title: In Vitro Chromatin Assembly: Strategies and Quality Control.
abstract: Chromatin accessibility is modulated by structural transitions that provide timely access to the genetic and epigenetic information during many essential nuclear processes. These transitions are orchestrated by regulatory proteins that coordinate intricate structural modifications and signaling pathways. In vitro reconstituted chromatin samples from defined components are instrumental in defining the mechanistic details of such processes. The bottleneck to appropriate in vitro analysis is the production of high quality, and quality-controlled, chromatin substrates. In this chapter, we describe methods for in vitro chromatin reconstitution and quality control. We highlight the strengths and weaknesses of various approaches and emphasize quality control steps that ensure reconstitution of a bona fide homogenous chromatin preparation. This is essential for optimal reproducibility and reliability of ensuing experiments using chromatin substrates.
authors.affiliation: University of Colorado at Boulder, Boulder, CO, United States.
authors.forename: U
authors.initials: U
authors.lastname: Muthurajan
authors.affiliation: University of Colorado at Boulder, Boulder, CO, United States.
authors.forename: F
authors.initials: F
authors.lastname: Mattiroli
authors.affiliation: University of Colorado at Boulder, Boulder, CO, United States.
authors.forename: S
authors.initials: S
authors.lastname: Bergeron
authors.affiliation: University of Colorado at Boulder, Boulder, CO, United States; Colorado State University, Fort Collins, CO, United States.
authors.forename: K
authors.initials: K
authors.lastname: Zhou
authors.affiliation: University of Colorado at Boulder, Boulder, CO, United States; Colorado State University, Fort Collins, CO, United States.
authors.forename: Y
authors.initials: Y
authors.lastname: Gu
authors.affiliation: Illinois Institute of Technology, Chicago, IL, United States.
authors.forename: S
authors.initials: S
authors.lastname: Chakravarthy
authors.affiliation: University of Colorado at Boulder, Boulder, CO, United States.
authors.forename: P
authors.initials: P
authors.lastname: Dyer
authors.affiliation: Illinois Institute of Technology, Chicago, IL, United States.
authors.forename: T
authors.initials: T
authors.lastname: Irving
authors.affiliation: University of Colorado at Boulder, Boulder, CO, United States; Howard Hughes Medical Institute, University of Colorado at Boulder, Boulder, CO, United States; Institute for Genome Architecture and Function, Colorado State University, Fort Collins, CO, United States. Electronic address: karolin.luger@colorado.edu.
authors.forename: K
authors.initials: K
authors.lastname: Luger
mesh_terms: D000818:Animals; D002843:Chromatin / Q000737:chemistry / Q000235:genetics; D042002:Chromatin Assembly and Disassembly; D004247:DNA / Q000032:analysis / Q000235:genetics; D005456:Fluorescent Dyes / Q000032:analysis; D006657:Histones / Q000032:analysis / Q000235:genetics; D006801:Humans; D008836:Micrococcal Nuclease / Q000378:metabolism; D018625:Microscopy, Atomic Force / Q000379:methods; D008958:Models, Molecular; D063347:Native Polyacrylamide Gel Electrophoresis / Q000379:methods; D009707:Nucleosomes / Q000737:chemistry / Q000235:genetics; D017510:Protein Folding; D055503:Protein Multimerization; D053838:Scattering, Small Angle; D014461:Ultracentrifugation / Q000379:methods; D014961:X-Ray Diffraction
chemical_list: D002843:Chromatin; D005456:Fluorescent Dyes; D006657:Histones; D009707:Nucleosomes; D004247:DNA; D008836:Micrococcal Nuclease
keywords: Analytical ultracentrifugation; Atomic force microscopy; Fluorescence labeling; Histone; Micrococcal nuclease digestion; Native polyacrylamide gel electrophoresis; Nucleosomal array; Nucleosome; SEC-MALS; Small-angle X-ray scattering