FIG. 3.
RB occupies a human U6 snRNA but not Ad VAI promoter during repression. (A) Affinity purification of GST-RB proteins and associated promoter-containing DNAs from in vitro RB repression assays. Portions of the human U6 snRNA and Ad VAI transcription assays shown in Fig. 2C were cross-linked with formaldehyde prior to affinity purification with glutathione-Sepharose to recover the GST-RB proteins. The concomitant recovery of associated promoter plasmid DNA was then measured by PCR. The recovered samples were analyzed for the presence of the U6 reporter construct (pU6/Hae/RA.2) or the Ad VAI reporter construct (M13-Ad VAI) with primers specific to the promoters contained in these plasmids. (B) RB occupies an endogenous human U6 snRNA (class 3) promoter but not a tRNALys (class 2) promoter in vivo. ChIP experiments were performed similarly to those described previously. A 10-fold serial dilution of input chromatin, from 10 to 0.01%, is shown in lanes 1 to 4. Immunoprecipitation reaction mixtures were prepared with IgG (lane 5), anti-SNAP43 (lane 6), anti-TBP (lane 7), or anti-RB (lane 8) antibodies, and any recovered DNA was analyzed with primers specific for the U6 snRNA, U2 snRNA, and tRNALys promoters or GAPDH exon 2 as a negative control.