title: Functional Characterization of Histone Chaperones Using SNAP-Tag-Based Imaging to Assess De Novo Histone Deposition.
abstract: Histone chaperones-key actors in the dynamic organization of chromatin-interact with the various histone variants to ensure their transfer in and out of chromatin. In vitro chromatin assembly assays and isolation of protein complexes using tagged histone variants provided first clues concerning their binding specificities and mode of action. Here, we describe an in vivo method using SNAP-tag-based imaging to assess the de novo deposition of histones and the role of histone chaperones. This method exploits cells expressing SNAP-tagged histones combined with individual cell imaging to visualize directly de novo histone deposition in vivo. We show how, by combining this method with siRNA-based depletion, we could assess the function of two distinct histone chaperones. For this, we provide the details of the method as applied in two examples to characterize the function of the histone chaperones CAF-1 and HIRA. In both cases, we document the impact of their depletion on the de novo deposition of the histone variants H3.1 and H3.3, first in a normal context and second in response to DNA damage. We discuss how this cellular assay offers means to define in a systematic manner the function of any chosen chaperone with respect to the deposition of a given histone variant.
authors.affiliation: Institut Curie, PSL Research University, CNRS, UMR3664, Equipe Labellisée Ligue contre le Cancer, Paris, France; Sorbonne Universités, UPMC Univ Paris 06, CNRS, UMR3664, Paris, France.
authors.forename: C
authors.initials: C
authors.lastname: Clément
authors.affiliation: Institut Curie, PSL Research University, CNRS, UMR3664, Equipe Labellisée Ligue contre le Cancer, Paris, France; Sorbonne Universités, UPMC Univ Paris 06, CNRS, UMR3664, Paris, France.
authors.forename: I
authors.initials: I
authors.lastname: Vassias
authors.affiliation: Institut Curie, PSL Research University, CNRS, UMR3664, Equipe Labellisée Ligue contre le Cancer, Paris, France; Sorbonne Universités, UPMC Univ Paris 06, CNRS, UMR3664, Paris, France.
authors.forename: D
authors.initials: D
authors.lastname: Ray-Gallet
authors.affiliation: Institut Curie, PSL Research University, CNRS, UMR3664, Equipe Labellisée Ligue contre le Cancer, Paris, France; Sorbonne Universités, UPMC Univ Paris 06, CNRS, UMR3664, Paris, France. Electronic address: genevieve.almouzni@curie.fr.
authors.forename: G
authors.initials: G
authors.lastname: Almouzni
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002843:Chromatin / Q000378:metabolism; D004249:DNA Damage; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D056488:Histone Chaperones / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D006657:Histones / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D009154:Mutation; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D061848:Optical Imaging / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism
chemical_list: D002843:Chromatin; D005456:Fluorescent Dyes; D056488:Histone Chaperones; D006657:Histones; D011993:Recombinant Fusion Proteins; D006147:Guanine; D019853:O(6)-Methylguanine-DNA Methyltransferase
keywords: CAF-1; H3.1; H3.3; HIRA; Histone chaperone; Histone deposition; Histone variant; SNAP-tag