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      Genetic Identification of a Network of Factors that Functionally Interact with the Nucleosome Remodeling ATPase ISWI

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          Abstract

          Nucleosome remodeling and covalent modifications of histones play fundamental roles in chromatin structure and function. However, much remains to be learned about how the action of ATP-dependent chromatin remodeling factors and histone-modifying enzymes is coordinated to modulate chromatin organization and transcription. The evolutionarily conserved ATP-dependent chromatin-remodeling factor ISWI plays essential roles in chromosome organization, DNA replication, and transcription regulation. To gain insight into regulation and mechanism of action of ISWI, we conducted an unbiased genetic screen to identify factors with which it interacts in vivo. We found that ISWI interacts with a network of factors that escaped detection in previous biochemical analyses, including the Sin3A gene. The Sin3A protein and the histone deacetylase Rpd3 are part of a conserved histone deacetylase complex involved in transcriptional repression. ISWI and the Sin3A/Rpd3 complex co-localize at specific chromosome domains. Loss of ISWI activity causes a reduction in the binding of the Sin3A/Rpd3 complex to chromatin. Biochemical analysis showed that the ISWI physically interacts with the histone deacetylase activity of the Sin3A/Rpd3 complex. Consistent with these findings, the acetylation of histone H4 is altered when ISWI activity is perturbed in vivo. These findings suggest that ISWI associates with the Sin3A/Rpd3 complex to support its function in vivo.

          Author Summary

          The eukaryotic genome is organized in a highly dynamic structure called chromatin. Access to DNA in the context of chromatin is granted by enzymatic activities that use the energy of hydrolysis of ATP to slide or covalently modify nucleosomes. ISWI is an evolutionarily conserved nucleosome-sliding factor that plays essential roles in transcription, DNA replication, and chromosome organization. Despite the wealth of data on ISWI function, little is known about how its activity is regulated and integrated in different physiological contexts in vivo. Using D. melanogaster as a model system, we conducted a genetic screen for factors regulating ISWI activity. One of the genes identified in our screen, Sin3A, encodes a subunit of a histone deacetylase complex that may regulate ISWI function by modifying its nucleosome substrate. Our genetic screen revealed that ISWI interacts with a network of cellular and nuclear factors that escaped previous biochemical analyses, indicating the participation of ISWI in a variety of biological processes not linked to date with known ISWI functions.

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          Gene Ontology: tool for the unification of biology

          Genomic sequencing has made it clear that a large fraction of the genes specifying the core biological functions are shared by all eukaryotes. Knowledge of the biological role of such shared proteins in one organism can often be transferred to other organisms. The goal of the Gene Ontology Consortium is to produce a dynamic, controlled vocabulary that can be applied to all eukaryotes even as knowledge of gene and protein roles in cells is accumulating and changing. To this end, three independent ontologies accessible on the World-Wide Web (http://www.geneontology.org) are being constructed: biological process, molecular function and cellular component.
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            A generic protein purification method for protein complex characterization and proteome exploration.

            We have developed a generic procedure to purify proteins expressed at their natural level under native conditions using a novel tandem affinity purification (TAP) tag. The TAP tag allows the rapid purification of complexes from a relatively small number of cells without prior knowledge of the complex composition, activity, or function. Combined with mass spectrometry, the TAP strategy allows for the identification of proteins interacting with a given target protein. The TAP method has been tested in yeast but should be applicable to other cells or organisms.
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              Histone H4-K16 acetylation controls chromatin structure and protein interactions.

              Acetylation of histone H4 on lysine 16 (H4-K16Ac) is a prevalent and reversible posttranslational chromatin modification in eukaryotes. To characterize the structural and functional role of this mark, we used a native chemical ligation strategy to generate histone H4 that was homogeneously acetylated at K16. The incorporation of this modified histone into nucleosomal arrays inhibits the formation of compact 30-nanometer-like fibers and impedes the ability of chromatin to form cross-fiber interactions. H4-K16Ac also inhibits the ability of the adenosine triphosphate-utilizing chromatin assembly and remodeling enzyme ACF to mobilize a mononucleosome, indicating that this single histone modification modulates both higher order chromatin structure and functional interactions between a nonhistone protein and the chromatin fiber.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                1553-7390
                1553-7404
                June 2008
                June 2008
                6 June 2008
                : 4
                : 6
                : e1000089
                Affiliations
                [1 ]Dipartimento di Scienze Biochimiche, Universita' degli Studi di Palermo, Palermo, Italy
                [2 ]Istituto Telethon Dulbecco, Universita' degli Studi di Palermo, Palermo, Italy
                [3 ]Department of Molecular, Cell, and Developmental Biology, University of California Santa Cruz, Santa Cruz, California, United States of America
                [4 ]Joint Science Department, Claremont McKenna, Scripps, and Pitzer Colleges, Claremont, California, United States of America
                [5 ]Molecular Biology Department, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen, The Netherlands
                European Molecular Biology Laboratory, Germany
                Author notes

                Conceived and designed the experiments: JA JT DC. Performed the experiments: GB GLR AS WA DDG ASS JA DC. Analyzed the data: GB SvH CL JT DC. Contributed reagents/materials/analysis tools: GLR AS WA MC JA. Wrote the paper: DC.

                Article
                08-PLGE-RA-0074R2
                10.1371/journal.pgen.1000089
                2390755
                18535655
                775fcefa-067a-431a-8163-d3784666db6b
                Burgio et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 22 January 2008
                : 2 May 2008
                Page count
                Pages: 15
                Categories
                Research Article
                Genetics and Genomics/Epigenetics

                Genetics
                Genetics

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