Inactivating mutation in histone deacetylase 3 stabilizes its active conformation

Histone deacetylases (HDACs), together with histone acetyltransferases (HATs), regulate gene expression by modulating the acetylation level of chromatin. HDAC3 is implicated in many important cellular processes, particularly in cancer cell proliferation and metastasis, making inhibition of HDAC3 a promising epigenetic treatment for certain cancers. HDAC3 is activated upon complex formation with both inositol tetraphosphate (IP4) and the deacetylase‐activating domain (DAD) of multi‐protein nuclear receptor corepressor complexes. In previous studies, we have shown that binding of DAD and IP4 to HDAC3 significantly restricts its conformational space towards its stable ternary complex conformation, and suggest this to be the active conformation. Here, we report a single mutation of HDAC3 that is capable of mimicking the stabilizing effects of DAD and IP4, without the presence of either. This mutation, however, results in a total loss of deacetylase activity, prompting a closer evaluation of our understanding of the activation of HDAC3.     

Protein Kinase CK2 Regulates the Dimerization of Histone Deacetylase 1 (HDAC1) and HDAC2 during Mitosis

Histone deacetylase 1 (HDAC1) and HDAC2 are components of corepressor complexes that are involved in chromatin remodeling and regulation of gene expression by regulating dynamic protein acetylation. HDAC1 and -2 form homo- and heterodimers, and their activity is dependent upon dimer formation. Phosphorylation of HDAC1 and/or HDAC2 in interphase cells is required for the formation of HDAC corepressor complexes. In this study, we show that during mitosis, HDAC2 and, to a lesser extent, HDAC1 phosphorylation levels dramatically increase. When HDAC1 and -2 are displaced from the chromosome during metaphase, they dissociate from each other, but each enzyme remains in association with components of the HDAC corepressor complexes Sin3, NuRD, and CoREST as homodimers. Enzyme inhibition studies and mutational analyses demonstrated that protein kinase CK2-catalyzed phosphorylation of HDAC1 and -2 is crucial for the dissociation of these two enzymes. These results suggest that corepressor complexes, including HDAC1 or HDAC2 homodimers, might target different cellular proteins during mitosis.     

Analysis of Human Nuclear Protein Complexes by Quantitative Mass Spectrometry Profiling

Analysis of protein complexes provides insights into how the ensemble of expressed proteome is organized into functional units. While there have been advances in techniques for proteome‐wide profiling of cytoplasmic protein complexes, information about human nuclear protein complexes are very limited. To close this gap, we combined native size exclusion chromatography (SEC) with label‐free quantitative MS profiling to characterize hundreds of nuclear protein complexes isolated from human glioblastoma multiforme T98G cells. We identified 1794 proteins that overlapped between two biological replicates of which 1244 proteins were characterized as existing within stably associated putative complexes. co‐IP experiments confirmed the interaction of PARP1 with Ku70/Ku80 proteins and HDAC1 (histone deacetylase complex 1) and CHD4. HDAC1/2 also co‐migrated with various SIN3A and nucleosome remodeling and deacetylase components in SEC fractionation including SIN3A, SAP30, RBBP4, RBBP7, and NCOR1. Co‐elution of HDAC1/2/3 with both the KDM1A and RCOR1 further confirmed that these proteins are integral components of human deacetylase complexes. Our approach also demonstrated the ability to identify potential moonlighting complexes and novel complexes containing uncharacterized proteins. Overall, the results demonstrated the utility of SEC fractionation and LC–MS analysis for system‐wide profiling of proteins to predict the existence of distinct forms of nuclear protein complexes.