FIGURE 2. SIRT1 upregulates SUV39H1 activity in vitro and in vivo through the SIRT1 N terminus.

a, Methyltransferase (HKMT) activity of recombinant SUV39H1 as a function of increasing amounts of recombinant baculovirus-expressed SIRT1 or bacterially expressed SIRT2. Endogenous histone octamers (purified from HeLa cells) used as substrates were stained with Coomassie blue. Quantifications of multiple experiments are represented below and compared to the HKMT activity of SUV39H1 alone (lane 2), which is considered to be 100%. b, As in a, but in the absence or presence of NAD⁺. c, As in a, but with increasing amounts of either wild type or SIRT1 deletion mutants (expressed in Escherichia coli) that either do not contain the N terminus (N) or only contain the N terminus (NT). d, As in a, but with increasing amounts of either wild-type SIRT1 or the catalytically inactive point mutant H363Y (expressed in baculovirus). e, Western blots from immunoprecipitations as in Fig. 1c, but using the Flag-tagged SIRT1 N-terminal region (SIRT1 NT)³ and Myc–SUV39H1 as indicated. f, Western blots performed on extracts from 293 cells transfected with either empty vector or vector expressing the SIRT1 N terminus using the specific antibodies indicated. Core histones are stained with Coomassie blue. The right side shows quantification of the increased levels of H3K9me3 on overexpression of the N-terminal 250 residues of SIRT1 (SIRT1 NT) relative to the vector control. Error bars represent s.d. n = 4 for a and c; n = 3 for f.