Figure 2.

BRG1-dependent restriction endonuclease chromatin accessibility within the MMTV promoter. A) Nucleosomal organization of the MMTV promoter. When stably integrated into the host genome the MMTV promoter is organized into a phased array of six positioned nucleosomes (A-F). The hormone inducible hypersensitivity region is encompassed by nucleosome B (Nuc-B). The expanded schematic of the proximal portion of the MMTV promoter shows the location of hormone-response elements (HREs), nuclear factor 1 (NF1), octamer transcription factors (OTFs) and the TATA binding protein (TBP) and identifies cleavage sites for restriction enzymes to test for induced hypersensitivity and chromatin remodeling (21). B) SW-13/MMTV cells, expressing GR, were transiently transfected with empty vector (-BRG1) or BRG1 expression plasmid (+BRG1). Nuclei, from untreated (-) or dexamethasone treated (10⁻⁷M) cells, were isolated and digested with restriction endonucleases targeting sequences within nucleosome-B (in vivo: Sst I, Mbo I, or Fok I). Purified genomic DNA was digested to completion with Hae III (In vitro). Fifteen micrograms of each sample was analyzed using linear Taq polymerase reiterative primer extension with a ³²P-labeled single-stranded specific for MMTV. Extension products were analyzed on a 6% polyacrylamide denaturing gel and exposed to PhosphorImage screen.