Circadian rhythms are daily molecular oscillations within cells ranging from prokaryotes to humans. This rhythm is self sustaining, and receives external cues in order to synchronize an organism's behavior and physiology with the environment. Many metabolites utilized in metabolic processes seem to follow a pattern of circadian oscillation. Iron, an essential component in cellular processes such as respiration and DNA synthesis, is obtained almost exclusively through diet, yet little is known about how the clock governs iron metabolism. The regulation of iron within the cell is very tightly controlled, as iron is highly reactive in the generation of oxidative stress and the excretion of excess iron is very limited. There are limited findings indicating that there are molecular ties between the circadian clock and the regulation of iron metabolism.
The first half of my dissertation focuses on the role of the circadian clock in modulating expression of iron metabolic components. We found that key components of iron import, in TFRC, and export, in SLC40A1, show altered expression in response to changes in the expression of clock transcription components. Furthermore, in circadian synchronized HepG2 hepatocytes TFRC and SLC40A1 showed rhythms in their mRNA expression, although expression of these genes was highly altered in conditions of high iron availability. We also examined IREB2, which expresses a master regulator of iron concentration in IRP2. IRP2 showed rhythms in phase with circadian component PER2, and IRP2's rhythmicity was lost under iron overload conditions. We observed that the ability of these three critical iron metabolic components to respond to sudden increases in available iron was mitigated in cells with clock impairment. Whole cistrome and transcriptome analysis was used to determine that rhythmicity in TFRC and SLC40A1 are not equal in their recruitment of circadian protein binding or in the stage of transcription in which circadian rhythms are generated. The cumulative effect of all of this regulation is that rhythmic variation in intracellular hepatic ferrous iron is clock controlled.
The second half of my dissertation focuses on understanding how iron uptake influences clock resetting. Initially, iron was added to the cells in the form of ferrous sulfate, or chelated out of the cells using 2-2'-dipyridyl and clock gene expression was monitored. Altered rhythmicity of these components was seen at both the mRNA and protein level in cells with disrupted iron homeostasis. Then, we measured changes in period, phase, and amplitude of these rhythms, ultimately using a luciferase reporter cell line to demonstrate that even slight changes in cellular iron produce an effect on rhythmic period. We find that the circadian clock and iron metabolism pathway are intimately related, and that the intracellular iron concentration plays a role in circadian clock behavior.
Overall, our research illustrates the importance of the circadian clock in liver metabolism and physiology. Improper iron metabolism due to genetic or dietary shortcomings is common in humans, and our work builds on the importance of chronotherapy in treatment of these conditions. Conversely, our research into the effect intracellular iron has on the clock contributes to the growing body of research into how circadian clocks, especially the peripheral clock of the liver, receive input from a range of metabolites in conjunction with signals from the master oscillator of the suprachiasmatic nucleus. / Ph. D. / The circadian clock is the system allowing the body to stay in synchrony with its environment. Clocks are found in organisms ranging from bacteria to humans, and use environmental cues such as light and temperature to coordinate important processes inside the cell. Many of these processes require enzymes which contain iron in order to function. Iron is obtained almost exclusively through feeding, and high iron levels are toxic to the cell. In this work, we looked at how the circadian clock helps maintain the amount of iron within the cell at healthy levels. We showed that the genes which are involved in managing iron are expressed in different amounts depending on the time of day, and that this causes the amount of iron within the cell to vary over time. We also examined how the amount of iron in the cells goes on to alter the circadian clock. The way the circadian rhythm oscillates is altered when either too much or too little iron is available to the cells. These findings have health impacts, especially in the context of the liver where poor management of the circadian clock or iron metabolism have been linked to the development of various forms of liver cancer.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/85398 |
| Date | 25 April 2017 |
| Creators | Schiffhauer, Samuel Peter |
| Contributors | Biological Sciences, Finkielstein, Carla V., Kojima, Shihoko, Kennelly, Peter J., Yang, Zhaomin |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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