Mouse Sir2 Homolog SIRT6 Is a Nuclear ADP-ribosyltransferase*

Members of the Sir2 family of NAD-dependent protein deacetylases regulate diverse cellular processes including aging, gene silencing, and cellular differentiation. Here, we report that the distant mammalian Sir2 homolog SIRT6 is a broadly expressed, predominantly nuclear protein. Northern analysis of embryonic samples and multiple adult tissues revealed mouse SIRT6 (mSIRT6) mRNA peaks at day E11, persisting into adulthood in all eight tissues examined. At the protein level, mSIRT6 was readily detectable in the same eight tissue types, with the highest levels in muscle, brain, and heart. Subcellular localization studies using both C- and N-terminal green fluorescent protein fusion proteins showed mSIRT6-green fluorescent protein to be a predominantly nuclear protein. Indirect immunofluorescence using antibodies to two different mSIRT6 epitopes confirmed that endogenous mSIRT6 is also largely nuclear. Consistent with previous findings, we did not observe any NAD+-dependent protein deacetylase activity in preparations of mSIRT6. However, purified recombinant mSIRT6 did catalyze the robust transfer of radiolabel from [32P]NAD to mSIRT6. Two highly conserved residues within the catalytic core of the protein were required for this reaction. This reaction is most likely mono-ADP-ribosylation because only the modified form of the protein was recognized by an antibody specific to mono-ADP-ribose. Surprisingly, we observed that the catalytic mechanism of this reaction is intra-molecular, with individual molecules of mSIRT6 directing their own modification. These results provide the first characterization of a Sir2 protein from phylogenetic class IV.