title: Tracking histone variant nucleosomes across the human cell cycle using biophysical, biochemical, and cytological analyses.
abstract: Histone variants such as H3.3, macroH2A, H2A.Z, and CENP-A are important epigenetic modifiers of the chromatin state in eukaryotic genomes. The centromeric histone H3 variant CENP-A/CENH3 epigenetically marks centromeres and is required for assembly of the kinetochore complex, a region of the chromosome that is responsible for proper genome segregation during mitosis. Several diverse techniques using biochemical, cell biology, and biophysical approaches have been utilized to study the nature of the CENP-A nucleosome across the cell cycle. In this chapter, we describe methods for CENP-A nucleosome purification and separation of CENP-A from other core histones using traditional SDS-PAGE and more resolving techniques such as Triton acid urea (TAU) and two-dimensional gels. We also discuss methods for observation of CENP-A on chromatin fibers using immunofluorescence. Finally, we provide a detailed description of analysis of chromatin structures using atomic force microscopy.
authors.affiliation: Chromatin Structure and Epigenetic Mechanisms Unit, Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, 20892, USA.
authors.forename: Marcin P
authors.initials: MP
authors.lastname: Walkiewicz
authors.forename: Minh
authors.initials: M
authors.lastname: Bui
authors.forename: Delphine
authors.initials: D
authors.lastname: Quénet
authors.forename: Yamini
authors.initials: Y
authors.lastname: Dalal
mesh_terms: D001324:Autoantigens / Q000032:analysis* / Q000302:isolation & purification; D015153:Blotting, Western / Q000379:methods; D002453:Cell Cycle; D000076247:Centromere Protein A; D002843:Chromatin / Q000648:ultrastructure; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005455:Fluorescent Antibody Technique / Q000379:methods; D006657:Histones / Q000032:analysis* / Q000302:isolation & purification; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D018625:Microscopy, Atomic Force / Q000379:methods; D009707:Nucleosomes / Q000737:chemistry*
chemical_list: D001324:Autoantigens; C000619700:CENPA protein, human; D000076247:Centromere Protein A; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D006657:Histones; D009707:Nucleosomes