SIRT7 Is Activated by DNA and Deacetylates Histone H3 in the Chromatin Context

Abstract

Mammalian sirtuins (SIRT1-7) are members of a highly conserved family of nicotinamide adenine dinucleotide (NAD(+))-dependent protein deacetylases that regulate many biological processes including metabolism, genome stability, and transcription. Among the seven human sirtuins, SIRT7 is the least understood, to a large extent due to the lack of enzymatic activity in vitro. Here, we reported that SIRT7 can be activated by DNA to hydrolyze the acetyl group from lysine residues in vitro on histone peptides and histones in the chromatin context. Both N- and C- termini of SIRT7 are important for the DNA-activated deacetylase activity. The regulatory mechanism of SIRT7 is different from that of SIRT6, which also showed increased activity on chromatin substrates, but the deacetylase activity of SIRT6 on a peptide substrate cannot be activated by DNA. This finding provides an improved enzymatic activity assay of SIRT7 that will promote the development of SIRT7 modulators. Further investigation into the activation mechanism of SIRT7 by DNA could provide new insights into its biological function and help the development of sirtuin activators.

Figure 1. SIRT7 deacetylase activity is significantly enhanced by DNA

(A) Overlaid HPLC traces showing SIRT7-catalyzed hydrolysis of H3K9 acetyl peptide with DNA. (B) HPLC traces showing SIRT7-catalyzed hydrolysis of H3K18 acetyl peptide with DNA. (C) Overlaid HPLC traces showing that other sirtuin proteins cannot be activated by DNA.

Figure 2. SIRT7 is activated by genomic DNA on chromatin substrates

(A) SIRT7 cannot be activated by exogenous DNA on histone proteins. (B) SIRT7 can deacetylate H3K18 in the chromatin context. (C) Genomic DNA is required for SIRT7 to remove H3K9 Ac from chromatin substrate. (D) Quantification of western blot results shown in (B & C). The levels of H3K9 Ac and H3K18 Ac were normalized by the amount of histone H3 proteins. The relative acetylation level was calculated by setting the negative control (SIRT7 minus reaction) level to 100%.

Figure 3. Both the N- and C- termini of SIRT7 are important for its activation by DNA

(A) Scheme showing the full length (FL, residue 1-400), N-terminal deletion (ΔN, residue 56-400), C-terminal deletion (ΔC, residue 1-364), and core domain (56-364) of SIRT7. (B) Coomassie blue-stained gel showing the purity of SIRT7 deletion mutants. (C) HPLC traces showing the activity of different SIRT7 deletion mutants with and without dsDNA on H3K18 Ac peptide. (D) The deacetylase activities of SIRT7 FL and deletion mutants on H3K18 Ac on chromatin substrates. Reactions without NAD were used as negative controls. (E) Quantification of the Western blot results in (D). The H3K18 Ac level in each reaction was normalized by the amount of histone H3 protein. The relative H3K18 Ac level was calculated by setting the level of negative control to 100%.