• Experimental and Molecular Medicine
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• v.50 no.12
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• pp.6.1-6.10
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• 2018

Bmal1 is one of the key molecules that controls the mammalian molecular clock. In humans, two isoforms of Bmal1 are generated by alternative RNA splicing. Unlike the extensively studied hBmal1b, the canonical form of Bmal1 in most species, the expression and/or function of another human-specific isoform, hBmal1a, are poorly understood. Due to the lack of the N-terminal nuclear localization signal (NLS), hBMAL1a does not enter the nucleus as hBMAL1b does. However, despite the lack of the NLS, hBMAL1a still dimerizes with either hCLOCK or hBMAL1b and thereby promotes cytoplasmic retention or protein degradation, respectively. Consequently, hBMAL1a interferes with hCLOCK:hBMAL1b-induced transcriptional activation and the circadian oscillation of Period2. Moreover, when the expression of endogenous hBmal1a is aborted by CRISPR/Cas9-mediated knockout, the rhythmic expression of hPer2 and hBmal1b is restored in cultured HeLa cells. Together, these results suggest a role for hBMAL1a as a negative regulator of the mammalian molecular clock.

• Animal cells and systems
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• v.16 no.1
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• pp.1-10
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• 2012

Most living organisms synchronize their physiological and behavioral activities with the daily changes in the environment using intrinsic time-keeping systems called circadian clocks. In mammals, the key molecular features of the internal clock are transcription- and translational-based negative feedback loops, in which clock-specific transcription factors activate the periodic expression of their own repressors, thereby generating the circadian rhythms. CLOCK and BMAL1, the basic helix-loop-helix (bHLH)/PAS transcription factors, constitute the positive limb of the molecular clock oscillator. Recent investigations have shown that various levels of posttranslational regulation work in concert with CLOCK/BMAL1 in mediating circadian and cellular stimuli to control and reset the circadian rhythmicity. Here we review how the CLOCK and BMAL1 activities are regulated by intracellular distribution, posttranslational modification, and the recruitment of various epigenetic regulators in response to circadian and cellular signaling pathways.

• Journal of Photoscience
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• v.9 no.2
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• pp.25-28
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• 2002

The chicken pineal gland has been used for studies on the circadian clock, because it retains an intracellular phototransduction pathway regulating the phase of the intrinsic clock oscillator. Previously, we identified chicken clock genes expressed in the gland (cPer2, cPer3, cBmal1, cBmal2, cCry1, cCry2, and cClock), and showed that a cBMALl/2-cCLOCK heteromer acts as a regulator transactivating cPer2 gene through the CACGTG E-box element found in its promoter. Notably, mRNA expression of cPer2 gene is up-regulated by light as well as is driven by the circadian clock, implying that light-dependent clock resetting may involve the up-regulation of cPer2 gene. To explore the mechanism of light-dependent gene expression unidentified in animals, we first focused on pinopsin gene whose mRNA level is also up-regulated by light. A pinopsin promoter was isolated and analyzed by transcriptional assays using cultured chicken pineal cells, resulting in identification of an 18-bp light-responsive element that includes a CACGTG E-box sequence. We also investigated a role of mitogen-activated protein kinase (MAPK) in the clock resetting, especially in the E-box-dependent transcriptional regulation, because MAPK is phospholylated (activated) in a circadian manner and is rapidly dephosphorylated by light in the gland. Both pulldown analysis and kinase assay revealed that MAPK directly associates with BMAL1 to phosphorylate it at several Ser/Thr residues. Transcriptional analyses implied that the MAPK-mediated phosphorylation may negatively regulate the BMAL-CLOCK-dependent transactivation through the E-box. These results suggest that the CACGTG E-box serves not only as a clock-controlled element but also as a light-responsive element.

• Biomolecules & Therapeutics
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• v.29 no.1
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• pp.31-40
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• 2021

All living beings on earth have an important mechanism of 24-h periodicity, which controls their physiology, metabolism, and behavior. In humans, 24-h periodicity is regulated by the superchiasmatic nucleus (SCN) through external and environmental cues. Peripheral organs demonstrate circadian rhythms and circadian clock functions, and these are also observed in cultured cell lines. Every cell contains a CLOCK: BMAL1 loop for the generation of circadian rhythms. In this review, we focused on cell autonomous circadian rhythms in immune cells, the inflammatory diseases caused by disruption of circadian rhythms in hormones, and the role of clock genes in inflammatory diseases.

• Molecules and Cells
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• v.43 no.3
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• pp.276-285
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• 2020

Circadian rhythm is an endogenous oscillation of about 24-h period in many physiological processes and behaviors. This daily oscillation is maintained by the molecular clock machinery with transcriptional-translational feedback loops mediated by clock genes including Period2 (Per2) and Bmal1. Recently, it was revealed that gut microbiome exerts a significant impact on the circadian physiology and behavior of its host; however, the mechanism through which it regulates the molecular clock has remained elusive. 3-(4-hydroxyphenyl)propionic acid (4-OH-PPA) and 3-phenylpropionic acid (PPA) are major metabolites exclusively produced by Clostridium sporogenes and may function as unique chemical messengers communicating with its host. In the present study, we examined if two C. sporogenes-derived metabolites can modulate the oscillation of mammalian molecular clock. Interestingly, 4-OH-PPA and PPA increased the amplitude of both PER2 and Bmal1 oscillation in a dose-dependent manner following their administration immediately after the nadir or the peak of their rhythm. The phase of PER2 oscillation responded differently depending on the mode of administration of the metabolites. In addition, using an organotypic slice culture ex vivo, treatment with 4-OH-PPA increased the amplitude and lengthened the period of PER2 oscillation in the suprachiasmatic nucleus and other tissues. In summary, two C. sporogenes-derived metabolites are involved in the regulation of circadian oscillation of Per2 and Bmal1 clock genes in the host's peripheral and central clock machineries.

• Proceedings of the Zoological Society Korea Conference
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• 2001.10a
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• pp.203.2-203
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• 2001

No Abstract, See Full Text

• Animal cells and systems
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• v.9 no.3
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• pp.153-160
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• 2005

Most living organisms exhibit the circadian rhythm in their physiology and behavior. Recent identification of several clock genes in mammals has led to the molecular understanding of how these components generate and maintain the circadian rhythm. Many reports have implicated the photic induction of either mPer1 or mPer2 in the hypothalamic region called the suprachiasmatic nucleus (SCN) to phase shift the brain clock. It is now established that peripheral tissues other than the brain also express these clock genes and that the clock machinery in these tissues work in a similar way to the SCN clock. To determine the role of the two canonical clock genes, mPer1 and mPer2, in the peripheral clock shift, stable HEK293EcR cell lines that can be induced and stably express these proteins were prepared. By regulating the expression of these proteins, it could be shown that induction of the clock genes, either mPer1 or mPer2 alone is not sufficient to cause clock phase shifting in these cells. Our real-time PCR analysis on these cells indicates that the induction of mPER proteins dampens the expression of the clock-specific transcription factor mBmal1. Altogether, our present data suggest that mPer1 and mPer2 may not function in clock shift or take part in differential roles on the peripheral circadian clock.

• Journal of Life Science
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• v.27 no.5
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• pp.501-508
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• 2017

Bone morphogenetic proteins (BMPs) are multifunctional cytokines that play important roles in a variety of cellular functions. Among BMP family members, BMP2 efficiently promotes osteoblast differentiation through Smad-mediated runt-related transcription factor 2 (Runx2) expression. Several recent studies suggest that BMPs are associated with clock genes, in particular Bmal1. Bmal1 protein heterodimerizes with Clock protein and then induces period 1 (Per1) expression. However, the role of Per1 on osteoblast differentiation remains unclear. In this study, we investigated whether Per1 is involved in osteoblast differentiation. MC3T3-E1 cells were treated with BMP2 for induction of osteoblastic differentiation. Osteogenic maker gene and Per1 mRNA expression were measured using real-time PCR. Interestingly, BMP2 treatment induced Per1 mRNA expression in MC3T3-E1 cells. To further investigate the function of Per1 on osteoblast differentiation, MC3T3-E1 cells were transiently transfected with pCMV-Per1. Per1 overexpression increased Runx2 mRNA and protein levels. Also, mRNA expression and promoter activity of osteocalcin were upregulated by Per1 overexpression. To investigate the effect of interaction between Per1 and osteogenic condition, MC3T3-E1 cells were cultured in osteogenic medium containing ascorbic acid and ${\beta}$-glycerophosphate. Osteogenic medium-induced ALP staining level and mineralization were synergistically increased by overexpression of Per1. Taken together, these results demonstrate that Per1 is a positive regulator of osteoblast differentiation.

• Journal of Integrative Natural Science
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• v.5 no.3
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• pp.198-210
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• 2012

Circadian rhythms govern a remarkable variety of metabolic and physiological functions. Accumulating epidemiological and genetic evidence indicates that the disruption of circadian rhythms might be directly linked to cancer. Intriguingly, several molecular gears constituting the clock machinery have been found to establish functional interplays with regulators of the cell cycle, and alterations in clock function could lead to aberrant cellular proliferation. In addition, connections between the circadian clock and cellular metabolism have been identified that are regulated by chromatin remodelling. This suggests that abnormal metabolism in cancer could also be a consequence of a disrupted circadian clock. Therefore, a comprehensive understanding of the molecular links that connect the circadian clock to the cell cycle and metabolism could provide therapeutic benefit against certain human neoplasias.

• Biomedical Science Letters
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• v.12 no.1
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• pp.53-56
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• 2006

The suprachiasmatic nucleus (SCN) of the anterior hypothalamus is the circadian pacemaker entrained to the 24-hr day by environmental time cues. Major circadian genes such as mPeriod ($mPer1{\sim}3$) and mCryptochrome ($mCry1{\sim}2$) are actively transcribed by the action of CLOCK/BMAL heterodimers, and in turn, these are being suppressed by the mPER/mCRY complex. In the study, the locomotor activity rhythms of mPer1 Knockout (KO) mice are measured, and the expression profiles of Heat Shock Protein 105kDa (HSP 105) genes in the SCN were measured by in situ hybridization. In agreement with previous reports, the locomotor activity rhythm of mPer1 KO mice was much shorter than that of wildtype. In addition, the total bout of activity of mPer1 KO was less in comparison to control mice. The expression of HSP 105 in the SCN of mPer1 KO mice was ranged from CT6 to CT22, with a peak level at CT14, implying that the gene are under the control of circadian clock. However, the expression of HSP 105 in the SCN of wildtype could not be detected in our study. Further analysis will reveal the direct or indirect regulation by mPer1 on the expression in the SCN and the role of the gene in the circadian clock.