Fig. 4: Histone desuccinylation by HDAC1/2/3 in vitro.

a, b Endogenous HDAC1 proteins were immunoaffinity purified from HeLa nuclear extracts by magnetic bead-conjugated HDAC1 antibody and then used for in vitro desuccinylation assay using either histone substrates (a) or synthetic H3K14su peptide (b). The increasing amount of HDAC1-associated beads is 2 μL, 4 μL, and 8 μL. Histone substrates were prepared from HeLa cells treated with 1 μM TSA for 12 h. TSA concentration in in vitro reaction was 10 μM and the reaction time was 2 h. c Analysis of desuccinylation of synthetic H3K14su peptide by HDAC1 by mass spectrometry analysis. Desuccinylation reaction was carried out with 2 μL HDAC1 beads and incubated for 30 min. Quadrate indicates the H3K14 succinylated peptide peak, rhombus the H3K14 unmodified peptide peaks; the succinyl (m/z 960.8681 [M + 3H]3 + ) and desuccinyl (m/z 927.5302 [M + 3H]3 + ) peptides were indicated. d In vitro histone desuccinylation by HA-HDAC1, FLAG-HDAC2, and HA-HDAC3 immunoaffinity purified from transfected HEK293T cells. Histone substrates were prepared from HeLa cells treated with 1 μM TSA for 12 h. e–g In vitro histone desuccinylation by HA-HDAC1 and HDAC1(H141A) mutant (e), FLAG-HDAC2 and HDAC2 (H142A) mutant (f), and HA-HDAC3 and HDAC3 (Y298F) mutant (g). All tagged HDAC1/2/3 and mutants were expressed and immunoaffinity purified from transfected HEK293T cells. A 2-fold increased series of HDAC1/2/3 and mutants were used in the reactions. WB analysis also showed that NCoR was copurified with HA-HDAC3, indicating that both wild-type and mutant HDAC3 were incorporated into the NCoR complex.