Novel Intronic Promoter in the Rat ERα Gene Responsible for the Transient Transcription of a Variant Receptor

Abstract

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.

Oestrogen Receptors, Receptor Variants and Oestrogen Actions in the Hypothalamic‐Pituitary Axis

“…In contrast, adult female rat pituitary expresses a unique truncated form of ERα called TERP‐1 (for truncated oestrogen receptor product‐1), transcribed from a unique promoter that is expressed in a tissue‐specific and physiologically regulated manner (34–36, 38, 77). TERP‐1 mRNA contains coding exons 5–8 of the full‐length rat ERα, and is detected primarily if not exclusively in pituitary cells of lactotrope and gonadotrope origin (35, 36, 77–80). TERP‐1 mRNA and protein are stimulated by oestrogen treatment in pituitary cells in vivo and in culture, and are generally insensitive to progesterone in the absence or presence of oestrogen (34–36, 45).…”

“…The ratio of TERP‐1 to full‐length ERα mRNA changes dramatically as a function of oestrous cycle stage, with undetectable TERP‐1 mRNA during the evening of oestrus and TERP : ER mRNA ratios of 4 : 1 or higher on proestrus (35). TERP‐1 protein is expressed at high levels, and is present in both the cytoplasm and the nucleus (79, 80). Because ERα protein levels are suppressed while TERP protein levels increase with oestrogen, the change in protein ratio can be even more dramatic (35, 36).…”

“…Because ERα protein levels are suppressed while TERP protein levels increase with oestrogen, the change in protein ratio can be even more dramatic (35, 36). Because TERP‐1 cannot bind DNA, it has no effect on gene transcription alone, but can modulate the effects of full‐length ERα and ERβ (78–80). TERP effects on ER activity depend on cell context, and both stimulatory and inhibitory effects have been noted (45, 78–80).…”

“…Because TERP‐1 cannot bind DNA, it has no effect on gene transcription alone, but can modulate the effects of full‐length ERα and ERβ (78–80). TERP effects on ER activity depend on cell context, and both stimulatory and inhibitory effects have been noted (45, 78–80). At low TERP : ER ratios, ER‐mediated transcription is stimulated, while at ratios of 1 : 1 or higher, ER transcription is suppressed or obliterated.…”

Structure, tissue distribution and estrogen regulation of splice variants of the sea bream estrogen receptor α gene

“…Perlman et al, 2005;Nott et al, 2008;Springwald et al, 2010). These transcripts may result from multiple mechanisms, the most common being alternative promoter usage and alternative mRNA splicing with deletion of one or more exons (Tiffoche et al, 2001;Hirata et al, 2003;Perlman et al, 2005;Ishunina and Swaab, 2008;Nott et al, 2008). While some variant mRNAs only differ from the wild type in their stability or translation efficiency (Kos et al, 2002), others produce variant proteins with different functional properties that may stimulate or inhibit the transcriptional activities of the wt-ERα and/or ERβ proteins, or may mediate non-classical estrogen actions (e.g.…”