Circadian clocks adapt the physiology of many different species to recurring changes in their environments—notably, to 24-hour cycles of daylight and darkness—in a proactive manner. In mammals, the circadian timing system is composed of a master pacemaker in the suprachiasmatic nucleus, which is located in the brain, and subsidiary clocks in virtually all body cells. However, these circadian clocks can measure time only approximately—the Latin words circa diem mean ‘about a day'—so they must be synchronized daily to ensure that they remain in time with the 24-hour day.
The process of synchronization begins with electrical signals from the retina causing an influx of calcium ions into postsynaptic neurons in the suprachiasmatic nucleus. This influx activates various protein kinases that increase the activity of a number of immediate early transcription factors which, in turn, enhance the expression of genes ensuring that the master pacemaker remains synchronized. The suprachiasmatic nucleus then synchronizes the subsidiary clocks via a large variety of signalling pathways that depend on feeding cycles, hormones, body temperature rhythms and neuronal cues (Dibner et al., 2010).
The molecular circuitry responsible for maintaining circadian rhythms is composed of two interlocked feedback loops. The major loop relies on two transcription factors, CLOCK and BMAL1, forming a complex that stimulates the expression of target genes by binding to regions of DNA called Enhancer-boxes (E-boxes) that are associated with the target genes (Figure 1). Four of these target genes—Per1, Per2, Cry1 and Cry2—produce proteins that counteract the effects of the CLOCK:BMAL1 complex and, as a consequence, establish a negative feedback loop that actually suppresses their own expression (Lowrey and Takahashi, 2000; Reppert and Weaver, 2001). In the other loop, CLOCK and BMAL1 control the transcription of the genes that code for various nuclear receptors that govern the cyclic transcription of the Bmal1 and Clock genes (Preitner et al., 2002; Ueda et al., 2002; Bugge et al., 2012; Cho et al., 2012). The circadian system also depends on a large number of post-translational processes. The overall effect of these two feedback loops, plus the various post-translational processes, is to determine the intrinsic period of the circadian cycle, which is subsequently synchronized to a period of 24 hours as described above.
In the 1990s, researchers led by Joseph Takahashi—then at Northwestern University, now at the University of Texas Southwestern (UTSW) Medical Center—identified a semi-dominant mutation in the Clock gene, ClockΔ19, that lengthened the circadian period in mice of the C57BL/6J strain, and caused most homozygous mutant mice to become arrhythmic when exposed to constant darkness over an extended time period (Antoch et al., 1997; King et al., 1997). Although the CLOCKΔ19 mutant protein can still form a complex with BMAL1 that is capable of binding to DNA (albeit with reduced affinity), it is not able to activate transcription. Takahashi and colleagues also noted that the penetrance of the clock mutant phenotype (that is, the fraction of mice in which these traits were evident) was much higher in some strains than in others. In order to identify the genes that might be responsible for this difference between strains, they performed a complex trait analysis on nearly 200 hybrid mice and identified 14 loci associated with possible ClockΔ19 modulator genes (Shimomura et al., 2001).
Now, in eLife, Takahashi and co-workers—including Kazuhiro Shimomura of Northwestern University as first author—report that they have extended this work by identifying a transcription factor that suppresses the ClockΔ19 mutation in one strain of these mice (a strain called BALB/cJ). To this end they used a combination of two genetic techniques (quantitative trait locus and haplotype block mapping) to narrow down the region of the genome that is responsible for the suppression of this mutation: this region is a DNA segment encompassing about 900 kilo base pairs and 22 protein encoding genes. Seven of these genes were expressed in the same tissues as Clock. However, only one of them, Usf1 (which codes for a transcription factor called upstream transcription factor 1), was more active in mice in which the ClockΔ19 mutation was suppressed. Moreover, a moderate overexpression of USF1 suppressed the ClockΔ19 mutant phenotype in C57BL/6J mice. This provides compelling evidence for the scenario proposed by the authors, namely that the increased accumulation of USF1 in BALB/cJ mice accounts for the suppression of the ClockΔ19 mutant phenotype in this mouse strain.
The region responsible for increased transcription of the Usf1 gene in BALB/cJ mice has been mapped to a DNA fragment containing about 1000 base pairs. Importantly, the analysis of protein-DNA complexes by biochemical assays and chromatin-immunoprecipitation experiments demonstrated that USF1 and CLOCK:BMAL1 can indeed bind to the same E-box motifs.
The intrinsic period of the circadian cycle can be determined by keeping the mice under conditions of constant darkness. Such experiments reveal that Usf1 knockout mice and wild-type mice have similar intrinsic periods. Thus, USF1 does not contribute significantly to determining the period of the circadian cycle in wild-type mice. However, compared to wild-type mice, the mutant mice are less active overall. Moreover, their levels of activity vary much less over the circadian cycle. This suggests that USF1 contributes to the robustness of circadian behaviour.
Based on an extensive body of genetic and biochemical work, Takahashi, Shimomura and co-workers have shown that USF1 is involved in the fine-tuning of the circadian system. Moreover, as USF1 does not appear to require BMAL1 to recognize E-box DNA elements, it is likely that the CLOCK:BMAL1 complex and USF1 cannot bind their cognate DNA elements at the same time, at least in target genes that depend on a single E-box (Figure 1). Shimomura et al. thus suggest that USF1 may facilitate the binding of CLOCK:BMAL1 complexes by maintaining an open chromatin configuration around the relevant E-box.
In addition to the impact that this work will have on research into circadian rhythms, it also addresses an issue—penetrance—that is of utmost importance for complex genetics. A large fraction of illnesses, including cardiovascular dysfunction, metabolic syndrome, cancer and degenerative brain diseases, are caused by complex interactions between multiple alleles and modifier genes. However, only a few of the genes that influence the penetrance of a mutant allele have been cloned and dissected at the molecular level. The work of Shimomura et al. provides a convincing example for how genetic penetrance can be modulated in different genetic backgrounds. Indeed, it goes far beyond the analysis of circadian clocks and is relevant for a large community of life scientists interested in genetic networks.
Histone methylation plays crucial roles in the development, gene regulation, and maintenance of stem cell pluripotency in mammals. Recent work shows that histone methylation is associated with aging, yet the underlying mechanism remains unclear. In this work, we identified a class of putative histone 3 lysine 9 mono/dimethyltransferase genes (met-2, set-6, set-19, set-20, set-21, set-32, and set-33), mutations in which induce synergistic lifespan extension in the long-lived DAF-2 (insulin growth factor 1 [IGF-1] receptor) mutant in Caenorhabditis elegans. These putative histone methyltransferase plus daf-2 double mutants not only exhibited an average lifespan nearly three times that of wild-type animals and a maximal lifespan of approximately 100 days, but also significantly increased resistance to oxidative and heat stress. Synergistic lifespan extension depends on the transcription factor DAF-16 (FOXO). mRNA-seq experiments revealed that the mRNA levels of DAF-16 Class I genes, which are activated by DAF-16, were further elevated in the daf-2;set double mutants. Among these genes, tts-1, F35E8.7, ins-35, nhr-62, sod-3, asm-2, and Y39G8B.7 are required for the lifespan extension of the daf-2;set-21 double mutant. In addition, treating daf-2 animals with the H3K9me1/2 methyltransferase G9a inhibitor also extends lifespan and increases stress resistance. Therefore, investigation of DAF-2 and H3K9me1/2 deficiency-mediated synergistic longevity will contribute to a better understanding of the molecular mechanisms of aging and therapeutic applications.
Major genomic deletions in independent eukaryotic lineages have led to repeated ancestral loss of biosynthesis pathways for nine of the twenty canonical amino acids1. While the evolutionary forces driving these polyphyletic deletion events are not well understood, the consequence is that extant metazoans are unable to produce nine essential amino acids (EAAs). Previous studies have highlighted that EAA biosynthesis tends to be more energetically costly2,3, raising the possibility that these pathways were lost from organisms with access to abundant EAAs in the environment4,5. It is unclear whether present-day metazoans can reaccept these pathways to resurrect biosynthetic capabilities that were lost long ago or whether evolution has rendered EAA pathways incompatible with metazoan metabolism. Here, we report progress on a large-scale synthetic genomics effort to reestablish EAA biosynthetic functionality in mammalian cells. We designed codon-optimized biosynthesis pathways based on genes mined from Escherichia coli. These pathways were de novo synthesized in 3 kilobase chunks, assembled in yeasto and genomically integrated into a Chinese Hamster Ovary (CHO) cell line. One synthetic pathway produced valine at a sufficient level for cell viability and proliferation, and thus represents a successful example of metazoan EAA biosynthesis restoration. This prototrophic CHO line grows in valine-free medium, and metabolomics using labeled precursors verified de novo biosynthesis of valine. RNA-seq profiling of the valine prototrophic CHO line showed that the synthetic pathway minimally disrupted the cellular transcriptome. Furthermore, valine prototrophic cells exhibited transcriptional signatures associated with rescue from nutritional starvation. 13C-tracing revealed build-up of pathway intermediate 2,3-dihydroxy-3-isovalerate in these cells. Increasing the dosage of downstream ilvD boosted pathway performance and allowed for long-term propagation of second-generation cells in valine-free medium at a consistent doubling time of 3.2 days. This work demonstrates that mammalian metabolism is amenable to restoration of ancient core pathways, paving a path for genome-scale efforts to synthetically restore metabolic functions to the metazoan lineage.