Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Anatomy and Cell Biology

Supervisor

Dr. Michael N. Lehman

Abstract

Circadian rhythms orchestrate physiological, behavioral and cognitive processes in order to anticipate and adapt organisms to key environmental cues. These endogenously driven oscillations are generated by a network of interlocked auto-regulatory transcriptional-translational feedback loops driven forward by the Bmal1/Clock heterodimer transcription factor. Given the ubiquitous and dynamic quality of circadian rhythms, the identification of factors involved in the coordination and regulation of the endogenous oscillations is central in broadening our understanding of biological timing systems. In an examination of gene expression in the mammalian central circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN), revealed a previously unreported rhythmic expression of runt-related transcription factor 2 (Runx2). The goal of the research described in the present dissertation was to determine the mechanistic basis of rhythmic Runx2 expression as well as determine whether Runx2 interacts with the core molecular machinery of the circadian clock. First, rhythmic gene expression Runx2 in the SCN, olfactory blub (OB) and paraventricular nucleus (PVN) of adult mice was demonstrated using quantitative real-time polymerase chain reaction and immunohistochemistry served to show rhythmic Runx2 protein expression in the SCN. Second, using SCN tissues from Bmal-/- mice or by transiently silencing BMAL1 expression in vitro it was shown that Runx2 expression is dependent on a functional core molecular clock network, namely the transcriptional activity of Bmal1. Chromatin immunoprecipitation was used to demonstrate that BMAL1 directly interacted with its putative binding sites within the RUNX2 promoter suggesting that the core clock machinery directly regulates its rhythmic gene expression. Next, Runx2 haplodeficient mice and transient silencing of RUNX2 expression in vitro revealed that decreases in Runx2 expression leads to a dampened amplitude of rhythmic Bmal1 expression and a lengthening of the period of molecular as well as running wheel behavioral rhythms. Finally, RUNX2 was found to interact with a putative binding site identified in the BMAL1 promoter suggesting that the influence of RUNX2 on the amplitude of BMAL1 expression was at least in part based on direct regulation at the gene level. Together, these findings serve to establish the foundational framework of the reciprocal relationship between the Runx2 transcription factor and the molecular network underlying circadian rhythms.

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