SIRT7-dependent deacetylation of CDK9 activates RNA polymerase II transcription

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

± SD (n = 3) ( * P < 0.05, ** P < 0.01). See also Supplementary Figure S4B. (B) snoRNA and pre-mRNA levels are decreased in SIRT7-deficient cells. HEK293T cells were transfected with non-targeting siRNA or SIRT7-specific siRNA. RNA levels were measured by RT-qPCR and normalized to actin mRNA. Bars represent means ± SD (n = 3) ( * P < 0.05, ** P < 0.01). See also Supplementary Figure S4B . (C) Ectopic SIRT7 rescues downregulation of U3 and U13 snoRNA in SIRT7-knockout cells. HEK293T/SIRT7 −/− cells were transfected with Flag-tagged SIRT7/WT, SIRT7/H187Y or SIRT7/ N78 and snoRNA levels were monitored by RT-qPCR. The bars represent means ±SD (n = 3) ( * P < 0.05, ** P < 0.01). See also Supplementary Figure S4C . (D) Overepression of SIRT7 increases the association of the transcription machinery with target genes. ChIP-qPCR monitoring Pol I and Pol II occupancy at selected target genes after overexpression of Flag-SIRT7 (WT) or mutant SIRT7/H187Y. Antibodies against RPA116 (Pol I) and RPB1 (Pol II, N20) were used for ChIP. Bars represent means ± SD (n = 3) ( * P < 0.05, ** P < 0.01). See also Supplementary Figure S4A . (E) ChIP-qPCR monitoring occupancy of Pol I (anti-RPA116) and Pol II (anti-RPB1, N20) at selected target genes after shRNA-(left panel) or siRNA-(right panels) mediated depletion of SIRT7. Cells transfected with non-targeting shRNA/siRNA served as control. The bars represent means ± SD (n = 3) ( * P < 0.05, ** P < 0.01). See also Supplementary Figure S4A and S4D. (F) SIRT7 and CDK9 occupancy is enriched at the promoter of target genes. ChIPs monitoring occupancy of endogenous CDK9 or stably expressed Flag-HA-SIRT7 at different regions of Brf2 and JMJD4 using antibodies against CDK9 or the Flag epitope. The bars represent mean values ± SD (n = 3). The scheme depicts the position of the amplified regions. See also Supplementary Figure S4E . (G) Knockdown of SIRT7 impairs CDK9 occupancy at target genes. ChIP-qPCR monitoring occupancy of endogenous CDK9 at target genes upon siRNA-mediated knockdown of SIRT7, cells transfected with non-targeting siRNA serving as control. Bars represent means ± SD (n = 3) ( * P < 0.05, ** P < 0.01). See also Supplementary Figures S4A and S4F. (H) Knockdown of SIRT7 abolishes Pol II occupancy at selected target genes. ChIPs showing occupancy of Pol II phosphorylated at CTD-Ser5 and CTD-Ser2 in cells transfected with SIRT7-specific siRNA or with non-targeting siRNA using antibodies against pSer5-CTD and pSer2-CTD. Bars represent means ± SD (n = 3) ( * P <0.05, ** P < 0.01). See also Supplementary Figures S4A and S4F . Model illustrating the role of SIRT7 in Pol II transcription. SIRT7-mediated deacetylation promotes the release of CDK9/cyclin T1 from the inactive P-TEFb/7SK snRNP complex and activates P-TEFb by deacetylation of CDK9, which leads to increased CTD-Ser2 phosphorylation and transcriptional processivity. tasis of cancer cells (5, 6) . Likewise, aberrant expression of CDK9 and cyclin T1/T2 has been observed in various tumors (41) , demonstrating that regulation of P-TEFb activity is crucial for controlled gene expression. Based on the available data we propose the following model of P-TEFbdependent transcription activation ( Figure 5 ). In normal cells, a significant amount of P-TEFb is sequestered in a large inactive complex containing 7SK/HEXIM1, activation requiring release from the 7SK ribonucleoprotein complex. Given that SIRT7 serves a vital role in the cellular stress response (12,