Figure 9. Depletion of TAF7 in cells increases the occupancy of LEC at Pol II-transcribed snRNA genes.

(a) Western blot showing TAF7 and Hsp90 at 48 h after transient transfection of non-targeting (control) siRNA pool or siRNA pool targeting TAF7. (b) Effect of TAF7 depletion on transcript levels of Pol II-transcribed snRNA genes detected by RT-qPCR performed with total RNA from cells transfected with siRNAs. Ct values were normalized to GAPDH. Data points are the average of three independent experiments, and error bars show s.d.. The P values for the indicated comparisons were determined by Student’s t test (*, P < 0.05; **, P < 0.01). (c) Schematic representation of immobilized oligo(dC)-tailed template assay. Templates immobilized on Streptavidin beads were incubated with purified recombinant protein EAF1 in the presence or absence of Mediator and/or TAF7 N243. After washing, bead-bound proteins were detected by Western blotting. (d) TAF7 interferes with EAF1 recruitment by Mediator in vitro. Upper panel shows immobilized oligo(dC)-tailed template assays. Assays were performed with an oligo(dC)-tailed template, EAF1, and Mediator in the presence or absence of TAF7 N243. Lower panel shows silver staining of Mediator and HA-tagged TAF7 N243 used in the assay. Mediator (x1) and HA-TAF7 N243 (x1) contain 1 pmol of the each protein or protein complex. (e) Model of immobilized oligo(dC)-tailed template assay. TAF7 N243 inhibits EAF1 recruitment to the oligo(dC)-tailed template by MED26-containing Mediator. (f) Depletion of TAF7 increased the occupancy of ICE1 (KIAA0947), EAF2, and total Pol II at U4-1 and U5B snRNA genes. Ct values of each ChIP were normalized to that of input. Data points are the average of three independent experiments; error bars show s.d.. The P values for the indicated comparisons were determined by Student’s t test (*, P < 0.05; **, P < 0.01).