FIG. 2.
Mapping of MNase cleavage sites and DNase I-hypersensitive sites on the active and inactive HPRT promoters in vivo. (A) Mapping of nucleosome positions on the inactive and active HPRT promoters by MNase digestion. All lanes are from the same gel and autoradiogram and are aligned accordingly. All designations and symbols are described below. (B) Mapping of DNase I-hypersensitive sites relative to MNase cleavage sites. All lanes are from the same gel and autoradiogram and are aligned accordingly. Lanes 1 to 16, MNase-treated samples; lanes 17 to 20, DNase I-treated samples. MNase-treated samples in panel B were digested with lower concentrations of MNase than were samples in panel A. Band BclI (lane 17), position of the full-length genomic BclI fragment containing the HPRT promoter in purified genomic DNA; band BclI/BamHI (lane 18), relative position of a BamHI site 100 bp downstream of the translation initiation site within the full-length BclI genomic DNA fragment (Fig. 1). Lanes 19 and 20 (active allele), relative positions of DNase I-hypersensitive sites on the active HPRT allele; the diagram to the right indicates the positions of the DNase I-hypersensitive sites relative to those of the transcription factor binding sites (small boxes) and the major transcription initiation sites of the HPRT promoter (bent arrow) and the direction of transcription (Fig. 1). All bands in the Southern blots (A and B) were visualized by indirect labeling with a radiolabeled 400-bp probe located just upstream of a reference BclI site 838 bp downstream of the translation initiation site of the HPRT gene (Fig. 1). The diagrams to the right of lanes from the active allele (A, lanes 10 to 18, and B, lanes 9 to 16) indicate the translational positions of nucleosomes (ovals) on the active HPRT promoter relative to transcription factor binding sites (small boxes) as determined by the in vivo MNase cleavage pattern. The dashed oval indicates that the first downstream nucleosome may be modified, shifted, or absent in a subpopulation of cells since an “intranucleosomal” MNase cleavage occurs at position +170 (see text). Numbers to the right of the autoradiogram indicate the positions of MNase or DNase I cleavage sites relative to the translation initiation site. Active allele, samples from 4.12 cells containing an active HPRT gene on the active human X chromosome; inactive allele, samples from 8121 cells containing an inactive HPRT gene on the inactive human X chromosome; cells, DNA from permeabilized cells; DNA, naked DNA treated with MNase. All position numbers are relative the translation initiation site of the HPRT gene.