3. Transcriptional regulation of the Bmal1 gene

acetyltransferases (HAT) [15, 19], and CLOCK itself might also have intrinsic HAT activity [20]. Levels of histone acetylation are also regulated by histone deacetylases (HDAC) as well as by HAT. Several HDAC are important in the control of circadian histone acetylation. For example, REV-ERBα represses transcription in part by recruiting the co-repressor complexes NCoR and/or SMRT to ROREs [21]. One major mechanism of transcriptional repression mediated by CRY and PER is the direct recruitment of the Sin3 complex, which contains HDAC1 and HDAC2 [17]. Another co-repressor complex containing HDAC1 and HDAC2 subunits, NuRD, binds PER-CRY and deacetylates nearby histones, thereby represses clock genes [22]. Sirtuins are another class of HDAC involved in the core clock mechanism that associate with the BMAL-CLOCK heterodimer, and levels of their common cofactor, nicotinamide adenine dinucleotide, are under tight circadian control in many physiological systems [23, 24]. In addition to being acetylated, lysine side chains can be methylated by methyltransferases, and their deacetylation often precedes and facilitates an acetylation-methylation switch. Histone H3 at Lys9 methylation (H3K9me) promotes heterochromatin formation and transcriptional repression. Rhythmic H3K9me near circadian E boxes is mediated by SUV39 methyltransferase and is antiphase to H3K9ac rhythms in the mouse liver [15]. The di- and trimethylation of H3 at Lys27 also proceed at Per1 and Per2 during the repressive phase [25]. The circadian clock regulates global transcriptional integrity and chromatin status by regulating RNA polymerase II, because circadian transcription is clustered in phase and accompanied by circadian control of RNA polymerase II recruitment and initiation [26]. The above individual mechanism is governed by the three-dimensional (3D) architecture of chromatin and its critical contributions to longdistance cis-acting mechanisms of gene regulation [27]. Regulatory elements such as enhancers, silencers and insulators built up functional 3D architectures in the nucleus and manage the transcription factory with specific properties [28]. Several looping factors, such as components of the Mediator complex, interact with clock transcription factors [29]. Deletion of one of the factors important for looping, Smc3, causes major disruptions to the clock [30]. Recently, the detailed 3D multi-loop aggregate/rosette chromatin architecture and functional dynamics have been revealed [31, 32], and this may explain how physiological functions are regulated with a tissue-specific rhythm in spite of the same core clock system. These results suggest that epigenetic regulation caused by the chromatin structure is important for circadian transcription, and further researches from the viewpoint of 3D chromatin structure are required to elucidate the

physiological function with circadian rhythm in the tissue.

The most common epigenetic modification is DNA methylation, which is a covalent chemical alteration that plays a crucial role in numerous biological processes. It occurs in mammals predominantly on cytosine residues in cytosine-guanine (CpG) dinucleotides, and tissuespecific genomic DNA methylation patterns play a fundamental role in establishing cell identity during differentiation. Generally, although about 70% of all CpG sequences in mouse and human genomes are methylated, CpG islands in promoter sequences are methylated at a relatively lower level [33]. Overall, DNA methylation exhibits no major rhythmic changes and the cellular function of DNA methylation depends on which gene is methylated. One of the most important issues regarding DNA methylation is how the machinery is directed towards

2.3. DNA methylation

128 Chromatin and Epigenetics

Bmal1 was originally characterised due to its high expression levels in brain and muscle cells [36]. The activity of Bmal1�/� mice immediately becomes arrhythmic in constant darkness; therefore, BMAL1 is apparently an essential and non-redundant component of the mammalian clock [37]. Among the core clock genes, BmalL1 expression oscillates in the SCN and in peripheral clock cells, in close association with circadian rhythms [38]. We evaluated the chromatin structure of the Bmal1 gene and discovered a unique structure within the Bmal1 promoter. The Bmal1 promoter region comprises mainly a general nucleosome structure upstream of a 5<sup>0</sup> SacI site, an open chromatin structure around RORE and a nuclear matrix-like structure at a 3<sup>0</sup> -flanking region (Figure 2). Oscillatory transcription of the Bmal1 gene requires the chromatin structure to undergo rhythmic alterations in vivo at the region around the ROREs and at the 3<sup>0</sup> -flanking region in response to SAF-A binding, indicating cooperative alteration of the chromatin structure between the 3<sup>0</sup> -flanking region and the ROREs [39]. The methylation of DNA on CpG islands results in transcriptional repression either by interfering with transcription factor binding or by including a repressive chromatin structure [40]. The methylation of CpG adjacent to the core Sp1 motif decreases Sp1/Sp3 binding [41], which might be associated with the repression of Bmal1 transcription by DNA methylation, because many putative Sp1-binding motifs are located around the Bmal1 promoter. The level of DNA methylation within a �1 kb region surrounding the transcription start site closely correlates with gene repression, and the promoter of clock genes including Bmal1 is usually unmethylated [39]. However, the hypermethylation of CpG islands in the promoter of Bmal1 transcriptionally silences its expression in haematological

Figure 2. Chromatin structure of Bmal1 promoter. Oval, unfilled boxes and arrow near BamHI indicate nucleosome, RORE and transcription start site, respectively. RORE: recognition motifs for retinoic acid receptor-related orphan receptor (ROR) and reverse Erb (REV-ERB) orphan nuclear receptors.

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 closely associated with various diseases.

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

Epigenetic Modulation of Circadian Rhythms: *Bmal1* Gene Regulation

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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

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

cells and the inhibition of apoptosis [55].

expressed [55].

We recently found that recovery from DNA methylation by 5-aza-2<sup>0</sup> -deoxycytidine (aza-dC) 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 for adaptation to environmental change.
