Biotinylation of lysine method identifies acetylated histone H3 lysine 79 in Saccharomyces cerevisiae as a substrate for Sir2

Abstract

Although the biological roles of many members of the sirtuin family of lysine deacetylases have been well characterized, a broader understanding of their role in biology is limited by the challenges in identifying new substrates. We present here an in vitro method that combines biotinylation and mass spectrometry (MS) to identify substrates deacetylated by sirtuins. The method permits labeling of deacetylated residues with amine-reactive biotin on the ϵ-nitrogen of lysine. The biotin can be utilized to purify the substrate and identify the deacetylated lysine by MS. The biotinyl-lysine method was used to compare deacetylation of chemically acetylated histones by the yeast sirtuins, Sir2 and Hst2. Intriguingly, Sir2 preferentially deacetylates histone H3 lysine 79 as compared to Hst2. Although acetylation of K79 was not previously reported in Saccharomyces cerevisiae, we demonstrate that a minor population of this residue is indeed acetylated in vivo and show that Sir2, and not Hst2, regulates the acetylation state of H3 lysine 79. The in vitro biotinyl-lysine method combined with chemical acetylation made it possible to identify this previously unknown, low-abundance histone acetyl modification in vivo. This method has further potential to identify novel sirtuin deacetylation substrates in whole cell extracts, enabling large-scale screens for new deacetylase substrates.

Lysine Acetylation Is Widespread on Proteins of Diverse Function and Localization in the Protozoan Parasite Toxoplasma gondii

“…Acetylation of K23 and K79 on H3 and K31 on H4 are novel histone acetylation marks that have yet to be described in Apicomplexa. In very recent reports, low levels of H3 K79 acetylation have been detected in HeLa cells and Saccharomyces cerevisiae yeast (3,14), but the role of this novel mark has yet to be defined. The majority of H3 K79 is methylated in other species by Dot1, but intriguingly, Toxoplasma does not appear to possess a Dot1 homologue (36).…”

Human OGG1 activity in nucleosomes is facilitated by transient unwrapping of DNA and is influenced by the local histone environment

“…Chemically acetylated H2B protein was prepared based on previously published methods 29, 30 , which provides solution-accessible lysine residues as potential substrates. Briefly, 0.3 µmol of purified H2B was dissolved in 1 mL of freshly prepared 7 M urea solution with 500 mM ammonium acetate and 50 mM ammonium bicarbonate (pH 8.5).…”

The evolution of human SIRT1 for multi-specificity is enabled by active-site adaptation

“…Several studies on the specificity of hSIRT1, using different types of peptide arrays (Blander et al, 2005;Garske and Denu, 2006), revealed no clear consensus sequence for acetyl-lysine recognition. In contrast to the multispecificity of hSIRT1, the substrate repertoire analysis of the ySir2 ortholog from S. cerevisiae revealed its narrow specificity toward conserved histone acetyl-lysine residues (Bheda et al, 2012). This observation suggests that multi-specificity in SIRT1 can be acquired or lost through evolution, nevertheless, its molecular basis is still unknown.…”