title: Approaches for Studying the Subcellular Localization, Interactions, and Regulation of Histone Deacetylase 5 (HDAC5).
abstract: As a member of the class IIa family of histone deacetylases, the histone deacetylase 5 (HDAC5) is known to undergo nuclear-cytoplasmic shuttling and to be a critical transcriptional regulator. Its misregulation has been linked to prominent human diseases, including cardiac diseases and tumorigenesis. In this chapter, we describe several experimental methods that have proven effective for studying the functions and regulatory features of HDAC5. We present methods for assessing the subcellular localization, protein interactions, posttranslational modifications (PTMs), and activity of HDAC5 from the standpoint of investigating either the endogenous protein or tagged protein forms in human cells. Specifically, given that at the heart of HDAC5 regulation lie its dynamic localization, interactions, and PTMs, we present methods for assessing HDAC5 localization in fixed and live cells, for isolating HDAC5-containing protein complexes to identify its interactions and modifications, and for determining how these PTMs map to predicted HDAC5 structural motifs. Lastly, we provide examples of approaches for studying HDAC5 functions with a focus on its regulation during cell-cycle progression. These methods can readily be adapted for the study of other HDACs or non-HDAC-proteins of interest. Individually, these techniques capture temporal and spatial snapshots of HDAC5 functions; yet together, these approaches provide powerful tools for investigating both the regulation and regulatory roles of HDAC5 in different cell contexts relevant to health and disease.
authors.affiliation: Department of Molecular Biology, Princeton University, 210 Lewis Thomas Laboratory, Princeton, NJ, 08544, USA.
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Guise
authors.affiliation: Department of Molecular Biology, Princeton University, 210 Lewis Thomas Laboratory, Princeton, NJ, 08544, USA. icristea@princeton.edu.
authors.forename: Ileana M
authors.initials: IM
authors.lastname: Cristea
mesh_terms: D002453:Cell Cycle; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D057809:HEK293 Cells; D006655:Histone Deacetylases / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational
chemical_list: C404500:HDAC5 protein, human; D006655:Histone Deacetylases
keywords: Cell cycle; HDAC5; Immunoaffinity purification; Immunofluorescence microscopy; In-gel digestion; Kinase inhibition; Phosphorylation; Protein complexes; Protein interactions; siRNA knockdown