FIG. 6.
GR binding recruits Oct-2 to octamer motifs adjacent to GR binding sites in the nucleus. (A) Nuclei prepared from CHO cells transfected with the MMTV promoter construct pHCWT, GR, and/or Oct-2 expression plasmids and treated with 10−6 M Dex or vehicle for 15 min were restricted with HindIII and digested with λ exonuclease (λexo) as indicated. Digestion was revealed by linear PCR extension of a primer extending from +74 to +52 of the MMTV LTR, and pause sites were positioned relative to A and T sequencing tracks amplified with the same primer. The Dex-, GR-, and Oct-2-specific λ pause site is indicated by the solid arrow, while the constitutive pause site generated by NF1 is indicated by the open arrow. The positions of transcription factor binding sites in the MMTV LTR are indicated schematically to the right of the autoradiogram. (B) Nuclei prepared from CHO cells transfected with pBluescript containing the strong GRE and octamer motif sequences from the MMTV LTR separated by 29 nucleotides (8) and GR and/or Oct-2 expression plasmids and treated with 10−6 M Dex or vehicle for 15 min were restricted with SmaI and digested with λ exonuclease as indicated. Digestion was revealed by linear PCR extension of a T3 polymerase primer, and pause sites were positioned relative to an A sequencing track amplified with the same primer. The positions of the octamer motif sequence and GRE sequences in the MMTV LTR are summarized schematically. The Dex-, GR-, and Oct-2-specific λ pause site is indicated by the arrow. (C) Nuclei prepared from CHO cells transfected with pBluescript containing either a Gal4 binding site separated by 8 nucleotides from the octamer motif sequence from the MMTV LTR (left) or a nonspecific oligonucleotide encoding an IAP enhancer core (right) along with Gal-GRWT, Gal-GRL501P, and/or Oct-2 expression plasmids were restricted with XhoI and digested with λ exonuclease as indicated. Digestion was revealed by linear PCR extension of a T7 polymerase primer, and pause sites were positioned relative to an A sequencing track amplified with the same primer. The positions of the octamer motif-IAP sequence and of the Gal4 sequence are summarized schematically. The Gal-GRWT-, Oct-2-, and octamer motif-dependent specific λ pause site is indicated by the arrow. Western blots of cellular extracts verified that Gal-GRWT and Gal-GRL501P were expressed to the same levels (73).