Functional and mass spectrometric analysis of histone deacetylase 5 (HDAC5) phosphorylation and protein-protein interactions

摘要

Chromatin remodeling is an important determinant of transcriptional regulation and cell fate. Histone deacetylases (HDACs) mediate transcriptional repression by removing acetyl groups from histone-bound DNA and carry out this function as components of larger protein complexes. Interestingly, HDAC5 has been shown to inhibit muscle differentiation through direct binding of myocyte enhancer factor-2 proteins, which activate skeletal myogenesis. This process is regulated by the phosphorylation-dependent binding of 14-3-3 and subsequent shift in subcellular localization of HDAC5 to the cytoplasm. To examine other cellular pathways that HDAC5 may modulate through signal-dependent export, 293 cells stably expressing EGFP-tagged HDAC5 were employed to evaluate protein localization and function, protein-protein interactions, and phosphorylation by fluorescence microscopy, co-immunoprecipitation, and mass spectrometry.