title: The scaffold/matrix attachment region binding protein hnRNP-U (SAF-A) is directly bound to chromosomal DNA in vivo: a chemical cross-linking study.
abstract: The protein heterogeneous nuclear ribonucleoprotein U (hnRNP-U, also known as scaffold attachment factor A, SAF-A) is an abundant component of hnRNP particles and of the nuclear matrix. Previous experiments have demonstrated that, in vitro, hnRNP-U specifically binds to scaffold/matrix attachment (S/MAR) region DNA elements and could thus be involved in higher order chromatin structure. In this paper we report on the use of chemical cross-linking to investigate whether the protein is also bound to DNA in vivo, which is a prerequisite for its presumed function in chromatin loop formation. We have improved published methods for cross-linking proteins to DNA with the aim to minimize unspecific fixation and possible contamination with RNA binding proteins. Our protocol is based on a limited cross-linking of living human cells with formaldehyde, followed by the purification of DNA/protein complexes by two consecutive cesium chloride density gradient centrifugations. Analysis of the protein constituents of these complexes shows a specific subset of cross-linked proteins with the histones as major components. By western blotting, we demonstrate that hnRNP-U is efficiently cross-linked to DNA under experimental conditions that yield DNA/protein complexes with a buoyant density equivalent to that of native chromatin. Dimethylsulfate cross-linking and limited protease digestion of the complexes was used to establish that hnRNP-U is bound directly to DNA and not via cross-linking to other proteins. This is the first direct demonstration of the in vivo DNA binding of a S/MAR specific protein and suggests a structural role of hnRNP-U in chromatin organization.
authors.affiliation: Division of Biology, University of Konstanz, 78434 Konstanz, Germany.
authors.forename: F
authors.initials: F
authors.lastname: Göhring
authors.forename: F O
authors.initials: FO
authors.lastname: Fackelmayer
mesh_terms: D001665:Binding Sites; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D002843:Chromatin / Q000737:chemistry; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D019175:DNA Methylation; D004591:Electrophoresis, Polyacrylamide Gel; D005557:Formaldehyde; D006367:HeLa Cells; D034722:Heterogeneous-Nuclear Ribonucleoprotein U; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D012313:RNA / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry / Q000378:metabolism*; D013463:Sulfuric Acid Esters
chemical_list: D002843:Chromatin; D003432:Cross-Linking Reagents; D034722:Heterogeneous-Nuclear Ribonucleoprotein U; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D012261:Ribonucleoproteins; D013463:Sulfuric Acid Esters; D005557:Formaldehyde; D012313:RNA; D004247:DNA; C007482:dimethyl sulfate