Paul Hardin (chronobiologist)

Paul Hardin (born September 14, 1960) is an American scientist in the field of chronobiology and a pioneering researcher in the understanding of circadian clocks in flies and mammals. Hardin currently serves as a distinguished professor in the biology department at Texas A&M University. He is best known for his discovery of circadian oscillations in the mRNA of the clock gene Period (''per''), the importance of the E-Box in ''per'' activation, the interlocked feedback loops that control rhythms in activator gene transcription, and the circadian regulation of olfaction in ''Drosophila melanogaster''. Born in a suburb of Chicago, Matteson, Illinois, Hardin currently resides in College Station, Texas, with his wife and three children.

Academic career

Hardin earned his B.S. in biology at Southern Methodist University (SMU) in 1982. He then continued to pursue a Ph.D in genetics from Indiana University Bloomington in 1987 with William H. Klein. He went on to conduct his postdoctoral research at Brandeis University under the supervision of chronobiologist Michael Rosbash. From 1991 to 1995, Hardin worked as a professor at Texas A&M University, and from 1995 to 2005 at the University of Houston. Since 2005, Hardin has worked as a professor and researcher in the biology department at Texas A&M University. He teaches courses on introductory biology, molecular cell biology, and a graduate level class on biological clocks. He also serves as the director of the Texas A&M's Center for Biological Clocks Research and as faculty for the Texas A&M Institute for Neuroscience and PhD program in genetics. In addition, Hardin was also actively involved in the Society for Research on Biological Rhythms; he served as the secretary in 2006, treasurer in 2010, and president in 2016.

Research

Discovery of ''per'' mRNA cycling

In 1971, Ron Konopka, a geneticist at the California Institute of Technology, discovered the Period gene, which he found to be involved in the circadian clock of ''Drosophila''. In 1999, Paul Hardin discovered that ''per'' mRNA underwent strong circadian oscillations by exposing isolated wild-type ''per'' mRNA to a series of light-dark (LD) cycles followed by cycles of constant darkness (DD). As a post-doctorate in the lab of chronobiologist Dr. Michael Rosbash, Hardin specifically noted that ''per'' mRNA levels in ''Drosophila'' brains fluctuate about 10-fold in a typical 24-hour light-dark cycle. Hardin further demonstrated that wild-type protein, PER, can rescue rhythmicity in the mRNA of an arrhythmic mutant of the ''per'' gene. His findings suggested that feedback of the PER protein regulates levels of ''per'' mRNA. Hardin ultimately published his seminal work on the rhythmic nature of ''per'' mRNA in ''Drosophila'' in the journal ''Nature''. This discovery led Hardin and other prominent members in the field of chronobiology to develop a model that describes the clock mechanism in ''Drosophila''. This model is referred to as the Transcription Feedback Loop, which suggests that the translated protein provides negative feedback on the mRNA transcription of itself.

Role of the E-box in ''per'' activation

In 1997, Hardin, with Haiping Hao and David Allen, analyzed the sequence of the ''per'' gene in ''Drosophila'' and found a 69-bp enhancer upstream of the gene. This enhancer sequence contained an E-box (CACGTG), which was determined to be necessary for high-level ''per'' transcription. As E-boxes are typically bound by proteins containing a basic helix-loop-helix (bHLH) protein structural motif, the presence of an E-box in ''per'' led to the hypothesis that the proteins involved in circadian rhythms may contain a bHLH domain. This proved to be vital in establishing the function of the previously discovered CLOCK protein, which was known to play a role in circadian rhythms and contained a bHLH domain as well. This discovery also aided in the identification of the BMAL1 and CYCLE proteins as critical players in the circadian rhythms of mammalian and ''Drosophila'' circadian systems respectively.

Circadian rhythms in olfaction

While teaching at the University of Houston, Hardin, along with fellow scientists Balaji Krishnan and Stuart Dryer, investigated circadian rhythms of olfaction in ''Drosophila''. Previous experiments had shown that ''Drosophila'' antennae demonstrate circadian rhythms. However, the mechanism for circadian rhythms in the antennae was unknown. To determine the mechanism of rhythms in antennae, Hardin and his team kept wild-type and mutant flies, ''per''⁰¹ and ''tim''⁰¹, in 12:12 light-dark (LD) cycles and measured olfaction in the antennae with an electroantennogram (EAG), that measures the average output of an insect antenna to its brain for a given odor, over a 24-hour period. Only the wild-type flies demonstrated rhythmicity in the electrical activity, which indicated that circadian rhythms were present in the olfactory response. In contrast, the mutants showed no cyclic activity. Therefore, Hardin's team discovered that circadian rhythms control the olfactory response in ''Drosophila'' antennae and his results were eventually published in ''Nature''.

Discovery of two interlocked feedback loops in circadian clock

In 1999, Hardin along with Nick Glossop and Lisa Lyons, conducted research on the specific role of ''Clk'' in the interlocked feedback loops present in ''Drosophila'' circadian oscillators. It was previously known that five genes ('' per'', '' tim'', '' dbt'', '' Clk'', and ''cyc'') controlled circadian rhythms in ''Drosophila''. The ''per''-''tim'' regulation mechanism was known at this time, though ''Clk'' regulation was not yet known.

Hardin and his team conducted a series of experiments to identify the two interlocked feedback loops in the circadian mechanism of ''Drosophila''. This means that the ''per''-''tim'' feedback loop connects to the ''Clk''-''cyc'' feedback loop, so that one loop has an effect on the other, and vice versa. They measured wild-type and mutant ''Clk'' mRNA levels to identify any changes in transcription levels. They observed that the PER-TIM complex suppresses transcription. They hypothesized that the ''Clk'' repressor was either the CLK-CYC complex itself or a repressor that was activated by CLK-CYC. They observed that the presence of active CLK and CYC resulted in the repression of ''Clk'', while arrhythmic ''per'' mutants exhibited low levels of ''Clk''. This evidence led them to propose the following model regarding two interlocked feedback loops:
# Late at night, PER-TIM dimers in the nucleus bind to and sequester CLK-CYC dimers. This interaction effectively inhibits CLK-CYC function, which leads to the repression of ''per'' and ''tim'' transcription and the de-repression of ''Clk'' transcription.
# As PER-TIM levels fall early in the morning, CLK-CYC dimers are released and repress ''Clk'' expression, thereby decreasing ''Clk'' mRNA levels by the end of the day.
# Concomitant with the drop in ''Clk'' mRNA levels (through CLK-CYC–dependent repression) is the accumulation of ''per'' and ''tim'' mRNA (through E-box–dependent CLK-CYC activation).
# The levels of CLK-CYC fall in the early evening, leading to a decrease in ''per'' and ''tim'' transcription and an increase in ''Clk'' mRNA transcription.
# A new cycle then begins as high levels of PER and TIM enter the nucleus and CLK starts to accumulate late at night.
In 2003, Hardin's team uncovered the second feedback loop associated with the circadian clock. ''vrille (vri)'' and ''Par Domain Protein 1'' (''Pdp1'') encode related transcription factors whose expression is directly activated by dCLOCK/CYCLE. They show that VRI and PDP1 proteins feed back and directly regulate ''dClock'' expression. Thus, VRI and PDP1, together with ''dClock'' itself, comprise a second feedback loop in the ''Drosophila'' clock that gives rhythmic expression of ''dClock'', and probably of other genes, to generate accurate circadian rhythms.

Summary of major research contributions

*1990: PER protein regulates rhythmic cycling in ''per'' mRNA of ''Drosophila''.
*1992: PER protein regulation of ''per'' mRNA occurs at the transcriptional level.
*1994: Circadian oscillations occur and behave differently in different body tissues such as the head, thorax, and abdomen in ''Drosophila''.
*1997: E-boxes, and particularly bHLH domains, are essential for ''per'' activation.
*1997: A 69 base-pair sequence of ''per'' gene contributes to circadian rhythms in gene expression.
*1999: Two interlocked negative feedback loops, PER-TIM loop and a CLK-CYC loop, regulate the circadian oscillator in ''Drosophila''.
*1999: The olfactory response in ''Drosophila'' is rhythmic.
*2000: PAR domain is an output of circadian rhythms in ''Drosophila''.
*2001: Circadian photoreceptor ''cryptochrome'' (''cry'') has a light-independent role in circadian oscillations under some circumstances.

Current research

Hardin's current research centers on the function of the circadian clock in ''Drosophila melanogaster''. One of Hardin's main research topics is understanding the mechanism behind the circadian rhythms in olfaction and gustatory physiology. His research also focuses on understanding the role of post-translational regulatory mechanisms in the feedback loop that set a 24-hour rhythm. Lastly, his lab has been working on identifying if the interlocked loops in the feedback mechanism function as a circadian oscillator or a clock output. His most recent article discusses the conservation of the transcription feedback loop in not only ''Drosophila,'' but also in other animal species as well.

Honors and awards

*John W. Lyons Jr. '59 Endowed Chair in Biology (2005)
*John and Rebecca Moores Professorship at the University of Houston (2004)
*Aschoff Honma Prize (2003)

References

{{DEFAULTSORT:Hardin, Paul
1960 births
Living people
21st-century American biologists
Chronobiologists
Texas A&M University faculty
Southern Methodist University alumni
Indiana University Bloomington alumni
Brandeis University alumni