Genes and rhythms

The smallest watch

Elena Kleshchenko, "Chemistry and Life" No. 11, 2017
The article has been published on the "Elements" website

The 2017 Nobel Prize in Physiology or Medicine was awarded to Jeffrey Hall, Michael Rosbash (Brandeis University, Boston) and Michael Young (Rockefeller University, New York) for the discovery of molecular mechanisms that control circadian rhythms.

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Circadian rhythms – literally "circadian" – help a living being to synchronize with the rotation of the Earth, become active during daylight hours and fall asleep in the dark (or vice versa, if the creature is at night). Observant people have long noticed that these rhythms are determined not only by the change of lighting. Mimosa in the famous experience of Jean-Jacques Dort de Merana (1729) was spreading and squeezing leaves in a dark room where daylight did not penetrate. Similar experiments were then repeated by many, for example, at the beginning of the twentieth century, the German plant physiologist Erwin Bunning attached the leaves of a bean sprout to a kimograph and recorded their movements during the normal change of day and night and under constant illumination. Animals also demonstrated circadian rhythms, and people who, as part of a similar experiment, lived for a long time in a cave or indoors, fell asleep and woke up at a rhythm quite close to 24-hour (although some overtook the terrestrial day, and others slightly lagged behind them). This means that both plants and animals have internal rhythm generators. After the DNA revolution in biology, it was natural to say, "Look for genes."

We have told the story of these genes at least twice (see "Chemistry and Life" № 2, 2000; № 6, 2011). But the clockwork mechanism of a living cell is so beautiful that you can tell it for the third time, fortunately there is a reason.

The first fundamentally important studies were carried out shortly after the discovery of the double helix. In the 60s and early 70s, Seymour Benzer studied the genetics of drosophila behavior at the California Institute of Technology. Not everyone at that time admitted that animal behavior could be rigidly determined by individual genes. Benzer, however, believed that the simplest elements of behavior could be the same phenotypic traits, depending on the genotype, as the color of the eyes or trunk of a fruit fly. (And if he hadn't died in 2007, he might well have been among the laureates.) He used chemical mutagenesis techniques to obtain drosophila lines with circadian rhythm failures. We managed to find three such lines: in one, the daily cycle was reduced to 19 hours, in the other it was extended to 28 hours, in the third line, the daily activity changed randomly, and even from the pupae these flies hatched not at a certain time of day, as in normal, but when they hit. Ronald Konopka, one of Benzer's students, investigating these lines, found that the mutant gene in all three is localized on the X chromosome (PNAS USA, 1971, 68, 2112-2116). The gene was named period, or per. Benzer and Konopka suggested (and, as it turned out later, they were right) that there is nonsense in the gene of arrhythmic flies-a mutation that interferes with the synthesis of the protein product, and mutations in the other two lines somehow change its properties.

Genes of circadian rhythms in drosophila and their products

The period gene was cloned and sequenced in 1984 by Michael Rosbash and Jeffrey Hall at Brandeis University, as well as Michael Young at Rockefeller University (PNAS USA, 1984, 81, 2142-2146; Nature, 1984, 312, 752-754; Cell, 1984, 38, 701-710; Cell, 1984, 39, 369-376). The protein encoded by this gene was named PER. It remained to understand how it works. There were several more or less speculative hypotheses about this, until the following important observations were made in the laboratories of Hall and Rosbash (they became possible due to the appearance of antibodies to PER). It turned out that the concentration of this protein in the nerve cells of drosophila varies throughout the day along the sinusoid, with a peak at night. Similarly, the concentration of the matrix RNA (mRNA) of the per gene changes, and it reaches its peak several hours earlier than the protein concentration. In arrhythmic mutants, the concentration of mRNA did not change, but the addition of a wild-type protein caused its expression. All this suggested some kind of feedback mechanism. And then it turned out that PER is a nuclear protein and moves from the cytoplasm to the nucleus. This suggested that it could be a transcription regulator (transcription, that is, mRNA synthesis, occurs in the nucleus).

In the 90s, Young's group found the timeless gene, or tim. The concentration of its mRNA also described a sinusoid with a period of 24 hours, and the product – the TIM protein – bound to PER, thereby blocking its destruction and contributing to its departure to the nucleus. Drosophila with mutations in the tim gene were found – they had broken the cyclicity of per expression, and the opposite was also true: the per mutants had broken the cyclicity of tim expression.

Then other participants in this process were discovered – the clock and cycle genes (the Rosbash group; however, the clock gene in mice was first discovered by Joseph Takahashi from the Howard Hughes Medical Institute). The products of these genes CLK and CYC interact with each other, and then sit on the promoters of the tim and per genes and turn on their transcription. When there are a lot of PER and TIM proteins, the PER:TIM dimer turns off the synthesis of mRNA from the clock and cycle genes, thereby the CLK and CYC proteins, and therefore, ultimately, its own mRNAs. The concentrations of PER and TIM, which have been increasing all this time up to the night peak, begin to fall, finally the "switch" – the dimer PER:TIM – disappears, and clock and cycle are activated again, so that their products turn on the tim and per genes again, – the daily cycle starts anew. This regulatory mechanism is called the "Transcription-Translation Feedback Loop" (TTFL).

The drop in PER and TIM concentrations after the peak is provided by other proteins. The product of the doubletime gene (DBT), which Yang and colleagues discovered, is a kinase enzyme; it phosphorylates PER, that is, attaches a phosphate group to it and thereby accelerates its degradation. And the product of the cryptochrome gene discovered by the Rosbash group, the CRY protein, is responsible for summing up the biological clock by the sun. Cryptochromes are flavoproteins (i.e. proteins containing riboflavin derivatives of nucleic acids) sensitive to blue light. The CRY protein is activated by light, interacts with TIM and triggers its degradation. And since TIM stabilizes the "partner" protein PER, its decay is also accelerated. A sleepy organism, illuminated by the sun, through the medium of CRY feels that it's time to get up, anyway.

These are not all genes of biological clocks, but their spring or pendulum is the main part that provides oscillations, that is, periodic oscillations. The mechanism described above, as it turned out, is very conservative, similar genes and feedback loops are found in many higher organisms, including humans. The "clock" genes of mammals and drosophila are homologous, but in plants the genes are different, but interact on the same principle. Cyanobacteria are an exception to the general rule: their circadian oscillator does not depend on transcription, but on protein phosphorylation. Interestingly, in human erythrocytes (mature erythrocytes are devoid of nuclei and DNA, they are sometimes impolitely called "bags with hemoglobin", respectively, there can be no transcription in them) there is an oscillation system based on redox cycles of peroxyredoxins, antioxidant enzymes. And these cycles are even regulated by external signals, such as temperature.

We should not forget about regulation at the highest levels. The fruit fly is small, and the sun doesn't really illuminate a person from the inside; "I think: it's black inside us." The main clock in mammals is located in the supraschiasmatic nucleus (SCN) of the hypothalamus. The retina of the eye transmits information about the illumination, synchronizing the clocks of its neurons with the sun. And at the command of the SHIA, the clock of the whole body is regulated through humoral factors (everyone has heard about the hormone melatonin) and the peripheral nervous system. According to the figurative expression of Professor Carlos Ibanez of the Karolinska Institute, the author of a popular science story about the discoveries of Hall, Rosbash and According to Young, posted on the Nobel website, "the circadian system of an animal is more like a watch store than a single watch." If you can imagine a store in which every morning the time on all the clocks is set by one, the most correct, but many watches are also summed up individually...

Indeed, there is evidence that the peripheral clock can adjust its time according to external factors, such as physical activity, air temperature or nutrition. (And when a grandmother wakes up her grandson to school in winter and gives him a piece of apple "to wake up" – this is not pampering, but a deep understanding of human physiology.) The peripheral clock, in turn, regulates the metabolism of fats and glucose, the release of hormones, and these signals are perceived by the CCH. The circadian clock influences our behavior, and by controlling our behavior, we help ourselves to cheer up or fall asleep... In general, our time perception is based on many feedback loops.

Daily changes in human physiology

Can mutations in clock genes make us owls or larks? They can, but such cases, apparently, are much rarer than the habit of a particular regime created by upbringing or sloppiness. For example, the family syndrome of early falling asleep – advanced sleep phase syndrome (ASPS) is described. Such people go to bed before dark and wake up after dark. This syndrome can be hereditary, and it can be caused by mutations in the gene hReg2 (Science, 2001, 291, 1040-1043; the letter h in the name of the gene comes from human). And the family syndrome of late falling asleep, delayed sleep-phase syndrome (DSPS), when a born "owl" falls asleep at three in the morning and cannot wake up in the morning, is associated with the hPer3 gene, and now also with Cry1, which again was shown by Yang's group (Cell, 2017, 169, 203-215.e213). Even the BHLHE41 gene, aka DEC2, has been found, a point mutation in which correlates with the phenotype of short sleep. Humans and mice – carriers of such a mutation – need less time to get enough sleep.

As for the connection between the biological clock and health – they are diverse. Violations of circadian rhythms cause not only sleep disorders (which is obvious), but also depression, bipolar disorders, memory disorders. And chronic lifestyle inconsistency with the indications of our internal clock can lead to serious diseases, including cancer, metabolic disorders and neurodegenerative diseases. It is assumed that research on circadian genes will help modern people find more delicate "screwdrivers" for our watches than melatonin, benzodiazepines and caffeine in all kinds, and learn to harmoniously combine work with rest.

Have a good night's sleep, everyone!

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