The location and sequence of androgen responsive elements (AREs) in the 5'-flanking DNA of the androgen-regulated rat probasin (PB) gene were determined. The DNA- and steroid-binding domains of the rat androgen receptor [glutathione-S-transferase (GST)-AR1] and the DNA-binding domain and hinge region alone (GST-AR2) were expressed in Escherichia coli as isopropyl-B-D-thioglactopyranoside-induced fusion proteins with GST and purified using glutathione affinity chromatography. Band shift assays indicated that the AR1 peptide was at least five times more effective than AR2 in binding to PB 5'-flanking DNA (-426 to +28), although both gave qualitatively similar patterns and were displaced by anti-AR antibodies. DNase I footprinting experiments revealed two putative AREs: one between positions -236 and -223 (ARE-1) and the other between -140 and -117 (ARE-2). Hormonal regulation of PB was determined by cotransfecting reporter constructions containing the PB 5'-flanking region (-426 to +28) linked to the bacterial chloramphenicol acetyl transferase (CAT) gene with androgen, glucocorticoid, or progesterone receptor expression vectors into human prostatic carcinoma cells (PC-3). PB-CAT gene expression was more effectively induced by androgens than by glucocorticoids or progestins. Both 5'- and 3'-deletion mapping of the PB 5'-flanking DNA revealed that ARE-1 and ARE-2 were required for androgen regulation. A single base mutation in either ARE resulted in a more than 95% loss of androgen induction of CAT. In comparable transfection experiments, the PB hormone-responsive elements showed a greater induction by androgens than did mouse mammary tumor virus or tyrosine aminotransferase elements. Thus, the preferential androgen regulation of the PB gene involves the participation of two different cis-acting DNA elements that bind AR.
The human (h) placental lactogenic hormone chorionic somatomammotropin (CS) is highly produced during pregnancy and acts as a metabolic adaptor in response to maternal insulin resistance. Maternal obesity can exacerbate this "resistance", and a >75% decrease in CS RNA levels was observed in term placentas from obese vs. lean women. The genes coding for hCS ( hCS-A and hCS-B) and placental growth hormone ( hGH-V) as well as the hCS-L pseudogene and pituitary growth hormone (GH) gene ( hGH-N) are located at a single locus on chromosome 17. Three remote hypersensitive sites (HS III-V) located >28 kb upstream of hGH-N as well as local hCS gene promoter and enhancer regions are implicated in hCS gene expression. A placenta-specific chromosomal architecture, including interaction between HS III-V and hCS but not hGH gene promoters, was detected in placentas from lean women (BMI <25 kg/m) by using the chromosome conformation capture assay. This architecture was disrupted by pre-pregnancy maternal obesity (BMI >35 kg/m), resulting in a predominant interaction between HS III and the hGH-N promoter, which was also observed in nonplacental tissues. This was accompanied by a decrease in hCS levels, which was consistent with reduced RNA polymerase II and CCAAT/enhancer-binding protein-β association with individual hCS promoter and enhancer sequences, respectively. Thus, pre-pregnancy maternal obesity disrupts the placental hGH/CS gene locus chromosomal architecture. However, based on data from obese women who develop GDM, insulin treatment partially recapitulates the chromosomal architecture seen in lean women and positively affects hCS production, presumably facilitating prolactin receptor-related signaling by hCS.
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