A region containing the DNase I HS located −14 kb upstream of the murine PU.1 gene directs cell type–specific expression in stably transfected cells.

(A) The DNase I HS region does not confer specificity (or activity) in transient transfections. The 3.5-kb HindIII fragment located at −14 kb (Figure 1B), containing a DNase I HS (Figure 3A), was placed in front of a 500-bp fragment containing 334 bp of 5′ upstream promoter sequence and 152 bp of 5′ untranslated sequences in front of a luciferase reporter gene in both normal and reverse orientation, as shown in the top diagram. These constructs were transiently transfected into myeloid and T-cell lines and luciferase activity measured (bottom panels) and compared with the activity of the promoter fragment or luciferase vector only. Also shown is the SD of at least 3 independent experiments. (B) The murine PU.1 upstream DNase I HS region confers activity in stable cell lines in myeloid cell lines but not a T-cell line. The PU.1 334-bp promoter constructs, with (top) or without (bottom) the 3.5-kb HindIII fragment containing the −14-kb DNase I HS, were stably transfected into myeloid U937 and 416B as well as T-cell BW5147 lines, and multiple independent clones assayed for luciferase activity normalized to the plasmid copy number as determined by Southern blot analysis. Shown is the SD of at least 10 independent clones for each construct in each cell line.