Approaches for studying the subcellular localization, interactions, and regulation of histone deacetylase 5 (HDAC5)

Amanda J. Guise, Ileana M. Cristea

Research output: Chapter in Book/Report/Conference proceedingChapter

3 Scopus citations

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages47-84
Number of pages38
DOIs
StatePublished - 2016

Publication series

NameMethods in Molecular Biology
Volume1436
ISSN (Print)1064-3745

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics

Keywords

  • Cell cycle
  • HDAC5
  • Immunoaffinity purification
  • Immunofluorescence microscopy
  • In-gel digestion
  • Kinase inhibition
  • Phosphorylation
  • Protein complexes
  • Protein interactions
  • siRNA knockdown

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    Guise, A. J., & Cristea, I. M. (2016). Approaches for studying the subcellular localization, interactions, and regulation of histone deacetylase 5 (HDAC5). In Methods in Molecular Biology (pp. 47-84). (Methods in Molecular Biology; Vol. 1436). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-3667-0_5