2. Transcriptional mechanism of the circadian clock

#### 2.1. Basic regulation of circadian transcription

The engine of the mammalian molecular clock consists of a transcription-translation feedback loop initiated by the transcription factor BMAL1-CLOCK heterodimer. BMAL1 and CLOCK have paralogs, known as BMAL2 and NPAS2, respectively. Heterodimers such as BMAL1- CLOCK bind to E-box enhancer sequences and activate the transcription of three Per (Per1, Per2 and Per3) and two Cry (Cry1 and Cry2) genes. The PER and CRY proteins subsequently repress the transcription at their own promoters through negative feedback by acting on the BMAL1-CLOCK heterodimer. The cellular circadian clock mediates the rhythmic output of the hundreds or thousands of CCG transcripts that are regulated by transcription factors or coregulators with rhythmic abundance that is a part of the cellular circadian clock [3]. The prominent transcription factors activated by BMAL1-CLOCK are REV-ERB α and β, which bind to ROREs, as well as DBP and E4BP4, which bind to D-boxes. E-box motifs contain a core CANNTG sequence, which is recognised by a basic helix-loop-helix (bHLH) domain that contains transcription factors. BMAL1-CLOCK binds tandem E boxes spaced 6 or 7 nucleotides (nt) apart with high affinity [6]. The bHLH containing the oncoprotein Myc also binds to E-boxes and directly activates the expression of multiple repressors of the clock, including Reverbα and Rev-erbβ [7]. In addition, USF1 binds to the E-box motifs of Dbp, Per1 and Per2 [8]. The RORE motif comprises an AT-rich sequence preceding a core (G/A) GGTCA motif. ROR and REV-ERB, respectively, activate and repress the transcription of genes by binding to ROREs [9]. They co-ordinately maintain robust circadian expression of core clock proteins, such as BMAL1. D-boxes are variants of basic leucine-zipper (bZIP) motifs and are 9- or 10-bp palindromes of two GTAA (C/T) half-site sequences [10]. The D-box motif is bound by the proline- and acidic

amino acid-rich bZIP (PAR-bZIP) transcription factor family, including DBP, E4BP4, HLF and TEF [11, 12]. A combination of three binding elements, E-boxes, ROREs and D-boxes, coordinates CCG transcription. Figure 1 shows that most core clock proteins including BMAL1, CLOCK, PER, CRY, REB-ERB, ROR and E4BP4 bind to many thousands of sites in the genome in a circadian manner [13].

#### 2.2. Epigenetic mechanism: effect of chromatin structure

based on interlocking transcriptional-translational feedback loops that have both positive and negative elements. The circadian oscillator orchestrates the rhythmic mRNA expression and output of hundreds or thousands of clock-controlled genes (CCG) that temporally coordinate many cellular functions [2]. Circadian transcriptional regulators are apparently involved in the initial stages of RNA polymerase II recruitment and initiation, as well as the histone modifications associated with these events to set the stage for gene expression [3]. The methylation of cytosine on CpG dinucleotides, which is also epigenetic regulation of gene expression, either directly interferes with the binding of transcriptional regulators or indirectly inactivates a gene by modulating chromatin to a repressive structure. About 43% of all protein-encoding genes in mice exhibit circadian rhythms of mRNA abundance somewhere in the body, largely in an organ-specific manner [4]. The temporal coordination of cellular functions is lost when circadian rhythms are disrupted by age, the environment or genetic mutation, with deleterious effects on health. For instance, the adrenal steroid hormone glucocorticoid that controls various physiological processes, such as metabolism, the immune response, cardiovascular activity and brain function, is under the control of the circadian clock [5], implying that several

The engine of the mammalian molecular clock consists of a transcription-translation feedback loop initiated by the transcription factor BMAL1-CLOCK heterodimer. BMAL1 and CLOCK have paralogs, known as BMAL2 and NPAS2, respectively. Heterodimers such as BMAL1- CLOCK bind to E-box enhancer sequences and activate the transcription of three Per (Per1, Per2 and Per3) and two Cry (Cry1 and Cry2) genes. The PER and CRY proteins subsequently repress the transcription at their own promoters through negative feedback by acting on the BMAL1-CLOCK heterodimer. The cellular circadian clock mediates the rhythmic output of the hundreds or thousands of CCG transcripts that are regulated by transcription factors or coregulators with rhythmic abundance that is a part of the cellular circadian clock [3]. The prominent transcription factors activated by BMAL1-CLOCK are REV-ERB α and β, which bind to ROREs, as well as DBP and E4BP4, which bind to D-boxes. E-box motifs contain a core CANNTG sequence, which is recognised by a basic helix-loop-helix (bHLH) domain that contains transcription factors. BMAL1-CLOCK binds tandem E boxes spaced 6 or 7 nucleotides (nt) apart with high affinity [6]. The bHLH containing the oncoprotein Myc also binds to E-boxes and directly activates the expression of multiple repressors of the clock, including Reverbα and Rev-erbβ [7]. In addition, USF1 binds to the E-box motifs of Dbp, Per1 and Per2 [8]. The RORE motif comprises an AT-rich sequence preceding a core (G/A) GGTCA motif. ROR and REV-ERB, respectively, activate and repress the transcription of genes by binding to ROREs [9]. They co-ordinately maintain robust circadian expression of core clock proteins, such as BMAL1. D-boxes are variants of basic leucine-zipper (bZIP) motifs and are 9- or 10-bp palindromes of two GTAA (C/T) half-site sequences [10]. The D-box motif is bound by the proline- and acidic

diseases are closely associated with disrupted circadian rhythms.

2. Transcriptional mechanism of the circadian clock

2.1. Basic regulation of circadian transcription

126 Chromatin and Epigenetics

Transcriptional regulation initially requires the coordinated control of chromatin and the genome structure [3]. In general, genetic information is packed into the chromatin structure, of which the nucleosome is the most basic unit; it determines the large-scale chromatin structure as a building block and influences transcription. Eukaryotic promoter regions are thought to have inactive states, assured by the tendency of nucleosomes to inhibit transcription by protecting protein-DNA interaction. Therefore, chromatin remodelling and loosening of the nucleosomal barrier including histone tail modifications are key steps in circadian modifications followed by sequence-specific, transcription factor binding that regulates gene expressions [14]. Distinct chromatin states are determined by unique histone post-translational modifications. First, histone acetylation levels fluctuate rhythmically at clock gene promoters and enhancers. Specifically, acetylated histone H3 at Lys27 (H3K27ac), a marker of active enhancers, and H3 at Lys9 (H3K9ac) are rhythmic and positively correlate with clock gene expression. For example, rhythmic BMAL1-CLOCK binding and H3K9ac are required as well as rhythmic histone H3 abundance at the start site for Dbp transcription [15]. Complexes of clock proteins such as PER contain various interactive partners with known catalytic activity towards chromatin [16, 17]. The acetylation of histone H3 (at Lys9 and Lys14) at Per1, Per2 and Cry1 and of H4 at Per1 during the transcriptional activation phase has been identified [18, 19]. Rhythmic histone acetylation at clock loci is largely mediated by p300 and CBP histone

Figure 1. Hierarchical regulation mechanism of circadian transcription. E, RORE and D indicate transcription factor recognition sites: E-box, RORE and D-box, respectively.

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.

and maintains specific genomic sequences. One mechanism might be the PML-RAT fusion protein in leukaemia, which induces DNA hypermethylation and gene silencing at specific target promoters [34]. Another is siRNA-mediated, RNA-directed DNA methylation, which is a stepwise process initiated by dsRNA that recruits DNMT to catalyse the de novo DNA methylation of specific regions [35]. Therefore, the susceptibility of individual CpG islands to de novo methylation might intrinsically differ, but the mechanism remains obscure. In any event, CpG methylation is strictly regulated and stable, and changes in methylation profiles are associated with diseases, indicating close relationships among DNA methylation sites, the

Epigenetic Modulation of Circadian Rhythms: *Bmal1* Gene Regulation

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

129

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

region (Figure 2). Oscillatory transcription of the Bmal1 gene requires the chromatin structure to

region in response to SAF-A binding, indicating cooperative alteration of the chromatin structure

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 recep-




open chromatin structure around RORE and a nuclear matrix-like structure at a 3<sup>0</sup>

undergo rhythmic alterations in vivo at the region around the ROREs and at the 3<sup>0</sup>

mechanism of methylation and biological functions.

tor (ROR) and reverse Erb (REV-ERB) orphan nuclear receptors.

between the 3<sup>0</sup>

3. Transcriptional regulation of the Bmal1 gene

#### 2.3. DNA methylation

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 and maintains specific genomic sequences. One mechanism might be the PML-RAT fusion protein in leukaemia, which induces DNA hypermethylation and gene silencing at specific target promoters [34]. Another is siRNA-mediated, RNA-directed DNA methylation, which is a stepwise process initiated by dsRNA that recruits DNMT to catalyse the de novo DNA methylation of specific regions [35]. Therefore, the susceptibility of individual CpG islands to de novo methylation might intrinsically differ, but the mechanism remains obscure. In any event, CpG methylation is strictly regulated and stable, and changes in methylation profiles are associated with diseases, indicating close relationships among DNA methylation sites, the mechanism of methylation and biological functions.
