Characterization of two promoters of a rat pregnancy-specific glycoprotein gene

Abstract

Pregnancy-specific glycoproteins (PSGs) are the major placental glycoproteins, that together with the carcinoembryonic antigens comprise a subfamily within the immunoglobulin superfamily. In order to develop an animal model for understanding the molecular mechanisms underlying the control of PSG expression, we isolated and characterized cDNA and genomic clones encoding a rodent PSG, rnCGM3. The rnCGM3 cDNA is 2761 bp in length and contains an open reading frame that encodes a 475 amino acid polypeptide with a domain arrangement of L1N1-L2N2-L3N3-A. The sequence in 5'-untranslated and L1 regions of rnCGM3 is identical to a previously identified cDNA, rnCGM6. The transcription initiation sites of both genes are located at nucleotide -197 upstream of the translation start site. In transient transfection assays using a chloramphenicol acetyltransferase (CAT) reporter gene, we demonstrated that DNA elements at nucleotides -326 to -185 (PI) and -147 and -86 (PII) relative to the translation start site of rnCGM3 could both function as promoters. The downstream promoter, PII, which is located within the first exon, shares high sequence identity with the minimal promoters of human PSG genes. Electrophoretic mobility shift assays (EMSAs) showed that protein factors in placental cell extracts formed three complexes (PIICI, PIICII, and PIICIII) with the PII promoter element. The PIICIII complex was also observed by DNase I footprinting analysis. Unlike PII, the upstream promoter, PI, contains a TATA box. DNase I footprinting analysis revealed two nuclear protein binding sites at nucleotides -311 to -290 (PISI) and -257 to -239 (PISII) in PI. EMSAs showed that protein factors in placental cell extracts bound to both sites and deletion of either site markedly reduced CAT expression. PISII contains a palindromic motif, TGTTGCTCAACA, and protein cross-linking and Southwestern hybridization analyses demonstrated that the protein factor binding to PISII had an apparent molecular mass of 40 kDa.