Figure 3: SIRT7 catalyses histone H3K122 desuccinylation in vitro.

(a) FLAG-SIRT5, FLAG-SIRT6, FLAG-SIRT7wt or FLAG-SIRT7H187Y was expressed in and purified with anti-FLAG M2 affinity gel from HEK293T cells and stained with Coomassie brilliant blue. (b) In vitro desuccinylation assays with synthesized H3K122succ peptides. Two micrograms of purified FLAG-SIRT7wt, FLAG-SIRT7H187Y, FLAG-SIRT5 or FLAG-SIRT6 were incubated with 500 ng H3K122succ peptides in the presence or absence of 1.0 mM NAD⁺. The reaction mixtures containing 8, 16 or 25 ng peptides were boiled and subjected to dot blot analysis with anti-H3K122succ or anti-H3. The dots were quantified by densitometry with ImageJ software. The numbers indicate the relative levels of the indicated modifications. (c) In vitro desuccinylation assays with calf thymus histones. Different amounts of purified FLAG-SIRT7wt, FLAG-SIRT7H187Y, FLAG-SIRT5wt or FLAG-SIRT6wt were incubated with 1 μg calf thymus histones in the presence of 1.0 mM NAD⁺ and/or 10 mM NAM. The reaction mixtures were boiled and analysed by western blotting with the indicated antibodies. (d) The base peaks of H3K122succ in control and SIRT7-treated calf thymus histones. The peak areas were used for the quantification of H3K122succ in the two samples. (e) The MS/MS spectra for the identification of H3K122succ. b and y ions indicate peptide backbone fragment ions containing the N and C terminal, respectively. ++ indicates doubly charged ions. (f) The quantification ratios of several succinylation and acetylation sites in histones by comparing the peak areas in SIRT7-treated and control samples. (g) In vitro desuccinylation assays with mononucleosomes. Different amounts of purified FLAG-SIRT7wt or FLAG-SIRT7H187Y were incubated with 1 μg HeLa cell-derived mononucleosomes in the presence or absence of 1.0 mM NAD⁺ and/or 10 mM NAM. The reaction mixture was analysed by western blotting with the indicated antibodies.