Oestrogens are capable of inducing rat prolactin gene transcription through liganded Oestrogen Receptor (ER) binding a degenerate oestrogen response element (ERE) sequence on the promoter 1440 bp upstream of the transcription start site. ER binding is thought to interact with DNA bound Pit-1 leading to chromatin looping bringing enhancer elements into proximity with the transcription start site. This ERE sequence, TGTCActaTGTCA, differs from the consensus GGTCAnnnTGACC by two nucleotides, one in each half site. Oestrogens have also been implicated in regulation of prolactin transcription in humans, however the mechanisms are not understood.
We have previously shown a small but consistent activation of the human prolactin promoter at 24 hours in GH3 derived D44 cells, which drive luciferase expression from a 5000 bp human prolactin promoter. Several putative EREs exist within this fragment. Deletion constructs of the promoter localised the oestrogen responsive sequence to be positioned within 828/1779. A likely ERE sequence, TGTCAcctTGTCC, is located at 1207/1220 bp, adjacent to a Pit-1 site. This sequence differs from the consensus ERE sequence by two nucleotides (and by three nucleotides compared to the rat prolactin ERE). Mutagenesis of this site abolished oestrogen induced promoter activation. Electromobility shift assays showed that this sequence bound ER but at a greatly reduced affinity compared to a consensus ERE sequence. Thus a degenerate, non-consensus ERE located adjacent to a Pit-1 binding site can regulate human prolactin gene transcription in a pituitary cell line. ChIP assays are presently being used to assess the chromatin modifying potential of ER at this site.