Identification of transcription factors interacting with the oxytocin gene promoter

Oxytocin (OT) and the related hormone vasopressin (AVP) are both expressed in the hypothalamus but are never co-expressed in the same neurons. The OT and AVP genes are believed to derive from one common gene by gene duplication and both genes are adjacent to each other on the chromosome in all species studied. Because of the lack of suitable cell lines expressing the OT gene, scientists have taken advantage of the availability of promoter sequences of numerous species to overcome this deficit. Alignment of the OT promoters available revealed stretches of high homology and further analysis of these stretches revealed that they are binding sites for the nuclear receptor superfamily of transcription factors such as the estrogen receptor (ER), retinoic acid receptor (RAR) and the thyroid hormone receptor (TR). Here, I identify that the orphan receptors, chicken ovalbumin up-stream promoter transcription factor II (COUP-TFII), ErbA-related factor 2 (Ear-2) and retinoid orphan receptor alpha1 (RORalphal) as potential regulators of the OT gene promoter.In contrast to the positive effect of the ER, RAR and TR on the OT promoter, COUP-TFII and Ear-2 have a negative effect on the human OT promoter. The effects of COUP-TFs on the OT promoter are two-fold; they are able to silence or actively repress basal promoter activity and secondly, inhibit the activation by other nuclear receptors via a mechanism of competitive binding. Two additive DNA elements were identified to mediate the effect of COUP-TFII and Ear-2. The first one is a direct repeat of the AGGTCA motif spaced by zero nucleotide (DR0) that is embedded in the OT/estrogen response element (OT/ERE). The second element, located downstream of the OT/ERE, consists of three AGGTCA motifs each spaced by four nucleotides. Inhibition of the estrogenic induction only requires the DR0 whereas both elements are required for full silencing of basal activity. DNaseI footprinting and gel shift assays show that recombinant COUP-TFII and Ear-2 are able to bind to the DR0 and the downstream repeats. Mutations that affect the ability of COUP-TFII and Ear-2 to bind to these elements also affect their activity in transient transfection studies. The importance of COUP-TFII and Ear-2 in the regulation of OT gene expression is supported by their expression in the epithelial layer of the rat parturient uterus. The uterus is a known site of OT expression where a dynamic regulation of OT is observed. (Abstract shortened by UMI.