Novel intronic promoter in the rat ER alpha gene responsible for the transient transcription of a variant receptor

• Published in: Endocrinology (Philadelphia) 2001-09, Vol.142 (9), p.4106-4119
• Main Authors: Tiffoche, C, Vaillant, C, Schausi, D, Thieulant, M L
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence - genetics; Cell Line; DNA - metabolism; Estradiol - metabolism; Estrogen Receptor alpha; Genes, Reporter - physiology; Genetic Variation; Humans; Introns - genetics; Molecular Sequence Data; Promoter Regions, Genetic - genetics; Rats; Rats, Wistar; Receptors, Estrogen - genetics; Receptors, Estrogen - metabolism; Receptors, Estrogen - physiology; Reference Values; Response Elements - physiology; Subcellular Fractions - metabolism; Tissue Distribution; Transcription, Genetic - physiology
• ISSN: 0013-7227
• DOI: 10.1210/en.142.9.4106

To analyze the molecular origin of an ER variant, the truncated ER product-1, transiently expressed at the proestrus in lactotrope cells, we generated a 2.5-kb sequence of a genomic region upstream and downstream the specific sequence truncated ER product-1. Genomic Southern blot analysis showed that truncated ER product-1 is spliced from a noncoding leader exon localized within the intron 4 of the ER alpha gene. Analysis of the promoter sequence revealed the presence of a major transcriptional start site, a canonical TATA box and putative cis regulatory elements for pituitary specific expression as well as an E-responsive element. In transient transfection, the truncated ER product-1 promoter was transcriptionally the most active in the lactotrope cell lines (MMQ). Analysis of truncated ER product-1 functionality showed that: 1) the protein inhibited ER alpha binding to the E-responsive element in electromobility shift assays, 2) inhibited the E2 binding to ER alpha in binding assays, 3) the truncated ER product-1/ER alpha complex antagonized the transcriptional activity elicited by E2, 4) nuclear localization of green fluorescent protein-ER alpha was altered in Chinese hamster ovary cell lines stably expressing truncated ER product-1. Collectively, these data demonstrated that the protein exerts full dominant negative activity against ER alpha. Moreover, truncated ER product-1/ER alpha complex also repressed the activity of all promoters tested to date, suggesting a general inhibitory effect toward transcription. In conclusion, the data suggest that truncated ER product-1 could regulate estrogen signaling via a specific promoter in lactotrope cells.