Molecular and Functional Characterization of Histone Deacetylase 4 (HDAC4).

Histone deacetylases (HDACs) regulate various nuclear and cytoplasmic processes. In mammals, these enzymes are divided into four classes, with class II further divided into two subclasses: IIa (HDAC4, HDAC5, HDAC7, HDAC9) and IIb (HDAC6 and HDAC10). While HDAC6 is mainly cytoplasmic and HDAC10 is pancellular, class IIa HDACs are dynamically shuttled between the nucleus and cytoplasm in a signal-dependent manner, indicating that they are unique signal transducers able to transduce signals from the cytoplasm to chromatin in the nucleus. Once inside the nucleus, class IIa HDACs interact with MEF2 and other transcription factors, mainly acting as transcriptional corepressors. Although class IIa HDACs share many molecular properties in vitro, they play quite distinct roles in vivo. This chapter lists methods that we have used for molecular and biochemical characterization of HDAC4, including development of regular and phospho-specific antibodies, deacetylase activity determination, reporter gene assays, analysis of subcellular localization, and determination of interaction with 14-3-3 and MEF2. Although described specifically for HDAC4, the protocols should be adaptable for analysis to the other three class IIa members, HDAC5, HDAC7, and HDAC9, as well as for other proteins with related properties.