SIRT7-dependent deacetylation of CDK9 activates RNA polymerase II transcription

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

CLIP assays have been performed essentially as described (13) . Briefly, nuclear lysates from UV-irradiated HEK293T cells (254 nm, 0.15 Jcm −2 ) expressing Flag-tagged SIRT7 were sonicated, precleared with protein G Sepharose, and protein-RNA complexes were immunoprecipitated using anti-Flag M2 beads (Sigma) or protein G Sepharose (GE Healthcare) coated with mouse IgGs as control. Beads were sequentially washed in IP buffer (20 mM Tris-HCl [pH 8.0], 200 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, protease inhibitors) and in IP buffer containing 400 mM KCl. After elution with the Flag peptide (200 g/ml) and proteinase K digestion (30 min, 55 • C), RNA was isolated, incubated with DNase I (Sigma) and subjected to RT-qPCR. For pulldown experiments, Flag-tagged proteins (1 g) were immobilized on M2 agarose and incubated with radiolabeled RNA for 1 h at room temperature in buffer AM-100 supplemented with 0.1% Triton X-100, 0.2 U/ml RNasin and protease inhibitors. After stringent washing, captured RNA was extracted, subjected to gel electrophoresis and visualized by PhosphorImaging. Alternatively, 5 g of biotinylated 5 ETS-RNA (+10/+389) were incubated with 50 l of streptavidin-coated magnetic beads (Thermo Fisher Scientific) for 30 min at room temperature in 5 mM Tris-HCl [pH 7.5], 0.5 mM EDTA, 100 mM NaCl, 0.05% Tween-20. After washing, 8 pmol of bead-bound RNA were incubated with 70 g of nuclear extract from HEK293T cells expressing Flag-tagged SIRT7 or with 20 pmol of purified GST-SIRT7/1-81 in buffer AM-100 supplemented with 0.1% Triton X-100, 0.2 U/ml RNasin (Promega) and protease inhibitors (Roche) for 4 h at 4 • C. After washing with 20 mM Tris-HCl [pH 8.0], 400 mM NaCl, 1 mM EDTA, 0.2% Tween-20 and protease inhibitors, bound proteins were analyzed on immunoblots.