SIRT7-dependent deacetylation of CDK9 activates RNA polymerase II transcription

Blank, Maximilian F.; Chen, Sifan; Poetz, Fabian; Schnölzer, Martina; Voit, Renate; Grummt, Ingrid

If CDK9 is deacetylated by SIRT7, CDK9 should be hyperacetylated after knockdown of SIRT7. Indeed, a marked increase in CDK9 acetylation was observed after depletion of SIRT7 ( Figure 3D ). To monitor deacetylation in vitro, HA-CDK9 was co-expressed with Flag-GCN5, incubated with recombinant SIRT7, and acetylation was monitored on immunoblots. Acetylation was significantly reduced upon incubation with wildtype SIRT7 in the presence of NAD + , confirming that SIRT7 is the enzyme that deacetylates CDK9 ( Figure 3E showing that the N-terminal region of SIRT7 mediates RNA binding in vivo. UV-crosslinked Flag-SIRT7-RNA complexes were captured on anti-Flag beads, and co-precipitated RNA was analyzed by RT-qPCR. The percentage of precipitated RNA relative to input RNA is shown. Error bars denote means ±SD (n = 3) ( * P < 0.05, ** P < 0.01, n.s.: not significant). (D) The N-terminal part is required for nucleolar localization of SIRT7. Direct fluorescence showing the cellular localization of GFPtagged SIRT7, SIRT7/ N32 and SIRT7/ N78. Indirect immunofluorescence and direct GFP fluorescence analysis was done as described (13) . Nucleoli were stained with anti-UBF antibodies. Scale bar, 10 m. (E) The N-terminal region of SIRT7 mediates protein interactions. Flag-SIRT7 or mutant N78 were immunoprecipitated and co-precipitated proteins were visualized on immunoblots. not interact with SIRT7, did not deacetylate CDK9 in vitro, underscoring the importance of the N-terminus for SIRT7 function (Supplementary Figures S3C and S3D) .