Figure 2.

SIRT7 is associated with the rDNA and RNA Pol I. (A) Results of ChIP analysis showing the localization of SIRT7 and Pol I within the rDNA gene repeats. Cross-linked chromatin from 293T cells was precipitated by the indicated antibodies and analyzed by PCR with the indicated primer pair. (B) Endogenous SIRT7 interacts with RNA Pol I. Partially purified human nuclear extract was immunoprecipitated with control nonspecific human antibodies (lanes 2,5) and α-Pol I antibodies (lanes 3,6). Before immunoprecipitation, the protein fraction used was treated with DNase (lanes 4–6) or left untreated (lanes 1–3). The precipitated proteins were analyzed by Western blotting with α-SIRT7 and α-RPA-116 antibodies as indicated. The input lanes contain 10% of the protein fraction used for IP. (C) Pol I is a component of the TAP-SIRT7 complex. U2OS cell lines harboring the empty vector or TAP-SIRT7 were established. TAP-tagged complexes were purified on IgG-Sepharose and Calmodulin-agarose resins, and the eluted protein complexes were tested for copurifying proteins by Western blot analysis. (Lane 1) Fifty micrograms of nuclear extract (10% of the Input). (Lane 2) Eluate from mock-transfected cells. (Lane 3) Eluate from a cell line expressing TAP-IRT7. The Western blot was probed with an antibody specific for RPA-116 (top panel) and α-SIRT7 (bottom panel). (D) SIRT7 interacts with histones. Histones were isolated from butyric acid-treated HeLa cells by ion exchange chromatography, incubated with GST-SIRT7-Sepharose and GST-Sepharose beads. Bound proteins were eluted and separated by SDS-PAGE and visualized by staining with Coomassie.