Abstract
Previous analyses of the uteroglobin promoter revealed seven distinct regions, which contribute to its overall activity in epithelial cells from endometrium and lung. Most significantly, a mutation of the promoter sequence around 65 base pairs upstream of the transcriptional start site severely impairs promoter activity. The transcription factor acting through this sequence has not been identified yet. Here, we report that members of the Sp transcription factor family specifically recognize this non-classical GC box, in addition to another functional motif located 230 base pairs upstream of the transcriptional start site. We have characterized in detail the interaction of recombinant Sp3 with both motifs by DNase I footprinting and methylation protection using the wild-type uteroglobin promoter and various linker scanning mutants as templates. Electrophoretic mobility shift analyses show that Sp1 and Sp3 both bind with similar affinity to these elements. We demonstrate that the DNA-binding proteins in the endometrial cell line Ishikawa which recognize these motifs are also Sp1 and Sp3. Gene transfer experiments into Drosophila Schneider cells that do not contain endogenous Sp factors revealed that both DNA motifs respond to transiently expressed Sp1 and Sp3. Our results show thus that the level of transcription from the uteroglobin promoter is controlled by members of the Sp transcription factor family through unusual Sp binding sites.
Highlights
Previous analyses of the uteroglobin promoter revealed seven distinct regions, which contribute to its overall activity in epithelial cells from endometrium and lung
To see whether the distal CACCC motif is recognized by members of the Sp family, we performed DNase I protection experiments with recombinant Sp3 using the wild-type uteroglobin promoter (WT) and the appropriate linker scanning mutants LS-232/-240 and LS-222/-229 (Fig. 1) as templates
With the promoter variant in which the proximal CACCC motif is mutated (LS-222/-229), no protection from DNase I cleavage is visible (Fig. 2A, compare lane with lanes and 13). These results show that region VI of the uteroglobin promoter contains only a single Sp binding site and that only the proximal CACCC box is part of the Sp recognition sequence
Summary
Vol 270, No 21, Issue of May 26, pp. 12737-12744, 1995 Printed in U.S.A. Members of the Sp Transcription Factor Family Control Transcription from the Uteroglobin Promoter*. We report that members of the Sp transcription factor family recognize this non-classical GC box, in addition to another functional motif located 230 base pairs upstream of the transcriptional start site. The DNA binding domains of all three proteins are highly conserved and recognize the GC box and a CACCC motif present in region VI of the uteroglobin promoter (14, 15). DNase I and dimethyl sulfate protection experiments as well as electrophoretic mobility shift analyses using the wild-type uteroglobin promoter and appropriate linker scanning mutants as templates revealed that, in addition to the proximal CACCC box of region VI, the transcriptionally essential region II of the uteroglobin promoter is recognized by Sp3 and Spl. We show that the proteins in nuclear extracts of the endometrial cell line Ishikawa which bind this motif are Spl and Sp3. The strong correlation between DNA binding data in vitro and our functional results suggests that (a) member(s) of the Sp multigene family control the activity of the uteroglobin promoter through binding to two non-GC box Sp binding sites
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