this sequence into a nucleosome. Inhibition can be overcome by ATP-dependent alterations in nucleosomal DNA structure mediated by hSWI/SNF, a putative human homologue of the yeast SWI/SNF complex. Additionally, the orientation of the TATA sequence relative to the surface of the histone core affects the access of TBP. We propose that the dynamic remodelling of chromatin structure to allow TBP binding is a key step in the regulation of eukaryotic gene expression.
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Affiliation(s)
- A N Imbalzano
- Department of Molecular Biology, Massachusetts General Hospital, Boston 02114
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