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 MT1 melatonin receptors that exhibit a highly restricted tissue distribution. Expression of MT1 receptors is subject to developmental and circadian control, which likely modulates the physiological actions of melatonin. To investigate the mechanisms controlling MT1 expression we cloned the proximal 1.5 kb region of the ovine MT1 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 MT1 expression, stimulated gene expression from a MT1 expression construct, indicating the presence of a functional promoter. Pitx-1 was strongly expressed in the ovine PT and stimulated MT1 promoter activity in transfection assays. Co-transfection with Egr-1 induced promoter-specific effects: Pitx-1-stimulated MT1 activity was inhibited, whereas βLH 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 MT1 promoter, transfected Clock and Bmal1 were unable to regulate either basal or Pitx-1-stimulated MT1 promoter activity.The current data, in conjunction with our previous study of the rat MT1 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 MT1 gene expression does not depend upon the direct action of circadian clock genes on E-box cis-elements.