Periodic exposure to light and dark as a result of rotation of the Earth have served as a major evolutionary pressure to partition divergent biological processes to different phases of the day. Mammals display periods of activity/inactivity, wake/sleep, and feeding/fasting during distinct portions of the day. In mammals, these activities are driven by a molecular timekeeping mechanism composed of activators (CLOCK/BMAL1) and repressors (PERs/CRYs) that constitute a transcription/translation feedback loop with approximate 24-hour periodicity. Circadian activator transcription factors bind at E-box DNA motifs throughout the genome and, together with collaborative transcription factors, drive oscillations in ~10% of the transcriptome in any given cell type. In 2005, it was found that metabolism is a major process that is regulated by the circadian clock as Clock mutant animals are predisposed to diet-induced obesity, diabetes, and metabolic syndrome. An important parameter of a molecular timekeeper is that it must integrate environmental signals in order to synchronize with changing external states. Desynchrony between the biological clock and the external environment during, for example, jet lag is associated with a temporary feeling of morose and metabolic dysfunction, but as the internal clock synchronizes with the external environment those phenotypes subside. At the molecular level, the clock repressor proteins are extensively phosphorylated, giving a mechanistic opportunity whereby the clock integrates changes in the external environment. New research has shown changes in the metabolic state serves as an important environmental cue of the external state as mice given ad libitum access to a high fat diet have disrupted molecular and behavioral rhythms. The overarching goal of the research within this dissertation is to characterize the molecular mechanisms whereby the clock senses changes in the metabolic state. A major clue concerning the link between the clock and metabolism came with the discovery that Nampt, the rate-limiting enzyme in the NAD+ salvage pathway, is a direct target of CLOCK/BMAL1 leading to 24-hour rhythms in Nampt mRNA are responsible for 24-hour rhythms in the energetic intermediate, NAD+. Discovery that the sirtuin family of class-III NAD+-dependent enzymes are capable of modulating clock function raised the possibility that metabolic state might modulate NAD+ and thus affect clock function through the major nuclear sirtuin, SIRT1. A gap has remained, however, in our understanding of whether NAD+ affects genome-wide circadian transcription and the exact mechanisms that mediate this effect. What remains unknown is whether changes in NAD(H) redox state through modulation of metabolic pathway flux are responsible for entraining circadian transcription to altered dietary states. A major pathophysiological state characterized by disruption of the circadian clock, NAD+ homeostasis, and sirtuin function involves organismal aging. Elderly humans display prominent circadian disruption characterized by advanced sleep phase (ASP) where they wake earlier and go to bed earlier than they did when they were young. Genetic ablation of Bmal1, sirtuins, or NAD+ synthetic pathways have profound effects on longevity. NAD+ levels themselves have been shown to decline with age and are thought to play a causative role in the aging process as pharmacologic repletion of NAD+ to youthful levels attenuates the development of age-associated morbidities. Finally, dietary interventions such as caloric restriction that influence NAD(H), sirtuin, and clock function promote longevity. A gap remains, however, in our understanding of the extent to which changes in NAD(H) constitute the mechanism for deterioration of clock transcriptional, behavioral, and metabolic cycles with age. The work herein seeks to understand the role of NAD(H) in regulating circadian transcriptional and behavioral cycles and the decline in circadian function during aging. I characterize the effect of NAD(H) on genome-wide circadian transcription, showing in chapters 2 and 3 that NAD(H) drives 50% of the hepatic oscillatory transcriptome and entrains phase-specific transcription to altered metabolic states. This is achieved by regulating recruitment of collaborative transcription factors to chromatin. For example, one such TF is HSF1, discussed in chapter 2, and another is FOXO1 discussed in chapter 3. I additionally characterize the requirement of the core clock for the transcriptional response to NAD+, demonstrating in chapter 2 that Bmal1 is necessary for opening chromatin at collaborative transcription factor binding sites and for recruitment of HSF1. In chapter 2, I characterize the mechanism whereby NAD+ influences the clock, through SIRT1-mediated deacetylation of PER2 on K680 which alters assembly and sub-cellular localization of clock repressors in turn modulating BMAL1 activity. In chapter 3, in collaboration with Drs. Kuo and Mrksich, we characterize a repressive effect of NADH uniquely on SIRT1 in vitro that drives acetylation patterns of histones and collaborative transcription factors such as FOXO1 in vivo. I investigate the extent to which the decline in NAD+ with age alters rhythmic transcription, behavior, and metabolic cycles, finding in chapter 2 that low NAD+ with age is associated with low amplitude in CLOCK/BMAL1 transcriptional activity and a reduction in late-night activity in mice through a mechanism similar to that of familial advanced sleep phase syndrome (FASPS). Finally, in investigating longevity-associated dietary interventions in mice in chapter 3, I describe a unique effect of caloric restriction in liver. Here, I show that time-of-day dependent changes in NADH:NAD+ levels drive altered transcription due to inhibition of SIRT1. Altogether, the studies herein shed light on the role of NAD+ and NADH in regulating activity of the core molecular clock and downstream oscillating genes, establishing insight into the mechanisms underlying nutrient sensing by the molecular clock during aging.

Creator

• Levine, Daniel C

DOI

• https://doi.org/10.21985/n2-6tkr-m025

Subject

• Molecular biology
• Medicine
• Endocrinology

Language

• en

Alternate Identifier

• http://dissertations.umi.com/northwestern:15034
• etdadmin_upload_727063

Date created

• 2019-01-01

Resource type

• Dissertation

Rights statement

• In Copyright