Human Class I Histone Deacetylase Complexes Show Enhanced Catalytic Activity in the Presence of ATP and Co-immunoprecipitate with the ATP-dependent Chaperone Protein Hsp70

Abstract

Antibodies to histone deacetylases (HDACs) have been used to immuno-isolate deacetylase complexes from HeLa cell extracts. Complexes shown to contain HDAC1, HDAC3, HDAC6, and HDAC1؉2 as their catalytic subunits have been used in an antibody-based assay that detects deacetylation of whole histones at defined lysines. The class II deacetylase HDAC6 was inactive in this assay, but the three class I enzymes deacetylated all histone lysines tested, although with varying efficiency. In comparison to HDAC1, HDAC3 preferentially deacetylated lysines 5 and 12 of H4 and lysine 5 of H2A. H4 tails in purified mononucleosomes were refractory to deacetylation by both HDAC1 and HDAC3, unless ATP was added to the reaction mix. Surprisingly, ATP also consistently enhanced cleavage of free, non-nucleosomal histones, but not small peptides, by both enzyme complexes. We found no evidence that ATP operates by phosphorylation of components of the HDAC complex, but have shown that HDACs 1, 2, and 3 all co-immunoprecipitate with the ATP-dependent chaperone protein Hsp70. Another common ATP-dependent chaperone, Hsp90, was absent from all HDAC complexes tested, whereas Hsp60 associated with HDAC1 only. We suggest that Hsp chaperone proteins enhance the deacetylase activity of HDAC complexes by ATP-dependent manipulation of protein substrates.

HDAC3: taking the SMRT-N-CoRrect road to repression

“…Although this approach does not provide pure enzymatic preparations, some differences in the preferred lysine residues of the individual complexes were observed. The in vitro results suggested that HDAC3 could completely deacetylate H2A, H4K5Ac and H4K12Ac, but only partially deacetylate H3, H2B, H4K8Ac and H4K16Ac (Johnson et al, 2002). Compared with HDAC1, HDAC3 deacetylated H4K8Ac, H4K16Ac and H2B at the same rate, but it deacetylated H4K5Ac, H4K12Ac and H2AK5Ac much more rapidly.…”

“…When targeted to preacetylated nucleosomal templates, Sin3/HDAC was found to deacetylate both H3 and H4, whereas the N-CoR/SMRT-HDAC3 complex displayed preferential activity toward H3 (Vermeulen et al, 2004). This result is somewhat surprising, considering the data discussed above (Johnson et al, 2002;Hartman et al, 2005). Future work is necessary to reconcile this difference.…”

“…Early studies showed that, like HDAC1 and HDAC2, HDAC3 isolated from mammalian cells can deacetylate both H3 and H4 in free histones or nucleosome substrates (Yang et al, 1997;Dangond et al, 1998;Emiliani et al, 1998). In a more detailed study, Johnson et al (2002) immunoprecipitated HDAC1, -2, -3 and -6 from cell extracts and compared their kinetics of cleavage of the acetyl group on different lysine residues of hyperacetylated free histones. Although this approach does not provide pure enzymatic preparations, some differences in the preferred lysine residues of the individual complexes were observed.…”

Histone deacetylases in acute myeloid leukaemia show a distinctive pattern of expression that changes selectively in response to deacetylase inhibitors

“…A mouse monoclonal antibody raised against HDAC3, but recognising HDAC1, 2 and 3, was from BD Transduction Laboratories; we have confirmed the unusual specificity of this antibody by comparison with our own antisera specific for HDAC1, 2 or 3. 21 Bound antibody was detected either with peroxidaseconjugated anti-rabbit antibody and Enhanced ChemiLuminescence (ECL, Amersham-Pharmacia, UK) or with IRDye-800-conjugated, affinity-purified goat anti-rabbit secondary antibody followed by quantitation with an infra-red dye imaging system (LI-COR Biosciences Ltd, UK). The latter was used for all experiments with primary blast cells from patients.…”

Upregulation of ABCG2 by Romidepsin via the Aryl Hydrocarbon Receptor Pathway

“…Hsp70 is an important cochaperone protein with Hsp90. It was reported that one of the class I HDACs is the deacetylase of Hsp70 and that romidepsin treatment can increase acetylation of Hsp70 (40). Of note, Hsp70 is required for the assembly of Hsp90-client protein complexes (41).…”