Identification of a 5′-UTR sequence modulating β₃ gene expression. (A) DNaseI footprint analysis. DNaseI footprint analysis was performed using a ³²P-labeled probe of the β₃ promoter from position −146 to +29 (shown above), incubated without (lane 1) and with (lane 2) K562 nuclear extracts. The protected region in the human β₃ gene is from position +13 to +22 relative to the transcription start site and corresponds to a region of high human-avian sequence homology. The sequence mutated for subsequent functional analysis (C) is indicated. (B) EMSA using the ³²P-labeled oligonucleotide probe incubated with nuclear extracts of K562 and K562 cells treated with PMA, Dami, HEL, HMEC-1, 293, or CHO cells. Equivalent amounts of labeled probe were added to each experiment per lane. The amount of probe associated with the shown K562 complex was approximately 20% of the total amount of free probe. The free probe was deliberately run off the gel to maximize resolution of the DNA-protein complex; no other gel shifts were observed. Competitors were used in a 50-fold molar excess and included both an unlabeled EMSA (c.c., cold competition) and an irrelevant (Irrel.) oligonucleotide. A lane with probe but no nuclear extract (lane 1) showed only the fast-migrating radiolabeled probe. (C) Functional assessment of the CCGCGGGAGG sequence. Sequence substitutions in the footprinted region (shown in [A]) were introduced into construct −146mutLuc. K562 and 293 cells were transfected with equivalent amounts of the promoterless (pGL2), −146Luc, and −146mutLuc constructs and assayed for luciferase activity, and all values were normalized for transfection efficiency within each cell line by cotransfection with pSV40-CAT. An approximately 2.6-fold difference between −146Luc and −146mutLuc was observed. To account for differences in transfection efficiency between K562 and 293 cells (the latter producing much higher activities with all three constructs), the data were normalized and expressed as fold activation over activity of the promoterless construct, pGL2. The −146mutLuc construct contained the mutated sequence shown in Fig 5B. Each experiment was performed four times.