Abstract
The regulation of insulin-like growth factor-binding protein-2 (IGFBP-2) gene transcription in specific cell and developmental contexts is not well understood. Here, we identified DNA regions that mediate IGFBP-2 gene transcription in two of the three major cell types of the uterine endometrium of the early pregnant pig. Two clusters of transcriptional start sites at nucleotides −109/−105 and −96/−87 (+1, translational initiation site) in the porcine IGFBP-2 gene were localized in uterine endometrium and in primary cultures of endometrial glandular epithelial (GE) and stromal (ST) fibroblastic cells. Upstream regions of this gene (spanning−1397/+73) were fused to a luciferase reporter gene, and the constructs were transiently transfected into endometrial GE and ST cells representative of pregnancy days 12 and 18 (day 115 = parturition). A short(110 bp) upstream region (−874/−765) stimulated the IGFBP-2 and heterologous SV40 promoters in the two cell types at both pregnancy days. Two noncontiguous copies of the novel sequence motif TCAGGG within the 110-bp fragment were implicated in transcriptional activity, since block mutation of these sequences led to a repression of SV40 basal promoter activity in endometrial cells. Southwestern blotting identified an endometrial nuclear protein of 34-kDa molecular weight that bound an oligonucleotide containing this motif, and EMSA suggested robust expression of this protein in early pregnancy endometrium and in ovary but at much reduced levels in endometrium at later pregnancy. A pair of E-box elements (CANNTG) within the 110 bp region was stimulatory to IGFBP-2 promoter activity; block mutation of these converted the 110-bp region into a potent transcriptional silencer in all but day 18 ST cells. Results identify novel DNA motifs that regulate the IGFBP-2 gene promoter in uterine endometrium in pregnancy-associated fashion.
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