In mammals, the suprachiasmatic nuclei (SCN) of the hypothalamus function as the major biological clock. Recent work has revealed that a program of rhythmic transcriptional regulation is required for endogenous SCN timekeeping and that light-induced changes in circadian timing result from alterations in this transcriptional program. Given this central role of inducible gene expression, a characterization of the intracellular signaling pathways and downstream transcription factors involved in biological timing and entrainment is critical for understanding the functional properties of the circadian clock. In our prior round of funding we collected a wealth of data supporting the hypothesis that the MARK pathway couples photic input to the clock timing process. What is lacking is an understanding of the precise routes by which the MARK pathway regulates the clock. We hypothesize that the MARK signaling cascade regulates timing at both a transcriptional and translational level, and we have assembled an array of novel tools to test this hypothesis.
In Aim 1 we will determine whether the MARK pathway-regulated kinases MSK1/2 and RSK2 function as cellular signaling intermediates that couple light to clock entrainment. Our published reports reveal that both kinases are activated by light in the SCN, and preliminary data indicate that MSK1 and RSK2 regulate clock entrainment. Behavioral studies will be complimented by studies that examine chromatin structure and gene expression in MSK and RSK null mice.
In Aim 2 we investigate the role of the CREB/CRE- transcription pathway (a target of the MARK pathway) in clock entrainment and clock rhythm amplitude. The CREB/CRE pathway has been implicated as a principal route by which entrainment cues impinge upon the core transcriptional timing process. However, there has yet to be an effective set of tools to test this idea in vivo. To this end, we have developed a novel set of CREB repressor and CREB activator transgenic mice that will be used to examine this question at a behavioral, biochemical and genetic level.
In Aim 3, we investigate the role of the MARK pathway as a regulator of light- and clock- dependent mRNA translation in the SCN. Given recent work implicating dysregulation of circadian timing/entrainment in disorders such as obesity, cardiovascular disease and cancer, there is a clear need to identify the intracellular signaling events that play a fundamental role in pacemaker entrainment.
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