Jay Dunlap

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.

Dunlap continues to investigate the circadian clock, using Neurospora and other organisms, such as Aspergillus fumigatus. As a result of the Neurospora crassa Genome Project, the results of which were published in 2003, and the development of knockouts for every gene, which are stored at the Fungal Genetics Stock Center, Dunlap believes the molecular basis for the circadian clock of Neurospora may be the first to be completely understood. Due to the highly conserved nature of biological clocks, clock mechanisms have evolved relatively few times and are similar between species. Knowledge of Neurospora systems may lead to applications with relevance to human health. The circadian nature of cellular processes in humans may be leveraged to target cancerous cells more effectively and treat sleep abnormalities.

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.

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.

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.

Dunlap and his colleagues have contributed greatly to advancements in the use of technology within the field of molecular biology. These methodological advancements have had major implications for both fungal biology and chronobiology and their future directions. For example, Dunlap's lab developed the first gene replacement for Neurospora in 1991. These technologies as well as Dunlap's support greatly contributed to the sequencing of the Neurospora genome (which was accomplished in 2002). Subsequently, Dunlap and his team improved gene replacements. He spearheaded the push to knock out all 10,000 genes in the Neurospora genome and construction of a high-density single nucleotide polymorphism map. Finally, Dunlap revolutionized the role of luciferase expression by examining codon bias and is using its implications in Neurospora and other organisms.

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.

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 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 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.

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.

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.

During Dunlap's time at Santa Cruz, one of the biology graduate students he met was Jennifer Loros. They forged a permanent relationship and were married on September 1, 1984. They have two children. When he is not conducting research, Dunlap enjoys gardening.

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.