FIG.6.

Chromatin remodeling of the MMTV promoter requires the binding of NF1. (A) Schematic of the MMTV promoter as a stable template, indicating the sites for restriction enzyme and PCR primer. (B) Stable cell lines containing Wt (pLS-Wt) or mNF1 constructs were treated with DEX for 1 h. Isolated nuclei were subjected to limited digestion with restriction endonucleases (DraI, SstI, and FokI). Purified DNA was digested to completion with AlwNI (for DraI- and SstI-digested samples) and with SstI (for FokI samples). Twenty micrograms of DNA was amplified by reiterative primer extension using Taq DNA polymerase and ³²P-labeled oligo TK-1. The PCR products were then separated on 6% denaturing polyacrylamide gels. (C) Schematic of MMTV promoter as a stable template showing the entry site for λ exonuclease, sites for restriction enzymes, and PCR primer. (D) As described in the legend for panel B, isolated nuclei were subjected to limited digestion with SstI with or without λ exonuclease, and purified DNA was digested to completion with AlwNI. Twenty micrograms of DNA was amplified by reiterative primer extension by using Taq DNA polymerase and ³²P-labeled oligo TK-1, and the products were analyzed on a 6% polyacrylamide gel. The 5′ boundary of NF1 is indicated.