Cell Research (2011) 21: 1262-1266. doi: 10.1038/cr. 2011.105; published online 21 June 2011 more, hMOF is also acetylated at Lys274 in vivo (see discussion later). Sequence alignment of hMOF with other members of the MYST family HATs reveals that among all the identified acetylation sites, Lys274 is the only strictly conserved residue, suggesting that acetylation of this site might be common and probably functionally important for the MYST family HATs. Lys274 and two other strictly conserved residues (Cys316 and Glu350) which are in the vicinity of the acetyl group of the cofactor acetyl-CoA were mutated to study their functional roles (Supplementary information, Data S1 and Figure S2). Strikingly, mutation of Lys274 to Arg (K274R) almost completely abolished the hMOF activity (0.1%), while the K274Q mutant retained about 2.9% activity (Supplementary information, Figure S2A), suggesting that the loss of the HAT activity by the K274R mutation could be due to the inability of Arg to be acetylated. To investigate whether Lys274 is autoacetylated, the wild-type and mutant hMOF proteins were incubated with acetyl-CoA and subsequently subjected to western blot with an antibody specific to acetylated lysine. Intriguingly, for all the hMOF proteins except the K274Q mutant, the acetylation levels prior to the acetyl-CoA incubation and the extents of the increase after the incubation correlated well with their respective HAT activities (Supplementary information, Figure S2), indicating that the acetylation of hMOF is catalyzed by hMOF itself. Although the K274Q mutant had an HAT activity comparable to that of the G327E mutant, its acetylation was undetected, which is in agreement with our MS data that Lys274 is the main acetylation site of the hMOF catalytic domain.

Next we investigated whether the autoacetylation of Lys274 of hMOF occurs inter-or intra-molecularly. The GST-tagged hMOF was incubated with either His-tagged wild-type or mutant hMOF in the presence or absence of acetyl-CoA (Figure 1B). Both GST-tagged and Histagged wild-type hMOF could autoacetylate themselves; however, the His-tagged K274R and G329E mutants exhibited no or very weak activity. The presence of any of the His-tagged hMOF proteins had no appreciable effect