Research efforts leading to a better understanding of biological rhythms will have a broad influence on all aspects of health and the treatment of disease. Twenty-four hour and seasonal rhythms are fundamental features of all forms of life, reflecting the adaptation of life to dominant features of the earth’s environment. Our daily 24-hour routines, including the sleep/wake cycle, are controlled by a complex neural system that has an endogenous master circadian oscillator and an important output signal, melatonin, the hormone of time. Light acts on this system to entrain the endogenous clock with environmental lighting. Circulating melatonin is produced in the pineal gland and is always elevated at night. Our primary objective is to advance our knowledge of vertebrate chronobiology and neuroendocrinology. Our efforts focus on understanding the regulation of the daily rhythm in melatonin production, which plays an essential role in mental health and in timing seasonal and circadian biology. Melatonin is used in humans with the intention of improving the entrainment of the biological clock in the blind and in individuals suffering from “jet lag” resulting from shift work and travel.

Post-Translational Regulation of the Melatonin Rhythm Enzyme: Arylalkylamine N-Acetyltransferase
Klein, Coon, Gaildrat, Ganguly, Morin, Kim, Weller; in collaboration with Aitken, Boutin, Cole, Zhen, Dyda, Hickman, Obsil, Falcón, Ho, Chik, Iuvone, Jaffe, Korf, Schomerus, Malpaux, Chemineau, Markey, Kowalak, Ron, Toyama, Dawid, Young
The enzyme that drives the day/night rhythm in melatonin production is arylalkylamine Nacetyltransferase (AANAT). Many regulatory mechanisms converge on this enzyme. Our studies have provided evidence for at least three post-translational mechanisms involved in cyclic AMP regulation of AANAT activity: phosphorylation-dependent prevention of proteolytic destruction, phosphorylation-dependent allosteric effects, and uncharacterized events that activate the protein without altering the phosphorylation state of AANAT. A highly conserved mechanism involved in regulating AANAT activity is prevention of proteolysis. Studies of several species, representing three vertebrate classes, have pointed to a close association between changes in AANAT activity and AANAT protein levels. We prepared antisera against AANAT from ungulates, primates, rodents, chicken, and fish. In all cases, the antisera demonstrated a close association between large changes in AANAT protein and activity, including the large in vivo day/night changes and the rapid reductions that occur when lights are turned on in the middle of the night. We established that the rapid decrease in AANAT protein is due to proteolysis by the proteasome, an extremely rapid and irreversible mechanism that turns off melatonin production, a mechanism that is regulated by cyclic AMP.

AANAT is typically phosphorylated in the cell. A hunt for phosphorylation-dependent AANAT binding identified 14-3-3 proteins. The binding to AANAT is triggered by phosphorylation of at least one and probably both PKA sites on the enzyme. The N-terminal PKA site is located in a sequence that becomes a consensus 14-3-3 binding motif upon phosphorylation (RRHTPLAN — RRHpTPLAN). Crystallo-graphic analysis of the AANAT/14-3-3 complex revealed that the PKA/14-3-3 motif forms multiple contacts with residues in the amphipathic binding groove of 14-3-3, which allowed us to propose a simple model of the complex. Other studies revealed that the binding could be disrupted by using a 16-residue peptide containing the PKA/14-3-3 binding motif. Although the 14-3-3/AANAT complex has been studied only in the ovine pineal gland, it is probable that, given the highly conserved nature of the PKA/14-3-3 binding motif of AANAT and the ubiquitous presence and highly conserved structure of 14-3-3 proteins, AANAT exists in this form in all vertebrates. In accordance with evidence that 14-3-3 prevents dephosphorylation by a phosphatase and clipping of the N-terminal tail by a protease, the mechanism through which proteolysis of AANAT is regulated appears to involve shielding by 14-3-3.

Phosphorylation-dependent binding of AANAT to 14-3-3 protein not only protects against proteolysis but also activates AANAT by increasing the affinity of AANAT for serotonin. Analysis of the crystal structures of AANAT, obtained with and without a bisubstrate inhibitor, indicates that a region of AANAT known as Loop 1 is involved in this allosteric effect. 14-3-3 stabilizes Loop 1 in a configuration that favors catalysis in general and strongly influences serotonin binding, causing a 10-fold increase in the affinity of the enzyme for serotonin. We created cell lines expressing Aanat and used them to study the effect of the level and phosphoryla-tion state of the enzyme on its function. Intracellular acetylation was activated by elevation of cyclic AMP levels without changing total AANAT protein, pointing to the effects of cyclic AMP on other factors involved in acetylation, such as the availability of AcCoA.

Pineal/Retinal Tissue-Specific Gene Expression
Klein, Coon, Shi, Weller; in collaboration with Baler, Carter, Humphries, Falcón, Gothilf, Ron, Toyama, Dawid
The pineal expresses an unusual set of genes, some of which are expressed in only one other tissue: the retina. To determine the molecular events involved in directing tissue-specific expression of these genes, we used regions of the rat and zebra fish Aanat promoter, first, to insert genes of interest into the pineal gland, which has allowed us to characterize promoters that drive expression in the pineal gland in both animals, and, second, to study the effects of over-expression of genes of interest. To probe the function of Fra-2, which is induced in coordination with AANAT in the rodent pineal gland, we developed a construct that knocks down expression of the transcription factor Fra2 the rat. However, the knockdown does not alter induction of AANAT. Work in zebrafish has led to the development of a line of fish that expresses the green fluorescent protein reporter gene in the pineal gland under control of 3' and 5' elements of the AANAT gene flanking the open reading frame. The line has potential use in analysis of the factors that regulate the developmental expression of the AANAT gene.

Evolution of AANAT
Klein, Coon, Shi; in collaboration with Falcón, Gothilf, Koonin
The discovery of two AANAT enzymes in fish triggered interest in the possibility that two genes exist in other vertebrates. We have so far failed to find evidence of a second AANAT in any vertebrate other than fish. Duplication of the fish genome appears to have occurred following the emergence of fish from the evolutionary line leading to higher vertebrates and to have allowed distinctly different enzymes to evolve in fish, with different kinetic characteristics, and, in some species, remarkable differences in tissue distribution. In trout, one Aanat gene is expressed in the pineal (Aanat-2) and the other in the retina (Aanat-1). Regulatory differences have also evolved; in the trout pineal gland, AANAT is high at night and suppressed by light, as seen in all vertebrates, but light increases AANAT in the trout retina. These differences may have evolved to meet the different pressures on melatonin function in the pineal gland (hormonal signal of time) as compared with that in the retina (paracrine control of photosensitivity).

Transcriptional Control of Aanat
Klein, Coon, Weller; in collaboration with Baler, Blackshaw, Carter, Humphries, Chong, Iuvone
Although post-translational mechanisms play an important role in regulating AANAT activity and protein levels, the abundance of mRNA encoding the enzyme is controlled in some vertebrates. For example, day/night changes in AANAT mRNA are large in birds and rodents, whereas day/night AANAT mRNA levels are similar in trout, sheep, cows, and primates. Thus, we have studied transcriptional control of AANAT by Fra-2, a transcription factor that has been thought to control expression of AANAT in the rat. The AANAT promoter has been used as a vehicle to confer pineal- and retinal-specific expression of genes. A genetically engineered rat has been produced in which Fra-2 protein is knocked down. However, suppression of Fra-2 does not alter AANAT expression, although it was found to influence expression of the orphan receptor NGFI-B (Nur77/TR3), a member of a nuclear hormone receptor subfamily that includes the related factors Nurr1 and NOR-1. This finding has opened new opportunities to examine the role that NGFI-B plays in biology and the role that Fra-2 plays in regulating this gene. Research on chicken Aanat has identified an E-box element, which mediates circadian expression of some clock-controlled genes such as the AANAT gene. Other studies establish that the circadian clock in the chicken pineal gland is directly linked to Aanat by transcription factors that are part of the molecular machinery that makes up the biological circadian clock, providing a direct link from the clock to chicken Aanat.

Primate Retinal Melatonin Synthesis
Klein, Coon, Weller; in collaboration with Young
We maintain an interest in the regulation of melatonin in the human pineal gland and have used the rhesus pineal gland and retina as models of the human tissues, making it was possible to study elements of the serotonin-to-melatonin pathway. As is the case in all vertebrates, the activity and protein levels of AANAT in the pineal and retina were elevated at night. However, as discussed above, there were no day/night differences in AANAT mRNA, indicating that AANAT activity is regulated in primates at a post-translational site, probably by the binding of 14-3-3 proteins. A striking finding was that HIOMT mRNA was low or undetectable in the retina, consistent with previous studies on post mortem human retinae and the bovine retina. Such low levels in the primate retina suggest that AANAT may not be dedicated to melatonin production and might have a somewhat different role in the retina, such as in the synthesis of Nacetylserotonin, which may act in place of melatonin as a ligand for melatonin receptors. Alternatively, N-acetylserotonin may have an anti-oxidant role. Another possible role for AANAT is that it acts to prevent the accumulation of toxic derivatives of aromatic amines.

Pineal Protein Hunt
Klein, Coon, Gaildrat, Ganguly, Morin, Kim, Weller; in collaboration with Baler, Blackshaw, Carter, Humphries, Malpaux, Chemineau, Markey, Kowalak
With the goal of defining a map of signal transduction and metabolic networks that describe pineal function, we are constructing a comprehensive list of proteins that are of special importance to pineal function. More specifically, we are working toward identifying and characterizing proteins that bind to AANAT in order to develop a better understanding of AANAT’s regulation. Fabrice Morin undertook protein-protein interaction screens of an ovine pineal cDNA library in yeast by using AANAT as the bait (yeast two-hybrid screen). The work revealed an interaction between AANAT and an enzyme that makes AcCoA, ATP-dependent citrate lyase (ACL). ACL was highly expressed in the pineal gland compared with other tissues and exhibited a day/night difference in phosphorylation state. Preliminary studies suggest that ACL and AANAT associate in the ovine pineal gland and that AcCoA generated by the ACL is preferentially channeled to AANAT. Two collaborations used gene chips/microarrays as a first step toward identifying pineal proteins that exhibit large day/night changes that are photically regulated and that are highly expressed in the pineal gland. With commercially available chips, we found a rhythm in the transcription factor Id1. We hope to characterize fully the rhythm in NGFI-B protein and, using microarray technology, to analyze the expression of genes in animals in which expression of Fra-2 was knocked down selectively in the pineal gland and retina. We are also using custom chips containing probes for genes expressed by retinal cones, brain genes, and ESTs from pineal and retinal libraries. With these, we have been able to detect daily rhythms in mRNA encoding the S-adenosylmethionine–synthesizing enzyme, methionine adenosyl transferase (MAT), and adrenergic regulation of MAT mRNA, with high levels occurring at night, which is when the demand for Sadenosylmethionine is highest.