Jay Clark Dunlap is an American chronobiologist and photobiologist who has made significant contributions to the field of chronobiology by investigating the underlying mechanisms of circadian systems in Neurospora, a fungus commonly used as a model organism in biology, and in mice and mammalian cell culture models. Major contributions by Jay Dunlap include his work investigating the role of frq and wc clock genes in circadian rhythmicity, and his leadership in coordinating the whole genome knockout collection for Neurospora. He is currently the Nathan Smith Professor of Molecular and Systems Biology at the Geisel School of Medicine at Dartmouth. He and his colleague Jennifer Loros have mentored numerous students and postdoctoral fellows, many of whom hold positions at various academic institutions.
Early life and education Born in Ludlow, Massachusetts on May 9, 1952, Jay Dunlap grew up in York, Pennsylvania as the third of four children. Dunlap became interested in biochemical oceanography during a high school summer program and decided to pursue this interest in college. He graduated with a B.S. in oceanography and a B.S. in chemistry from the University of Washington in 1974.
Dunlap originally planned to pursue oceanography in his graduate studies. However, after meeting with John Woodland Hastings, who studied the circadian regulation of bioluminescence in marine organisms, Dunlap decided to study biology in graduate school at Harvard University. While studying with Hastings, Dunlap changed his field of study to circadian biology.
Career and research For his postdoctoral fellowship, Dunlap attended the University of California, Santa Cruz and started working with Jerry Feldman, who had successfully isolated clock gene mutants in Neurospora that have abnormally long or short circadian-oscillation periods. Dunlap was unable to clone frequency, a gene that has an important role in the transcription-translation negative feedback-loop (TTFL) that drives circadian rhythms in Neurospora, as the Santa Cruz lab did not have the molecular tools necessary to study Neurospora's molecular biology in depth. Dunlap learned basic molecular techniques as he worked alongside fellow biology graduate students in other labs. At one point, Dunlap worked with Harry F. Noller, a renowned biochemist whose lab had "unofficially adopted" Dunlap.
In 1984, Dunlap secured a junior faculty position at the Department of Biochemistry at Geisel School of Medicine at Dartmouth. He became a professor of biochemistry in 1994 before being named the Inaugural Chair of the Department of Genetics in 1999. In 2010, Dunlap was named Nathan Smith Professor, and in 2016, he was appointed inaugural chair of the Department of Molecular and Systems Biology which subsumed Genetics and other departments.
Working closely with Jennifer Loros' laboratory, Dunlap's research has primarily focused on the molecular basis of circadian rhythms using Neurospora as a model system to further understand the mammalian circadian clock. Although clock gene mutations were also identified in Drosophila and Chlamydomonas, Dunlap studied Neurospora in his postdoctoral work, as a wider array of biochemical and genetic tools were applicable to the species at the time. Neurospora was a simple model organism and a powerful tool to study molecular genetics; its then-unknown molecular clock presented a great opportunity for exploration.
Identifying Neurospora clock components and mechanisms Based on the work of Dunlap and others, clock genes are now understood to encode proteins that participate in a self maintaining negative feedback loop: transcriptional activators drive expression of specific clock gene mRNAs, which are translated into clock proteins, which enter the nucleus and act to depress the activity of the transcriptional activators driving the expression of the clock genes. However, clock genes were not yet cloned when Dunlap began his research as an assistant professor in 1984. Dunlap correctly predicted that single cells, including mammalian cells, can act as autonomous oscillators with their own intrinsic circadian rhythms.
Dunlap deciphered the circadian system by framing and addressing three problems in cellular metabolism:
How is the clock put together: what are the gears and cogs, how do they mesh, what regulates them, and how do they regulate one another so the collective output is a molecular/biochemical cycle with all the circadian characteristics? How do abrupt and transient changes in the environment, chiefly ambient light or temperature, reset the phase of the clock and align the internal clock of an organism with the external time? How is an intracellular molecular cycle used to regulate the behavior of the cell?
Prior to the adoption of transcriptional reporters such as luciferase, studies of the Neurospora circadian clock utilized the rhythmic development of asexual spores (conidia), assayed using a race tube. Conidial production peaks in the subjective night—a behavioral phenotype lacking in arrhythmic strains. During her graduate work, Jennifer Loros observed mutant frq9 as a recessive, arrhythmic, and phenotypically null allele at the frq gene. Her observation, combined with the ability to transform Neurospora with exogenous DNA, provided the basis for a novel strategy to clone frq, namely by transformation-based rescue of the null mutant behavioral phenotype. Utilizing a bidirectional chromosome walk beginning at oli, a gene on the same linkage group as frq, Dunlap and colleagues walked over 200kb across frq. The location of frq was verified in 1986 through transformation of cosmids into frq9 and by rescuing the circadian rhythm. frq was thus the second clock gene to be cloned, following Drosophila per. Furthermore, the lab manually sequenced roughly 9kb and conducted transcript mapping on the frq genomic region; the results were published in Nature in 1989. In subsequent work, Dunlap and colleagues showed that frq was rhythmically expressed and were able to manipulate the expression of frq sufficiently to create a null mutant. They implemented a system in which a heterologous promoter—induced in a manner that did not affect the clock—could be used to drive regulated expression of frq. Using this system, they demonstrated that the product of frq acted to repress its own synthesis; it was autoregulatory. Dunlap and colleagues observed that the continual over-expression of frq resulted in arrhythmicity, and they defined the phase of clock's rhythm to be the time at which the cell returned to normal expression levels of frq. They concluded, in a Science article in 1994, that the core pacemaker of the Neurospora clock is regulated via negative feedback by clock proteins, and frq determines its own expression through auto-regulation via negative feedback, demonstrating that intracellular, auto-regulatory negative feedback is the basis of a circadian oscillator.
Dunlap's work on the auto-regulatory mechanism included modeling the circadian clock's negative feedback loop and discovering the roles and connections between activators (which he identified as proteins with PAS domains) and repressors (products of the clock genes). Additionally, Dunlap demonstrated the role of protein phosphorylation in the clock mechanism and has done research involving the role of these proteins (namely Casein Kinase 2) on the temperature compensation mechanism. In 2009, Dunlap and colleagues showed that the FRQ protein is phosphorylated at over 100 sites in a highly reproducible and time-of-day-specific manner and that casein kinase 2 establishes and maintains temperature compensation within the circadian clock. Four years later in 2013, Dunlap and colleagues found that FRQ is an Intrinsically Disordered Protein whose stability is determined by its interaction with partner protein FRH. Additionally, Dunlap and colleagues discovered that the daily phosphorylation of FRQ governs its ability to interact with the proteins in the negative element complex. The kinetics of these circadian processes, Dunlap discovered, are heavily influenced by progressive phosphorylation of FRQ.
Mechanism of entrainment
After identifying frq as a clock gene whose product's abundance tends to be variable and rhythmic, Dunlap, Loros, and colleagues showed how environmental regulation of its expression led to understanding the molecular basis of circadian entrainment by light: through the induction of expression of frq by light.
In 1995, Loros and Dunlap worked to uncover the molecular basis underlying how light resets the clock, a mechanism later shown in collaborative work with Hitoshi Okamura to be conserved in mammals. The daily cycle in frq transcript levels, combined with the ability of light to acutely induce frq expression, explained light resetting (the advances and delays seen on a phase response curve). If light was provided and induced frq-mRNA when it was rising to peak levels (late subjective night), light would rapidly bring frq-mRNA levels to peak values, resulting in an advance. If light induced frq-mRNA while its levels were falling (early subjective night), frq-mRNA would rapidly go back to peak levels causing a phase delay. The results of this investigation led to the conclusion that the light induction of frq is responsible for the phase-specific advances and delays observed in Neurospora and provided a general explanation for how the unidirectional response of a clock component to an environmental signal (light) could result in a bidirectional time-of-day specific clock response (advances or delays): the basis for circadian entrainment. These experiments eventually led to the universal recognition of entrainment via light-induced changes in a specific variable of the circadian oscillator, later observed in Drosophila and mammals.
Identification of PAS-PAS heterodimers as activators in the circadian feedback loop
The mechanism through which frq is induced by light was unknown at the time that entrainment was explained, and studies aimed at identifying the proteins responsible for light-induction of frq led to the identification of White Collar-1 and White Collar-2 as components of the circadian activator complex. Work by Giuseppe Macino had shown White Collar-1 to associate via PAS domains with White Collar-2 to create the White Collar Complex; Dunlap, Loros and colleagues showed how this heterodimeric complex is the transcription factor that acts in the dark to drive expression of frq, thereby acting as the activator in the circadian negative feedback loop. This observation associated specific biochemical activities, DNA binding and transcriptional activation, with known clock proteins, allowing the formulation of the oscillator as a single step transcription-translation negative feedback loop. Later, in 1997, the first mammalian clock gene (CLOCK) was shown to encode a protein similarly having PAS domains and, later, to associate via PAS domains with a different protein, BMAL1, again forming a heterodimeric protein complex that acted as a transcriptional activator; similar proteins were identified in 1998 in Drosophila. This confirmed a common model for the transcription-translation negative feedback loops in fungi and animals: a positive element composed of two different proteins interacting via PAS domains drives expression of negative elements such as FRQ or PER that, in association with other proteins, represses the activity of heterodimeric activators: negative feedback. These observations contributed to the naming of Circadian Rhythms as first runner up to Breakthrough of the Year in Science magazine in 1997.
Identification of a circadian photoreceptor