Regulation of the ovine MT1 melatonin receptor promoter: interaction between multiple pituitary transcription factors at different phases of development.

Pineal secretion of melatonin provides a neuroendocrine representation of the light-dark cycle, which is used to synchronise daily and annual rhythms of physiology and behaviour. In mammals, melatonin primarily acts through MT(1) melatonin receptors that exhibit a highly restricted tissue distribution. Expression of MT(1) receptors is subject to developmental and circadian control, which likely modulates the physiological actions of melatonin. To investigate the mechanisms controlling MT(1) expression we cloned the proximal 1.5kb region of the ovine MT(1) promoter. Sequence analysis revealed putative cis-elements for transcription factors involved in pituitary development, namely Pitx-1 and Egr-1, and multiple putative E-boxes, which are involved in both circadian and developmental gene regulation. Nuclear protein from ovine pars tuberalis (PT) cells, a site of high endogenous MT(1) expression, stimulated gene expression from a MT(1) expression construct, indicating the presence of a functional promoter. Pitx-1 was strongly expressed in the ovine PT and stimulated MT(1) promoter activity in transfection assays. Co-transfection with Egr-1 induced promoter-specific effects: Pitx-1-stimulated MT(1) activity was inhibited, whereas betaLH promoter activity was enhanced. In addition to Pitx-1 the circadian clock genes Clock and Bmal1 were also expressed in the PT. However, despite multiple putative E-boxes in the MT(1) promoter, transfected Clock and Bmal1 were unable to regulate either basal or Pitx-1-stimulated MT(1) promoter activity. The current data, in conjunction with our previous study of the rat MT(1) promoter, suggests a general model in which melatonin receptor expression in the mammalian pituitary is determined by the developmentally changing balance between stimulatory and inhibitory transcription factors. Furthermore, our data suggest that circadian variation in MT(1) gene expression does not depend upon the direct action of circadian clock genes on E-box cis-elements.