Analyses of Cis ‐Acting and Trans ‐Acting Elements that are Crucial to Sustain Pregnancy‐Specific Glycoprotein Gene Expression in Different Cell Types

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Abstract
Pregnancy-specific β1 glycoprotein genes (PSG) are mainly expressed during human placental development, though their expression has been reported in other normal and pathological tissues, e.g. hydatidi-form mole (HM), of distinct origins. However, the molecular components implicated in the regulation of PSG are not well understood. To identify some of the regulatory elements involved in the transcriptional control of PSG expression, the DNA-protein interactions and the basal activities of the TATA-box-less PSG5 promoter were determined in different tissues and cell types. In DNAse-I protection assays, DNA-binding proteins from human term placenta (HTP) protected a region of 27 bp located from nucleotides –150 to –124, overlapping the farthest 5′ upstream cap site and resembling an initiator-like element. In electrophoretic mobility shift assays (EMSA), three complexes were detected using nuclear extracts from HTP and an oligonucleotide containing the 27-bp motif. In situ ultraviolet crosslinking analysis of the specific complexes revealed that two proteins of 78.0 kDa and 53.0 kDa are involved in such interactions, in accordance with the bands of 80.0 kDa and 57.5 kDa observed by Southwestern blotting. Competitive EMSA using mutant oligonucleotides with the substitution of 5′ACCCAT3′ by 5′GATATC3′ within the 27-bp motif revealed that this sequence is fundamental for the formation of the specific DNA-protein complexes. We show in transient transfection experiments performed in HeLa, COS-7 and JEG-3 cells, that such mutation completely abolished the transcriptional activity of the PSG5 promoter, independently of the cell type. Moreover, this mutation disrupted the formation of the specific DNA-protein complexes which were essentially the same as those displayed by HTP. We also determined the binding activities of nucleopro-teins derived from placental tissues in earlier developmental and pathological stages, i.e. first trimester placenta (1-TRIM) and HM, respectively, showing that the DNA-binding patterns were different from each other and distinct from those elicited by HTP. Our results indicate that the cis -acting and trans -acting elements analyzed are indispensable to support PSG5 promoter activity in cell lines which do or do not produce PSG. In addition, these elements appear to play a role in the mechanisms involved in PSG basal expression during placental development and differentiation.