Coupling of histone deacetylation to NAD breakdown by the yeast silencing protein Sir2: Evidence for acetyl transfer from substrate to an NAD breakdown product

Abstract

The Saccharomyces cerevisiae silencing protein Sir2 is the founding member of a universally conserved family of proteins that have been shown to possess NAD-dependent histone deacetylation and ADP-ribosylation activities. Here we show that histone deacetylation by Sir2 is coupled to cleavage of the high-energy bond that links the ADP-ribose moiety of NAD to nicotinamide. Analysis of the NAD cleavage products revealed the presence of nicotinamide, ADP-ribose, and a third product that appeared to be related to ADP-ribose. With the use of label transfer experiments, we show that the acetyl group in the histone substrate is transferred to this NAD breakdown product during deacetylation, forming a product that we conclude to be O-acetyl-ADP-ribose. Detection of this species strongly argues for obligate coupling of histone deacetylation to NAD breakdown by Sir2. We propose reaction mechanisms that could account for this coupling via acetyl-ADP-ribose formation. The unprecedented coupling of amide bond cleavage to cleavage of a high-energy bond raises the possibility that NAD breakdown by Sir2 plays an important role in silencing that is independent of its requirement for deacetylation.

Inflammatory stimuli induce inhibitory S-nitrosylation of the deacetylase SIRT1 to increase acetylation and activation of p53 and p65

“…The in vitro ADP-ribosylation assay was performed as previously described (108) with minor modifications, using FLAG-tagged wild-type and mutated SIRT1 and immunopurified from COS-7 cells as described above. The reaction was carried out in SIRT1 activity assay buffer (Biomol) containing immunopurified SIRT1, 32 P-labeled NAD + (10 μCi, Amersham Biosciences), unlabeled NAD + (10 μM), and histone H3 (3 μg) for 1 hour at 37°C in the presence of a PARP inhibitor, PJ34 (3 μM, EMD Biosciences), and an inhibitor for arginine mono-ADP-ribosyltransferases, m -iodobenzylguanidine (0.5 mM, MIBG, Sigma).…”

New Insights into the Regulation of Heterochromatin

“…Sir2 is a histone deacetylase with activity mainly towards H4K16 [29–31], the acetylation status of which directly regulates higher-order chromatin folding in vitro [32] and plays a major role in heterochromatin function in vivo [6, 33]. Sir3 and Sir4 preferentially interact with histone tails devoid of H4K16ac [34–36].…”

Chromosome boundary elements and regulation of heterochromatin spreading

“…Sir2 is a histone deacetylase with main activity on H4K16 [26–28], the acetylation of which directly regulates higher-order chromatin folding in vitro [14] and plays a major role in heterochromatin function in vivo [13, 29]. Sir3 and Sir4 preferentially interact with histone tails devoid of H4K16ac [30–32].…”