4. Disease

Appropriate circadian gene expression is necessary for the normal cell development. That is, distorted clock gene expression leads to various diseases. This chapter focuses on cancers and some other diseases.

### 4.1. Cancer

Close relationships between clock gene expression and the initiation and progression of cancer are obvious from the findings of many studies. Clock gene expression is altered in many types of malignancies including breast, lung, haematopoietic, pancreatic and skin cancers. Clock genes are apt to be downregulated in many cancer types, as shown in Table 1. These phenomena imply that clock genes have some anti-tumour effects. The physiological disruption of circadian


Table 1. Clock genes and their possible functions for cancer suppression.

rhythms and the genetic loss of Per2 or Bmal1 promote tumorigenesis in lung cancer [47], and such disruptions are associated with upregulated c-Myc levels. The expression of Bmal1 is suppressed in pancreatic cancer, and this gene activates the p53 tumour suppressor pathway, playing an important role in cancer suppression [48]. Fu et al. found that PER2 is an important factor for tumour suppression and the DNA damage response [49]. The overexpression of Per1 or Per2 can lead to the apoptosis of cancer cells [50, 51]. Mao et al. reported that Cry2 expression is decreased in breast cancer, resulting in an altered methylation pattern in CpG islands [52]. The findings of another study support this observation, and CRY2 suppression is closely associated with risks for breast cancer [53]. From a mechanistic viewpoint, one of the main factors in such disrupted circadian gene expression might be MYC. According to a report by Altman et al., this gene directly activates REV-ERB, which suppresses Bmal1, and their constitutive expression suspends clock mechanisms [7]. These findings suggest that the appropriate expression of clock genes is necessary to maintain normal tissues. On the other hand, leukaemia stem cells in acute myeloid leukaemia (AML) have intact circadian expression. Furthermore, knockdown studies have shown that Bmal1 and Clock are required for AML cell growth and that disrupted circadian rhythm machinery is an anti-leukaemic factor that leads to leukaemia stem cell differentiation [54]. In addition, upregulated Clock plays critical roles in the proliferation of colorectal carcinoma cells and the inhibition of apoptosis [55].

malignancies [42, 43]. Relationships between the DNA methylation of clock genes and diseases have been identified. The ROREs, which are critical elements for Bmal1 oscillatory transcription [44], are embedded in a unique GC-rich open chromatin structure. We also found that DNA demethylation of the Bmal1 promoter enhances Bmal1, and then Per2 and Cry1 transcription that function in the circadian oscillation of Bmal1 transcription recover, suggesting that the circadian rhythm is restored [42, 43]. Furthermore, DNA methylation might contribute to the developmental expression of clock genes [45]. These lines of evidence suggest that the DNA methylation of clock genes, in particular, Bmal1, plays a key role in the disruption of circadian rhythms that are

differs between the Bmal1 and Rpib9 genes, suggesting that the release of methylation depends on the locus/gene or sequence and that methylation status is specific to the DNA site [46]. Taken together, these findings imply that methylation is specific to gene function and that an early response to the aza-dC demethylation of sites in Bmal1 might be functionally important

Appropriate circadian gene expression is necessary for the normal cell development. That is, distorted clock gene expression leads to various diseases. This chapter focuses on cancers and

Close relationships between clock gene expression and the initiation and progression of cancer are obvious from the findings of many studies. Clock gene expression is altered in many types of malignancies including breast, lung, haematopoietic, pancreatic and skin cancers. Clock genes are apt to be downregulated in many cancer types, as shown in Table 1. These phenomena imply that clock genes have some anti-tumour effects. The physiological disruption of circadian

Gene Expression Mechanism Cancer type DNA methylation References Per1 Downregulated Apoptosis Colon, lung, breast — [38] Per2 Downregulated Apoptosis Lung, lymphocyte — [36, 37]

Cry2 Downregulated Unknown Breast Hypermethylation [39, 40] Bmal1 Downregulated p53 pathway Pancreas — [35]

Table 1. Clock genes and their possible functions for cancer suppression.

Downregulated MYC-downregulation Lung, breast — [34, 36, 37] Downregulated p53-upregulation Lung, breast — [36, 37]

Downregulated p300, CAT activation Leukaemia Hypermethylation [29, 33, 34]


closely associated with various diseases.

for adaptation to environmental change.

4. Disease

130 Chromatin and Epigenetics

4.1. Cancer

some other diseases.

We recently found that recovery from DNA methylation by 5-aza-2<sup>0</sup>

The roles of clock genes seem to differ among stages or tissues in patients with cancer. In addition to classical genetic mutations, the epigenetic landscapes of cancer cells are rather contorted. From an epigenetic perspective, clock genes functionally associate with histone modifying genes that are responsible for cancer progression and maintenance. Mixed lineage leukaemia (MLL) genes were originally discovered through detailed analyses of leukaemogenic rearrangement but they are now thought to be responsible for histone H3K4 methyltransferase activity and promoters of target gene transcription. Mutations of MLL genes literally trigger mixed lineage leukaemia and are necessary to maintain malignancy through aberrant epigenetic gene regulation [56]. The relationship between MLL genes and circadian rhythm maintenance through histone modification has been studied in detail. According to Katada et al., MLL1 has CLOCK-associated histone modifying activity, and it is necessary to generate circadian rhythms in fibroblasts [57]. Kim et al. found that MLL3 and 4 are factors that regulate circadian rhythmic homeostasis in the liver [58]. In addition, MLL3 contributes to circadian rhythm generation in mouse embryonic fibroblasts (MEFs) [59]. The histone modifying enzyme EZH2 is another histone-lysine N methyl transferase that is responsible for histone H3K27 methyl transfer. This modification results in transcription repression. Ezh2 also promotes tumorigenesis by altering the expression of numerous tumour suppressor genes [60]. EZH2 interacts with CLOCK-BMAL1 complexes and is necessary for circadian rhythm maintenance [25]. Although CLOCK per se is not considered to be an oncogene, it might affect cancer cell proliferation if it is atypically expressed [55].

Considering the altered methylation patterns of the promoter regions of clock genes, the features of epigenetic abnormalities of cancer cells comprise highly methylated CpG islands of specific genes accompanied by low methylation status of other genes [61]. Some studies have indicated that this phenomenon is true for clock genes. The Cry2 promoter tends to be highly methylated in patients with breast cancer, resulting in lower Cry2 expression compared with controls [43]. Taniguchi et al. reported that CpG islands of the Bmal1 promoter are hypermethylated in diffuse large B-cell lymphoma and in acute lymphocytic and myeloid leukaemia [42]. We also reported this phenomenon and that the methylation pattern of the Per2 promoter region does not change in RPMI8402 cells [46]. The aberrant methylation pattern of the Bmal1 promoter was restored, and the intrinsic rhythm was revived after 1 day of aza-dC treatment. These findings indicate that active mechanisms in leukaemia cells maintain the promoters of hypermethylated Bmal1 gene status.

more appropriate model of mania in humans. However, precisely how these CLOCK disruptions affect the upregulated dopamine content in the VTA remains obscure. The expression of monoamine oxidase A (MAOA), which inactivates monoamine neurotransmitters including dopamine, serotonin and norepinephrine, is regulated by circadian clock genes including Bmal1, Npas2 and Per2 [75]. However, in this mechanism, CLOCK, unlike NPAS2, does not work as a transcriptional activator. Therefore, the absence of CLOCK directly results in downregulated MAOA activity, and consequent dopamine upregulation cannot be concluded. Some other CLOCK functions including histone modification activity or an indirect action of CLOCK might

Epigenetic Modulation of Circadian Rhythms: *Bmal1* Gene Regulation

http://dx.doi.org/10.5772/intechopen.79975

133

Patients with depression frequently have insomnia and abnormal circadian rhythms that could reasonably relate to altered clock gene expression. Circadian clock gene expression has been compared between post-mortem brain samples from patients with major depressive disorder (MDD) and age-matched controls [76]. The findings showed abnormal clock gene phasing and decreased Bmal1 and Per2 oscillation in most brain regions of the patients. These findings provided direct evidence that clock gene expression is altered in the central nervous system of patients with MDD. On the other hand, depression states and anti-depressant effects are often tested in experimental animal models such as laboratory mice that are suspended by the tail or forced to swim to mimic short duration stress or exposed to social defeat to mimic chronic stress [77]. The volume of the hippocampus is reduced in both patients and in a model that develops depressive pathophysiology after exposure to chronic stress, and this volume is restored by administering anti-depressant medicine. Brain-derived neurotropic factor (BDNF) plays a very important role in the hippocampus as an anti-depressant and for adaptation to stress. Antidepressants enhance BDNF expression in the mouse brain [78]; BDNF infused into the hippocampus has anti-depressant effects in behavioural mouse models of depression [78, 79] and the action of the anti-depressant desipramine is attenuated mice with a BDNF deletion in the forebrain [78, 80]. Expression of Bdnf gene is rhythmic in rat brain regions including hippocampus. However, its downregulation in an animal model of depression was due to the methylation status of the promoter region of the Bdnf gene [81]. Tsankova et al. found that the expression of two BDNF variants, Bdnf III and IV, is downregulated and that the promoter regions of corresponding variants are hypermethylated in laboratory mice exposed to defeat stress [82]. Furthermore, chronic imipramine administration increased histone acetylation on the corresponding promoters, and this downregulation was reversed. The findings of the above studies indicate that Bdnf gene expression is rhythmically maintained under normal conditions but is epigenetically regulated under stress. However, precisely how Bdnf expression is rhythmically maintained

The circadian clock controls the daily oscillations of gene expression and physiological function at the cellular level, indicating that the control of circadian rhythms at the cellular level is important for human health. After we elucidated the transcriptional mechanism of the nonredundant essential unique clock gene, Bmal1, we developed a circadian functional assay system that consists of luminescent reporter cells and the application of Bmal1 findings. We found that

be involved in dopamine upregulation, and investigations into this are underway.

remains unclear and awaits further investigation.

5. Assays of Bmal1 transcription modulators

As noted above, many studies have emphasised close relationships between epigenetic modification and circadian clock genes in cancer proliferation and progression. However, the precise mechanisms seem highly complex and remain obscure. Further investigation is required to elucidate these mechanisms.

### 4.2. Other diseases

Circadian rhythms are also associated with diseases other than cancer through effects on the cardiovascular, renal, immune, endocrine, neuropsychiatric and metabolic systems [5, 62–67]. Many physiological processes cannot be harmonised when the intrinsic rhythm is aberrant and such dyssynchrony leads to many diseases.

Here, we consider neuropsychiatric disorders. Disrupted sleep-wake cycles, depression, Alzheimer's disease and mood disorders among neuropsychiatric disorders are notably linked to altered circadian rhythms. However, circadian epigenomics have received less consideration in studies of neuropsychiatric disorders compared with cancers.

Alzheimer's disease is an age-dependent neurodegenerative disorder that is associated with severe cognitive impairment, and its incidence is increasing, particularly in developed countries due to extended life spans. The typical clinical symptoms are disordered circadian rhythms and abnormal sleep patterns. Amyloid beta is a key molecule in this neurodegeneration [68], and it reportedly degrades BMAL1 protein [69]. The lack of this powerful rhythm generator disrupts circadian rhythms in many patients. Furthermore, the methylation rhythm of the Bmal1 promoter changes in the neocortex of patients with this disease. These phenomena imply that the aberrant methylation of the Bmal1 promoter and rapid BMAL1 degradation together affect behavioural changes or cognitive impairments. Furthermore, a methylome study of the neocortex of brains at autopsy revealed attenuated methylation rhythms in samples from patients with Alzheimer disease compared with controls [70]. The neocortex is very rare in terms of tissues with circadian methylation rhythms.

According to many studies, contorted clock gene expression patterns and mood disorders are closely associated in experimental animal models. Genetic experiments have found that CLOCK is a key factor in maniac states because Clock mutant mice (ClockΔ19) develop clear features [71–73] of mania, circadian rhythm disruption, hyperactivity and decreased sleep. The physiological features of these mutant mice include altered gene expression patterns and excited neurons due to upregulated dopamine content in the ventral tegmental area (VTA) [74]. Notably, knockdown of CLOCK in the VTA using RNA interference results in concomitant mania-like (hyperactivity and decreased anxiety) and depression-like behaviours in mice. Since patients with mania often experience depressive episodes, this knockdown mouse is a more appropriate model of mania in humans. However, precisely how these CLOCK disruptions affect the upregulated dopamine content in the VTA remains obscure. The expression of monoamine oxidase A (MAOA), which inactivates monoamine neurotransmitters including dopamine, serotonin and norepinephrine, is regulated by circadian clock genes including Bmal1, Npas2 and Per2 [75]. However, in this mechanism, CLOCK, unlike NPAS2, does not work as a transcriptional activator. Therefore, the absence of CLOCK directly results in downregulated MAOA activity, and consequent dopamine upregulation cannot be concluded. Some other CLOCK functions including histone modification activity or an indirect action of CLOCK might be involved in dopamine upregulation, and investigations into this are underway.

with controls [43]. Taniguchi et al. reported that CpG islands of the Bmal1 promoter are hypermethylated in diffuse large B-cell lymphoma and in acute lymphocytic and myeloid leukaemia [42]. We also reported this phenomenon and that the methylation pattern of the Per2 promoter region does not change in RPMI8402 cells [46]. The aberrant methylation pattern of the Bmal1 promoter was restored, and the intrinsic rhythm was revived after 1 day of aza-dC treatment. These findings indicate that active mechanisms in leukaemia cells main-

As noted above, many studies have emphasised close relationships between epigenetic modification and circadian clock genes in cancer proliferation and progression. However, the precise mechanisms seem highly complex and remain obscure. Further investigation is required to

Circadian rhythms are also associated with diseases other than cancer through effects on the cardiovascular, renal, immune, endocrine, neuropsychiatric and metabolic systems [5, 62–67]. Many physiological processes cannot be harmonised when the intrinsic rhythm is aberrant and

Here, we consider neuropsychiatric disorders. Disrupted sleep-wake cycles, depression, Alzheimer's disease and mood disorders among neuropsychiatric disorders are notably linked to altered circadian rhythms. However, circadian epigenomics have received less consideration

Alzheimer's disease is an age-dependent neurodegenerative disorder that is associated with severe cognitive impairment, and its incidence is increasing, particularly in developed countries due to extended life spans. The typical clinical symptoms are disordered circadian rhythms and abnormal sleep patterns. Amyloid beta is a key molecule in this neurodegeneration [68], and it reportedly degrades BMAL1 protein [69]. The lack of this powerful rhythm generator disrupts circadian rhythms in many patients. Furthermore, the methylation rhythm of the Bmal1 promoter changes in the neocortex of patients with this disease. These phenomena imply that the aberrant methylation of the Bmal1 promoter and rapid BMAL1 degradation together affect behavioural changes or cognitive impairments. Furthermore, a methylome study of the neocortex of brains at autopsy revealed attenuated methylation rhythms in samples from patients with Alzheimer disease compared with controls [70]. The neocortex is very rare in terms of tissues

According to many studies, contorted clock gene expression patterns and mood disorders are closely associated in experimental animal models. Genetic experiments have found that CLOCK is a key factor in maniac states because Clock mutant mice (ClockΔ19) develop clear features [71–73] of mania, circadian rhythm disruption, hyperactivity and decreased sleep. The physiological features of these mutant mice include altered gene expression patterns and excited neurons due to upregulated dopamine content in the ventral tegmental area (VTA) [74]. Notably, knockdown of CLOCK in the VTA using RNA interference results in concomitant mania-like (hyperactivity and decreased anxiety) and depression-like behaviours in mice. Since patients with mania often experience depressive episodes, this knockdown mouse is a

tain the promoters of hypermethylated Bmal1 gene status.

in studies of neuropsychiatric disorders compared with cancers.

elucidate these mechanisms.

such dyssynchrony leads to many diseases.

with circadian methylation rhythms.

4.2. Other diseases

132 Chromatin and Epigenetics

Patients with depression frequently have insomnia and abnormal circadian rhythms that could reasonably relate to altered clock gene expression. Circadian clock gene expression has been compared between post-mortem brain samples from patients with major depressive disorder (MDD) and age-matched controls [76]. The findings showed abnormal clock gene phasing and decreased Bmal1 and Per2 oscillation in most brain regions of the patients. These findings provided direct evidence that clock gene expression is altered in the central nervous system of patients with MDD. On the other hand, depression states and anti-depressant effects are often tested in experimental animal models such as laboratory mice that are suspended by the tail or forced to swim to mimic short duration stress or exposed to social defeat to mimic chronic stress [77]. The volume of the hippocampus is reduced in both patients and in a model that develops depressive pathophysiology after exposure to chronic stress, and this volume is restored by administering anti-depressant medicine. Brain-derived neurotropic factor (BDNF) plays a very important role in the hippocampus as an anti-depressant and for adaptation to stress. Antidepressants enhance BDNF expression in the mouse brain [78]; BDNF infused into the hippocampus has anti-depressant effects in behavioural mouse models of depression [78, 79] and the action of the anti-depressant desipramine is attenuated mice with a BDNF deletion in the forebrain [78, 80]. Expression of Bdnf gene is rhythmic in rat brain regions including hippocampus. However, its downregulation in an animal model of depression was due to the methylation status of the promoter region of the Bdnf gene [81]. Tsankova et al. found that the expression of two BDNF variants, Bdnf III and IV, is downregulated and that the promoter regions of corresponding variants are hypermethylated in laboratory mice exposed to defeat stress [82]. Furthermore, chronic imipramine administration increased histone acetylation on the corresponding promoters, and this downregulation was reversed. The findings of the above studies indicate that Bdnf gene expression is rhythmically maintained under normal conditions but is epigenetically regulated under stress. However, precisely how Bdnf expression is rhythmically maintained remains unclear and awaits further investigation.
