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Genetic and molecular approaches have been critical for elucidating the mechanism of the mammalian circadian clock. Here, we demonstrate that the ClockΔ19 mutant behavioral phenotype is significantly modified by mouse strain genetic background. We map a suppressor of the ClockΔ19 mutation to a ∼900 kb interval on mouse chromosome 1 and identify the transcription factor, Usf1, as the responsible gene. A SNP in the promoter of Usf1 causes elevation of its transcript and protein in strains that suppress the Clock mutant phenotype. USF1 competes with the CLOCK:BMAL1 complex for binding to E-box sites in target genes. Saturation binding experiments demonstrate reduced affinity of the CLOCKΔ19:BMAL1 complex for E-box sites, thereby permitting increased USF1 occupancy on a genome-wide basis. We propose that USF1 is an important modulator of molecular and behavioral circadian rhythms in mammals. DOI:http://dx.doi.org/10.7554/eLife.00426.001.

Most laboratory mouse strains including C57BL/6J do not produce detectable levels of pineal melatonin owing to deficits in enzymatic activity of arylalkylamine N-acetyltransferase (AANAT) and N-acetylserotonin O-methyl transferase (ASMT), two enzymes necessary for melatonin biosynthesis. Here we report that alleles segregating at these two loci in C3H/HeJ mice, an inbred strain producing melatonin, suppress the circadian period-lengthening effect of the Clock mutation. Through a functional mapping approach, we localize mouse Asmt to chromosome X and show that it, and the Aanat locus on chromosome 11, are significantly associated with pineal melatonin levels. Treatment of suprachiasmatic nucleus (SCN) explant cultures from Period2(Luciferase) (Per2(Luc)) Clock/+ reporter mice with melatonin, or the melatonin agonist, ramelteon, phenocopies the genetic suppression of the Clock mutant phenotype observed in living animals. These results demonstrate that melatonin suppresses the Clock/+ mutant phenotype and interacts with Clock to affect the mammalian circadian system.

The mouse Clock gene encodes a basic helix-loop-helix-PAS transcription factor, CLOCK, that acts in concert with BMAL1 to form the positive elements of the circadian clock mechanism in mammals. The original Clock mutant allele is a dominant negative (antimorphic) mutation that deletes exon 19 and causes an internal deletion of 51 aa in the C-terminal activation domain of the CLOCK protein. Here we report that heterozygous Clock/+ mice exhibit high-amplitude phase-resetting responses to 6-h light pulses (Type 0 resetting) as compared with wild-type mice that have low amplitude (Type 1) phase resetting. The magnitude and time course of acute light induction in the suprachiasmatic nuclei of the only known light-induced core clock genes, Per1 and Per2, are not affected by the Clock/+ mutation. However, the amplitude of the circadian rhythms of Per gene expression are significantly reduced in Clock homozygous and heterozygous mutants. Rhythms of PER2::LUCIFERASE expression in suprachiasmatic nuclei explant cultures also are reduced in amplitude in Clock heterozygotes. The phase-response curves to changes in culture medium are Type 0 in Clock heterozygotes, but Type 1 in wild types, similar to that seen for light in vivo. The increased efficacy of resetting stimuli and decreased PER expression amplitude can be explained in a unified manner by a model in which the Clock mutation reduces circadian pacemaker amplitude in the suprachiasmatic nuclei.

We performed genome-wide mutagenesis of C57BL/6J mice using the mutagen N-ethyl-N-nitrosourea (ENU) and screened the third generation (G3) offspring for visual system alterations using electroretinography and fundus photography. Several mice in one pedigree showed characteristics of retinal degeneration when tested at 12-14 weeks of age: no recordable electroretinogram (ERG), attenuation of retinal vessels, and speckled pigmentation of the fundus. Histological studies showed that the retinas undergo a photoreceptor degeneration with apoptotic loss of outer nuclear layer nuclei but visual acuity measured using the optomotor response under photopic conditions persists in spite of considerable photoreceptor loss. The Noerg-1 mutation showed an autosomal dominant pattern of inheritance in progeny. Studies in early postnatal mice showed degeneration to occur after formation of partially functional rods. The Noerg-1 mutation was mapped genetically to chromosome 6 by crossing C57BL/6J mutants with DBA/2J or BALB/cJ mice to produce an N2 generation and then determining the ERG phenotypes and the genotypes of the N2 offspring at multiple loci using SSLP and SNP markers. Fine mapping was accomplished with a set of closely spaced markers. A non-recombinant region from 112.8 Mb to 115.1 Mb was identified, encompassing the rhodopsin (Rho) coding region. A single nucleotide transition from G to A was found in the Rho gene that is predicted to result in a substitution of Tyr for Cys at position 110, in an intradiscal loop. This mutation has been found in patients with autosomal dominant retinitis pigmentosa (RP) and results in misfolding of rhodopsin expressed in vitro. Thus, ENU mutagenesis is capable of replicating mutations that occur in human patients and is useful for generating de novo models of human inherited eye disease. Furthermore, the availability of the mouse genomic sequence and extensive DNA polymorphisms made the rapid identification of this gene possible, demonstrating that the use of ENU-induced mutations for functional gene identification is now practical for individual laboratories.

A step towards annotating the mouse genome is to use forward genetics in phenotype-driven screens to saturate the genome with mutations. The purpose of this article is to highlight the new projects in North America that are focused on isolating mouse mutations after ENU mutagenesis and phenotype screening.

The amplitudes of many circadian rhythms, at the behavioral, physiological, cellular, and biochemical levels, decrease with advanced age. Previous studies suggest that the amplitude of the central circadian pacemaker is decreased in old animals. Recently, it has been reported that expression of several circadian clock genes, including Clock, is lower in the master circadian pacemaker of old rodents. To test the hypothesis that decreased activity of a circadian clock gene renders animals more susceptible to the effects of aging, we analyzed the circadian rhythm of locomotor activity in young and old wild-type and heterozygous Clock mutant mice. We found that the effects of age and the Clock mutation were additive. These results indicate that age-related changes in circadian rhythmicity occur equally in wild-type and heterozygous Clock mutants, suggesting that the Clock mutation does not render mice more susceptible to the effects of age on the circadian pacemaker.