Figure 4. SIRT1 deacetylates HDAC1 at residue K432 and stimulates its enzymatic activity.

a, Recombinant SIRT1 was titrated over a fixed amount of p300 and HDAC1 and the acetylation of HDAC1 was assessed using western blotting (* p < 0.05, one-way ANOVA). b, HDAC1-flag, p300-HA, and SIRT1-myc were expressed in HT22 cells. HDAC1 was immunoprecipitated, and its acetylation status was probed using an anti-acetyl lysine antibody. c, HDAC1 was pre-incubated with either p300 or with SIRT1 as in (A) and (B), and its enzymatic activity was examined using a fluorescence based HDAC enzymatic activity assay (Supplementary Fig. 3b) (** p < 0.01, one-way ANOVA). d, Sirt1 F/F neurons were infected with Cre-eGFP and eGFP lentiviral vectors, following which HDAC1 was immunoprecipitated, and its activity was measured as in c (*p < 0.05, unpaired t-test). e, Diagram depicting the acetylated lysine residues in HDAC1 and HDAC2. f, Annotated MS/MS spectrum of Lys acetylated peptide “NSSNFK_acK_acAK_acR” that identified lysine acetylation sites on K438, K439 and K441 of HDAC1 upon reaction with p300. b and y ions represents collision-induced peptide fragment ions containing N- or C-terminal respectively. “*” indicates fragment ions with neutral loss of amine. g, Label-free quantification for each lysine acetylation site using protein-abundance normalized peptide precursor ion intensity shows that lysine acetylation abundance on p300-treated HDAC1 decreased upon the addition of SIRT1 to the reaction. h, Recombinant SIRT1 was incubated together with p300 and HDAC1 as in a, and acetylation of HDAC1 was assessed using an antibody specific to acetylated K432. (Arrowhead shows a non-specific cross-reacting band in the lanes containing recombinant SIRT1; * p < 0.05, unpaired t-test). i, HEK293T cells were treated with the indicated concentrations of SIRT1 activator (Compound#10) for 12 h and the HDAC1 acetylation at K432 was assessed.