Identification of Cis-acting elements in the human oxytocin gene promoter mediating cell-specificity, estrogen stimulation, and retinoic acid induction

Oxytocin (OT) is a nonapeptide produced in hypothalamic neurons. This neuropeptide hormone plays important roles in the physiology of reproduction. The gene encoding OT has been cloned in several species including human, rat, cow and, most recently, mouse. Although the gene sequences were known for some time, no information about the mechanisms regulating the OT gene promoter in any of the species has become available. This has been due, in part, to the absence of a cellular system expressing the endogenous OT gene. As a first step, the present thesis research was oriented towards the identification of a suitable cellular system for the investigation of OT gene expression. The cell line which responded to these needs was the mouse neuroblastoma cell line, Neuro-2a. In these cells, the transfected human OT gene promoter was highly active as opposed to all the other neuronal and non-neuronal cell lines tested. Using this system, we began investigating the molecular mechanisms underlying hormone induced and cell-specific expression of the OT gene. First, we determined that the OT gene promoter is regulated by estrogens. By 5$ sp prime$ and 3$ sp prime$ deletion analyses, we identified an estrogen response element (ERE) at position $-$164 to $-$152 which bears resemblance to the ERE consensus sequence. This ERE is necessary and sufficient for mediating the estrogen induction of the human OT gene promoter. The sequence of the ERE is GGTGACCTTGACC, and the underlined nucleotide represents the deviation from the perfectly palindromic ERE consensus sequence.By testing the effect of other members of the steroid hormone receptor family on the OT gene promoter, we found that the promoter responded strongly to retinoic acid (RA). Mapping of the RA response element (RARE) showed that four direct repeats of TGACC motifs were required for optimal RA induction. Interestingly, two of these TGACC repeats are contained within the ERE, whereas the other TGACC repeats are located downstream of the ERE. When the estrogen and RA treatments were performed simultaneously, additive and interfering interactions were observed. Additive effects resulted when the ERE and RARE were intact in the human OT promoter. By contrast, RA repressed estrogen induction by 50% when one of the two downstream TGACC repeats was mutated. Therefore, we speculate that in the latter case, the RA receptor forms in the presence of RA, an inactive complex with the two TGACC motifs that overlap the ERE and thus, prevents the estrogen receptor from forming a transcriptionally active complex with the ERE.The specific expression of the human OT promoter in Neuro-2a cells was shown to involve three cell-specific DNA elements located in the proximal promoter region between $-$49 to +36. These cell-specific elements, referred to as PPE 1, 2 and 3, are crucial for the elevated baseline expression observed in Neuro-2a cells. All these three elements contain highly conserved regions among species. Of note, PPE 2 contains the purine rich sequence GAGAGA which resembles the binding site for the GAGA factor and for the transcription factor PU.1. Each of three proximal promoter elements bound a specific factor(s) from Neuro-2a nuclear extracts, as assayed by gel-retardation. Moreover, PPE 1, 2 and 3 could synergize with the ERE or with the human metallothionein II$ sb{ rm A}$ glucocorticoid response element