In vivo footprinting of the proximal CD20 promoter by amplifying either the coding or the noncoding strand. (A) Naked genomic DNA (lane 1) or intact cells from the B-cell line HS-Sultan (lane 2) were treated with DMS and in vitro or in vivo footprints were visualized by ligation mediated PCR using primer set 1. The numbers on the left correspond to the sequence of the CD20 promoter as shown in panel D. The in vivo protected residues are designated site 1 and are surrounded by a bracket. (B) In vivo footprinting using primer set 1 over the region indicated was performed using in vivo methylated DNA from the following cell lines: lane 1, CD20positive B-cell line BJA-B; lane 2, CD20 negative pre–B-cell line PB697. (C) In vivo footprinting on the opposing strand of the CD20 promoter was performed using primer set #2. Naked DNA (lane 1) or intact cells from the B-cell line HS-Sultan (lane 2) or the cervical carcinoma cell line HeLa (lane 3) were used. (D) Nucleotide sequence of the first 230 base pairs of the CD20 promoter. Known transcriptional start sites are marked with an arrow (⇓), a nucleotide residue within the most 3′ cluster of transcriptional start sites is designated +1. Binding sites for regulatory proteins as determined by EMSA and in vivo footprinting are surrounded by a bracket. In vivo protected nucleotides are indicated by a triangle (▾), nucleotides rendered hypersensitive by in vivo methylation by a diamond (♦).