Melatonin Mediated Transcriptional Mechanisms in the Ovine Pars Tuberalis

In seasonal mammals the nocturnal secretion of melatonin acts as an internal representation of the environmental photoperiod. A specialised pituitary tissue known as the pars tuberalis (PT) is autonomously capable of integrating the photoperiodic information encoded in melatonin duration through the melatonin G-coupled protein receptor (MT1). The PT then communicates this information to the hypothalamus and pars distalis (PD) which control downstream photoperiodic changes in physiology and behaviour. Recent studies have revealed that two transcriptional cohorts are acutely coupled to melatonin’s onset and offset in the ovine PT. Melatonin suppresses cAMP signalling in the PT through MT1 Gαi protein coupling. However melatonin’s withdrawal at dawn causes a spike in intracellular cAMP. This is believed to be stimulated by a currently unknown agent dubbed StimX. The accumulation of cAMP stimulates the transcription of Per1 and ICER whose expression closely tracks the onset of dawn in the PT. At dusk, the onset of melatonin acutely induces a second transcriptional cohort (including Cry1, Nampt, NeuroD1 and Npas4) via the MT1 receptor. Hence melatonin controls two transcriptional cohorts acutely coupled to its onset (dusk) and offset (dawn) whose phase relationship could hypothetically form a mechanism by which photoperiod is determined. This thesis aims to identify the mechanism responsible for acute gene induction by melatonin downstream of the MT1 receptor, critical for the understanding of acute melatonin action and the process behind downstream control of seasonal processes. Transcriptomic, bioinformatic and in vitro cell and explant culture techniques are used to approach this area of interest. NPAS4’s potential role as an immediate regulator of melatonin-mediated transcriptional circuits is explored through the transactivation of candidate target promoter reporters.Work in this thesis has shown that in vivo melatonin regulates 219 transcripts by at least 1.5 x after 1.5 h perfusion in the ovine PT (115 induced, 104 repressed). Promoter analysis identified the induced gene cohort to be enriched for GC content and CpG islands suggesting the involvement of epigenetic mechanisms of transactional control. Predicted transcription factor binding site analysis of the induced cohort identified the potential importance of ZFP161 and PAX5 as factors employed by melatonin to communicate the acute inductive response. MCF7 oMT1 and NES2Y cell lines were shown to respond to melatonin treatment through Gαi signalling. However these cell lines was unable to elicit an inductive response from candidate ovine promoter reporters in response to melatonin treatment. Subsequently ovine PT explant culture recapitulated in vivo transcriptional profiles of candidate genes through a 6.5 h time course via qPCR. NPAS4 was shown to form functional heterodimeric partnerships with ARNT and ARNTL and transactivate both Cry1 and Nampt promoter reporters through novel binding sites. These data indicate NPAS4 to act as an immediate activator of melatonin regulated circuits.