American chronobiologist and photobiologist
Jay 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 presently 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:
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 "frq"9 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 "frq"9 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.
Although it was established that heterodimeric WC-1/WC-2 transcription factor was required for light-induction of "frq", researchers believed that WC-1 and WC-2 did not have a direct role in the process of photoreception. WC-1/WC-2 transcription factor was instead assumed to be the final target of a signal transduction cascade initiated by the action of light on a distinct blue light photoreceptor. In 2002, Dunlap and colleagues biochemically studied WC-1/WC-2 in vitro to show that WC-1 bound FAD as a cofactor (also shown independently by Yi Liu), and analysis of binding to DNA by the WC-1/WC-2 complex showed that light resulted in a structural change in the heterodimer. The dose response and action spectrum for this in vitro structural change in WC-1 was FAD-dependent and matched the in vivo dose response and action spectrum for light-suppression of circadian banding determined by Briggs and colleagues in 1967. These findings revealed that WC-1 is a blue light photoreceptor and a circadian photoreceptor; the signal transduction cascade from photoreceptor to transcription factor happens all within the same protein. WC-1 is the founding member for the family of blue-light photoreceptors common to all fungi. Circadian photoreceptors were later identified in animals and green plants and shown to be distinct from WC-1.
Circadian output.
In 1989, Dunlap's work with Jennifer Loros led to the first targeted screen for genes regulated by the circadian clock, paving the way for the systematic dissection of clock output pathways. The term "clock-controlled genes" (CCGs) was coined in this study. CCGs are defined as genes whose level of expression is regulated by the circadian clock but whose activities do not impact the operation of the clock. Circadian control of gene expression is now widely thought to be the principal means through which clocks control the biology of the cells in which they operate. Subsequent work expanded the universe of CCGs in "Neurospora", and later in mammalian cells, and revealed the connection between the circadian and cell cycles in which the clock regulates the DNA damage response which, in turn, can regulate the clock. The search for CCGs finally culminating in the complete description of the circadian transcriptome of "Neurospora" where as much as 40% of the genome is controlled on a daily basis by the clock.
Studies on bioluminescence.
 Jay Dunlap’s graduate work at Harvard with J.W. Hastings focused on bioluminescence in the marine organism "Gonyaulax". Their work uncovered the structure of Gonyaulax