The ability to know when one must eat, sleep, and wake throughout the day is determined by a cycle known as the circadian rhythm. Many behavioral and physiological activities controlled by the circadian rhythm allow a plethora of organisms to evolve and survive through the years. Disturbances to this rhythm significantly increase one’s susceptibility to cancer, aging and other metabolic diseases. Recent studies have shown that the regulation of the circadian rhythm has a strong link between its circadian clock genes and epigenetic regulators. We show that CLOCK protein, a product of the Clock gene has histone acetyltransferase activity (HAT), which leads to chromatin remodeling and mutations to the protein can cause diseases such as cancer and aging. These changes are inherited through several generations and are therefore considered to be part of an epigenetic process.
Introduction
In 2017, one of the most prestigious awards in the scientific world, the Nobel Prize in Physiology or Medicine was awarded to Jeffrey C. Hall, Michael Rosbash and Michael W.Young for their work on the molecular mechanisms that regulate circadian rhythm. The sleeping and feeding patterns for most organisms on Earth ranging from cyanobacteria to complex organisms like animals and humans have been dependent on an approximately 24-hour internal clock system1.
The circadian clock manages many physiological and behavioral processes including body temperature, sleep/wake, hormone secretion, and metabolism. Being a diurnal creature, human beings tend to conduct most of their daily activities during the day and rest at night. Nocturnal creatures like raccoons, on the other hand, sleep during the day and hunt for food at night. The ability to know ‘what to do’ during the day or night requires the circadian clock to provide cues at specific times throughout the day for various activities while synchronizing metabolic reactions with the anticipated activity cycles. The master clock that is located in the hypothalamic suprachiasmatic nuclei (SCN) works in a hierarchal system. A disruption to the cycle caused by night shifts at work or jet lag could affect a person’s metabolic rate at various levels2. It has been observed that such disruptions have been increasingly common in modern society and is known to be a contributing factor to metabolic diseases, cardiovascular diseases, and metabolic syndrome3.
Recent research has focused on the molecular pathway that links metabolism and circadian clock and have discovered that the core mammalian circadian clocks function through epigenetic mechanisms. Activators and repressors of transcription factors related to the circadian clock that is influenced by environmental factors which in turn affects the level of hormones and metabolites in the body has a direct effect on metabolic activities. The purpose of this paper is to review the use of circadian clock as an ideal model to uncover the role of epigenetics and post-translational modifications in transcription regulation. By understanding the mechanism that underlies circadian rhythm and its regulation, a targeted approach can be made to treat metabolic diseases.
EPIGENETICS REGULATION
The term ‘epigenetics’ was first coined by Conrad Waddington in 1942 and was defined as ‘a branch of biology which studies the causal interaction between genes and their products which bring the phenotype into being'. Since then, the definition of epigenetics has varied depending on who defines it and what context it is based on4. In this review paper, epigenetics is simply defined as a heritable gene regulation that does affect the original DNA sequence.
Epigenetic regulations include DNA methylation, RNA coding mechanisms, histone modifications and nuclear organization affects gene expression that is encoded within the chromatin. DNA methylation is the addition of methyl group on the CG dinucleotide of specific genes by DNA methyltransferases (DNMTs) which can either activate or repress gene expression based on which amino acid residue is being methylated5. RNA coding mechanisms that involve non-coding RNAs (ncRNAs) such as long non-coding RNAs (lncRNAs) and microRNA (miRNA) interacts with histone modifying complexes to influence gene expression by regulating transcription6. Histone modifications are covalent post-translational modifications to histone proteins in the form of ubiquitylation, acetylation, phosphorylation, methylation, and sumoylation. As a result of these modifications, the chromatin state can be altered from either euchromatin or heterochromatin, which regulates for gene transcription. The epigenetic regulation that will be the focus of this paper, histone acetylation is the addition of acetyl groups on lysine residues of histones by histone acetyltransferases (HATs). The addition is carried out using acetyl-CoA as the acetyl donor.
As discussed, these epigenetic regulations provide a form of plasticity in cellular functions and gene expression allowing a reversible change based on internal responses to the environment including diet, temperature and lifestyle habits.
CIRCADIAN RHYTHM AND ITS CIRCADIAN CLOCK
Over billions of years, multicellular organisms have evolved under a 24-hour cycle as a result of the Earth’s rotation around the Sun. The circadian rhythm does not only provide an innate sense that tells an organism when to eat or sleep but it also has allowed them to survive based on these advantages with light as its guiding principle. A core set of circadian clock genes found in most cells throughout our body code for proteins that are responsible for clock output genes and other pathways involving the circadian rhythm.
With the discovery of the period (per) locus in Drosophila melanogaster, analysis of its mutants was identified and demonstrated that this machinery consists of several transcriptional and translational autoregulatory feedback loops. The main core element of the basic clock machinery is a gene encoding a basic helix-loop-helix transcription factor-Per/Arnt/Sim (bHLH-PAS) known as circadian locomotor output cycles kaput (CLOCK)7. The domain organization of CLOCK had many similarities with members of the PAS family. Clock regulates the intrinsic circadian period and the persistence of circadian rhythmicity, which is both essential properties of the circadian clock system. A study conducted by Vitaterna et al in 1994, suggests that Clock is necessary for sustained circadian rhythmicity and is essential for behaviors involving circadian rhythm. Behavioral assays performed on mice with mutant Clock genes had irregular sleeping patterns and eating habits along with shortening of their sleeping periods. Wild-type mice with a functional Clock gene had regular sleeping patterns and eating habits with no significant changes in the number of hours spent on sleeping8.
The Clock gene is a fairly large transcriptional unit with 24 exons spanning about 100,000 base pairs found on chromosome 5 and is evolutionarily conserved as an important feature of the circadian clock mechanism. A single gene associated with a spontaneous mutation is known as tau that was isolated from a golden hamster can affect the mammalian circadian rhythm and has since provided a better understanding of the physiology of the rhythm. tau is an autosomal mutation that is known to reduce the circadian period by two hours in heterozygous mutants and four hours in homozygous mutants9.
CLOCK, a protein that aids in the regulation of the circadian rhythm is a transcription factor that acts as a positive regulator in the primary feedback loop. Experiments conducted with mice showed that mCLOCK forms a heterodimer with mBmal1 and activates the transcription of Period (PER1, PER2, and PER3) and Cryptochrome genes (CRY1 and CRY2) through E box elements located in the promoter regions10. A negative feedback mechanism then represses transcription by acting on the mCLOCK-mBmal1 complex which drives gene expression cycles of other circadian output genes also known as the clock-controlled genes (CCGs).
CLOCK AND ITS HAT ACTIVITY
In the quest to understand the transcriptional activation function of CLOCK, a study conducted by Doi et al demonstrated that the carboxy-terminal region of the CLOCK protein displayed a sequence homology with the carboxy-terminal domain of ACTR, which has known intrinsic HAT activity. Both proteins shared sequence similarity in about six amino acid regions and are evolutionarily conserved. Besides that, CLOCK and ACTR also share other structural similarities such as the nuclear receptor interaction domain (NRID) and serine-rich regions at the middle portion of the proteins. Sequence comparisons of acetyl-CoA binding motifs of several HATs revealed that CLOCK contains a motif within the carboxy-terminal glutamine-rich region. Overall, the organizations of both proteins are strikingly similar and thus led researchers to further analyze the ability of CLOCK protein to acetylate histones. Using immunoprecipitation assay with Myc antibodies, CLOCK was found to exhibit HAT activities in cells that expressed Myc-tagged mCLOCK11. In gel-HAT assays that were performed with purified histones as substrates, acetylation was found to have taken place at regions where Myc-mCLOCK migrated. Histones that were primarily acetylated were H3 and H4 and such specificity is also found to be true with ACTR. Further research led to the discovery of CLOCK being able to acetylate its binding partner Bmal1, which allows for CRY dependent repression12.
With the functional and structural knowledge of CLOCK, researchers further delved into the function of CLOCK as HAT that is necessary for circadian rhythm. Mouse embryonic fibroblasts (MEFs) derived from a homozygous mutant CLOCK mouse showed no expressions of clock genes and ectopic expression of mCLOCK successfully restored the circadian expression of mPer1 gene and a circadian output gene, Dbp13. On the other hand, ectopic expression of HAT deficient mCLOCK was not able to restore the circadian expression of both mPer1 and Dbp genes. Thus, it is obvious that the HAT activity of CLOCK is necessary for the circadian rhythm involving the regulation of mPer1 and Dbp genes. CLOCK being a master regulator of the circadian rhythm conducts chromatin remodeling in the form of histone acetylation to either express or repress the circadian genes based on external and internal cues within the body of an organism.
RECRUITMENT OF CHROMATIN MODIFIERS TO CCG
In earlier sections of the paper, CCGs were mentioned as circadian output genes that are regulated through a feedback mechanism. In this section, we are going to look at how recruitment of chromatin modifiers to the promoter sites of CCGs influences circadian rhythm. CLOCK is known to bind to the E box regions on the DNA that leads to selective chromatin remodeling. SIRT1 and HDAC3 are two examples of histone deacetylases (HDACs) that modulates the circadian clock mechanisms14. SIRT1 is a class III HDAC that depends on the availability of NAD+ and is directly linked to metabolism and the process of aging. The role of SIRT1 includes regulating CCGs and proteins involved in metabolic pathways by histone deacetylation. According to a study conducted by Nakahata et al, the activity of SIRT1 proteins varies and oscillates in a circadian manner throughout the day15. SIRT1 protein is recruited to the promoter sites of CCGs such as Nampt upon interaction with the mCLOCK-mBmal1 complex. In SIRT1 mutant mice, the circadian gene expressions and the acetylation of mBmal1 are significantly affected suggesting that SIRT1 modulates the amplitude of CLOCK-mediated acetylation. The CLOCK protein levels are also significantly reduced in SIRT1 mutant mice, which are assumed to cause changes in the circadian rhythm.
Histone deacetylase 3 (HDAC3) was found to be modulating histone acetylation of CCGs that are involved in lipid metabolism. Nuclear receptor corepressor 1 (NCoR1) recruits HDAC3 to mediate transcriptional repression of genes (Bmal1). The circadian rhythm, as well as the metabolic process, was significantly affected in mice that had disrupted NCoR1-HDAC3 genes16. These mutant mice had a shorter day period and were resistant to diet-induced obesity. The recruitment of HDAC3 in the liver was reported to be higher during the day and lower at night.
Based on the results that were obtained through the experiments that were mentioned above, both SIRT1 and HDAC3 possess intrinsic histone deacetylation activities and when recruited to the promoter regions of the CCGS, they are able to alter the chromatin state causing a change in the circadian cycle mechanism.
EFFECTS OF CLOCK MUTATION ON AGING AND CARCINOGENESIS
The circadian rhythm is essential for a healthy life and disruption to its cycle; gene expression and protein levels of the circadian clock can lead to various diseases such as cancer and progressive aging. With increasing amount of disruptions to the circadian cycle in such modern lifestyles and the increasing levels of cancer within the population, epidemiologists have been wondering if there was a correlation between these two events. A number of gene expression studies were conducted to test the hypothesis: A disruption in circadian rhythm will lead to a higher risk of cancer susceptibility.
In breast cancer cells, gene expressions of all three Per (Per1, Per2, and Per3) were downregulated. A study on the link between breast cancer and circadian clock demonstrated that PER2 is able to cause instability in estrogen receptor α (ERα). The role of ERα is to promote the growth of mammary epithelial cells and a disruption to the receptor is known to cause breast cancer. Further investigation showed that Per1 and Per2 have tumor suppressor activities and mPer2 mutant mice were more cancer-prone with increased tumor development and a decrease in apoptosis and cell cycle arrest17. The mPer2 gene is seen to function as a tumor suppressor through the regulation of DNA damage-responsive pathways. Other cancer cell lines such as leukemia and lung cancer have decreased mRNA levels of both PER1 and PER2.
By utilizing DNA array, a large percentage of genes that were expressed in either liver or muscle of wild-type mice was identified as circadian rhythm-related transcription factors18. Clock mutant mice had a significant decrease in the expressions of those rhythmic genes, which in turn affects cell proliferation and apoptosis. To assess the role of CLOCK in the regulation of cell growth, LLC1 cell lines that are essentially malignant tumors obtained from a mouse pulmonary system were generated with suppressions to the CLOCK protein by small interfering RNA (siRNA). Out of four cell lines, half of them had a much slower growth rate and when those cell lines were inoculated into wild-type mice, tumors were formed.
Besides carcinogenesis, advanced aging was also observed in organisms with disrupted levels of CLOCK proteins. Mice with homozygous mutant Clock showed early signs of hair graying after radiation as early as six weeks old compared to wild-type mice that would usually show signs of hair graying only after 30 weeks. Alopecia, a severe form of hair loss leading to permanent baldness, which is usually observed in only older mice of 37 weeks, was found to occur in Clock mutant mice at an early 13 weeks19. These Clock mutant mice also suffered from cataracts and eye inflammation after radiation at 40 weeks instead of 80 weeks as observed in wild-type mice.
Together, all the results demonstrated that a mutation in the CLOCK protein or Clock gene could possibly lead to the development of cancer and even advanced aging. Thus, it can be said that a disruption in the circadian rhythm that then affects the level of CLOCK is partly responsible for the development of such diseases.
THE HERITABILITY OF CIRCADIAN RHYTHM
To be described as an epigenetic regulation, changes that have occurred in the chromatin or histones of a parent should be inherited by future offspring. For many years, behavioral studies have been used as an approach to understanding the genetic nature of many complex behaviors including human circadian rhythm sleep disorders (CRSD). CRSD is an abnormality that involves a person’s sleep/wake timing due to difficulty in initiating or ending sleep. There are several types of CRSD such as advanced sleep phase disorder (ASPD), delayed sleep phase disorder (DSPD), irregular sleep-wake disorder (ISWD) and jet lag disorder20.
The Familial Advanced Sleep Phase Disorder (FASPD), an autosomal dominant human sleep trait that was studied using linkage analysis in a family that suffered from it showed a mutant allele on the chromosome 2q telomeric regions for all members of the family21. The region was then identified to be a single base change in the Per2 gene causing a missense mutation whereby the wild-type amino acid serine was substituted to a glycine. Mouse models representing FASPD that were engineered were subjected to various tests and analysis. Under controlled environmental conditions, FASPD mice had a two-hour shorter circadian period compared to wild-type mice suggesting that the speed of the circadian clock within the organism was affected. The speed control of the circadian clock is known to be regulated by phosphorylation associated with Per2. A mutation on Per2 will, therefore, affect phosphorylation and subsequently affect the speed of the circadian clock.
Using Genetic Association Studies, two families that are not related by any means were used as subjects of this study, one with a history of FASPD and one without. The results obtained showed that three generations from the family with FASPD had a mutation on the Per2 gene while those from the family without FASPD had no mutations on Per2 and had no significant changes in the level of phosphorylation throughout family members. Studies on DSPD and ISWD have also shown similar results in terms of heritability and thus make a convincing theory that the epigenetic changes of the circadian rhythm especially the circadian clock can be inherited through several generations.
Conclusion
Circadian rhythm is a complex ubiquitous feature that is essential for the physiological balance of our body in terms of sleep/wake, metabolic activities, feeding, hormone secretion and cell growth. The daily cycle of light and dark has been a core regulator of organisms from different phyla throughout years of evolution. In mammals, the central pacemaker located in the SCN is responsible for the control of CCGs. Products of these CCGs are used in feedback mechanisms to either allow for activation of transcription factors or repression of their expressions.
Crosstalks between various pathways are highly important to ensure the healthy state of an organism and specific genes that are known to play a role in this cycle such as Clock genes are found to be tightly regulated. Epigenetic changes that occur to Clock have a profound effect on downstream pathways and are most likely to be heritable. CLOCK protein that possesses an intrinsic HAT activity is one of the major epigenetic changes that oscillate throughout the day based on environmental cues like sunlight. Disruption to CCGs caused by a random mutation or other external factors could lead to severe abnormalities and diseases such as cancer, aging other metabolic disorders.
Future directions should include understanding the molecular mechanism that underlies the heritability of epigenetic changes that are present in individuals with circadian rhythm disorders. Studies should focus on the molecular basis of how these epigenetic changes could lead to cancer and since a link has been found between CLOCK levels and carcinogenesis, possible therapeutics could be developed to target these damaging changes. In conclusion, the circadian rhythm is a great model to study epigenetics and could be used as a possible target for various diseases.