| Popis: |
Using an array of in silico and proteomics techniques I identified potential regulators of Cry1 expression linked with melatonin. The results presented here suggest that melatonin de-represses Cry1 expression by inhibiting EGR1, a transcriptional repressor. Due to the permissive action of a pars tuberalis specific factor called PITX1, upon de-repression the expression of Cry1 increases over the levels dictated by the circadian clock in this tissue. Next, to explore how the circadian phase of the core clock gene Cry1 is controlled, I coupled promoter fragments of different lengths from the ovine Cry1 gene to a luciferase reporter. Real time bioluminescence recording of 3T3 cells transfected with our constructs demonstrated that a 60 bp region, lying from -64 to -121 relative to the transcription start site, was necessary and sufficient for the phase conservation and the amplitude. This region, well conserved among mammalian species, contains a canonical E-box and an atypical E’-box, as well as other unidentified elements. Importantly, no retinoid related response elements (ROREs) were present in this region, or in any of the larger constructs. These data contradict current models proposing that differential phasing of clock genes Period and Cry1 expression rhythms depends on phase-delaying effects of ROREs on Cry1 transcription, and emphasise the role of accessory elements flanking proximal E-boxes in modulating the phase of circadian gene expression. |
