title: Recovery of transcriptionally active chromatin restriction fragments by binding to organomercurial-agarose magnetic beads. A rapid and sensitive method for monitoring changes in higher order chromatin structure during gene activation and repression.
abstract: The unfolding of nucleosomes along transcriptionally active DNA sequences uncovers previously shielded cysteinyl-thiol groups of histone H3 molecules located at the center of the nucleosome core. This change in conformation and SH reactivity of nucleosomes along transcribed DNA sequences makes it possible to separate active from inactive nucleosomes by mercury affinity chromatography. The binding of thiol-reactive nucleosomes to an organomercurial-agarose column has been shown previously to reflect, with accuracy, both the timing and extent of transcription of the associated DNA sequences (Chen, T. A., and Allfrey, V. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5252-5256). Here, we extend this experimental approach to the analysis of higher order chromatin structures. Large chromatin fragments released by treating isolated nuclei with restriction endonucleases are fractionated on mercurated agarose magnetic beads that capture nucleosomes with accessible histone H3 thiols, but do not react with the hidden H3 thiols of the compactly beaded nucleosomes of inactive genes. The SH-reactive domains of c-myc and other genes are rapidly separated from the non-SH-reactive restriction fragments by the magnetic bead technique. The new method also overcomes a major limitation of mercurated agarose column chromatography, which is not suitable for studies of higher order chromatin structure because large chromatin fragments occlude the mercury column; occlusion is not a problem in magnetic separations using suspended mercurated agarose beads. Here, we describe the synthesis of mercurated agarose magnetic beads with high capacity for SH groups and test their application to the recovery of chromatin restriction fragments of c-myc and the growth arrest gene gas1.
authors.affiliation: Laboratory of Cell Biology, Rockefeller University, New York, New York 10021.
authors.forename: T A
authors.initials: TA
authors.lastname: Chen-Cleland
authors.forename: L C
authors.initials: LC
authors.lastname: Boffa
authors.forename: E M
authors.initials: EM
authors.lastname: Carpaneto
authors.forename: M R
authors.initials: MR
authors.lastname: Mariani
authors.forename: E
authors.initials: E
authors.lastname: Valentin
authors.forename: E
authors.initials: E
authors.lastname: Mendez
authors.forename: V G
authors.initials: VG
authors.lastname: Allfrey
mesh_terms: D000818:Animals; D002087:Butyrates / Q000494:pharmacology; D018797:Cell Cycle Proteins; D002460:Cell Line; D002843:Chromatin / Q000648:ultrastructure*; D002846:Chromatography, Affinity; D058851:GPI-Linked Proteins; D005786:Gene Expression Regulation; D016259:Genes, myc; D006367:HeLa Cells; D006657:Histones / Q000737:chemistry / Q000648:ultrastructure; D006801:Humans; D066298:In Vitro Techniques; D008280:Magnetics; D008562:Membrane Glycoproteins / Q000235:genetics; D008565:Membrane Proteins; D051379:Mice; D008854:Microscopy, Electron; D009941:Organomercury Compounds; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D015183:Restriction Mapping; D029701:Saccharomyces cerevisiae Proteins; D014158:Transcription, Genetic; D015533:Transcriptional Activation
chemical_list: D002087:Butyrates; D018797:Cell Cycle Proteins; D002843:Chromatin; C066709:GAS1 protein, S cerevisiae; C096192:GAS1 protein, human; D058851:GPI-Linked Proteins; C501180:Gas1 protein, mouse; D006657:Histones; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009941:Organomercury Compounds; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins