A Novel <i>Bmal1</i> Mutant Mouse Reveals Essential Roles of the C-Terminal Domain on Circadian Rhythms

<div><p>The mammalian circadian clock is an endogenous biological timer comprised of transcriptional/translational feedback loops of clock genes. <i>Bmal1</i> encodes an indispensable transcription factor for the generation of circadian rhythms. Here, we report a new circadian mutant mouse from gene-trapped embryonic stem cells harboring a C-terminus truncated <i>Bmal1</i> (<i>Bmal1</i>^<i>GTΔC</i>) allele. The homozygous mutant (<i>Bmal1</i>^{<i>GTΔC/GTΔC</i>}) mice immediately lost circadian behavioral rhythms under constant darkness. The heterozygous (<i>Bmal1</i>^{<i>+/GTΔC</i>}) mice displayed a gradual loss of rhythms, in contrast to <i>Bmal1</i>^<i>+/-</i> mice where rhythms were sustained. <i>Bmal1</i>^{<i>GTΔC/GTΔC</i>} mice also showed arrhythmic mRNA and protein expression in the SCN and liver. Lack of circadian reporter oscillation was also observed in cultured fibroblast cells, indicating that the arrhythmicity of <i>Bmal1</i>^{<i>GTΔC/GTΔC</i>} mice resulted from impaired molecular clock machinery. Expression of clock genes exhibited distinct responses to the mutant allele in <i>Bmal1</i>^{<i>+/GTΔC</i>} and <i>Bmal1</i>^{<i>GTΔC/GTΔC</i>} mice. Despite normal cellular localization and heterodimerization with CLOCK, overexpressed BMAL1^GTΔC was unable to activate transcription of <i>Per1</i> promoter and BMAL1-dependent CLOCK degradation. These results indicate that the C-terminal region of <i>Bmal1</i> has pivotal roles in the regulation of circadian rhythms and the <i>Bmal1</i>^<i>GTΔC</i> mice constitute a novel model system to evaluate circadian functional mechanism of BMAL1.</p></div

Structures and genotyping of <i>Bmal1</i>^<i>GTΔC</i> mice.

<p>(A) The genomic structure of ES cells harboring <i>Bmal1</i>^<i>GTΔC</i> allele. Black and grey colors indicate the exon (E) and intron (I) region, respectively. Above panel represents intact organization of Bmal1 DNA and below panel show the insertion site of the gene-trap vector. Black triangle indicates the location of the primer sequence. (B) Domains of WT and BMAL1^GTΔC proteins. (C) The genotypes were determined by using three primers PCR method (Bmal1^wt-F, Bmal1^WT-R and Bmal1^GTΔC-R). (D) The genotypes were also determined by western blotting of β-GAL.</p

The representative protein expression profiles of the liver.

<p>Mouse liver tissues were harvested at the indicated circadian times and analyzed by western blotting. The circadian expression profiles of clock proteins in WT, <i>Bmal1</i>^{<i>+/GTΔC</i>}, <i>Bmal1</i>^{<i>GTΔC/GTΔC</i>}, <i>Bmal1</i>^<i>+/—</i>and <i>Bmal1</i>^<i>-/-</i> mice (n = 3).</p

Underlying molecular mechanisms of <i>Bmal1</i>^<i>GTΔC</i>.

<p>(A) Effects of <i>Bmal1</i>^<i>GTΔC</i> on Per1 promoter activity. (B) BMAL1^wt, BMAL1^GTΔC and CLOCK were tagged with fluorescence proteins and the localizations were examined. The heterodimerization and cellular localization of CLOCK:BMAL1^wt and CLOCK:BMAL1^GTΔC were examined by BiFC assays (C) and IP experiments (D). (E) The dose dependent degradation of CLOCK by BMAL1^wt and BMAL1^GTΔC (n = 3).</p

The circadian profiles of MEFs derived from WT, <i>Bmal1</i>^{<i>+/GTΔC</i>}, <i>Bmal1</i>^{<i>GTΔC/GTΔC</i>}, <i>Bmal1</i>^<i>+/-</i> and <i>Bmal1</i>^<i>-/-</i> mice.

<p><b>(A)</b> The representative real-time luminescence profiles of each genotype. Cells were synchronized by 2hr treatment of DEX and monitored by the real-time bioluminescence device. (B) FRP of WT, <i>Bmal1</i>^{<i>+/GTΔC</i>} and <i>Bmal1</i>^<i>+/-</i> cells. Asterisks indicate significant differences (*<i>p</i><0.05) compared with WT mice. Data are represented as the mean ± S.E.M. (n = 3).</p

Altered circadian gene expression of WT, <i>Bmal1</i>^{<i>+/GTΔC</i>} and <i>Bmal1</i>^{<i>GTΔC/GTΔC</i>} mice.

<p>(A) <i>In situ</i> hybridization results of <i>Per2</i> and <i>Bmal1</i> mRNA expression in the SCN of WT, <i>Bmal1</i>^{<i>+/GTΔC</i>} and <i>Bmal1</i>^{<i>GTΔC/GTΔC</i>} mice. (B) The mRNA expression in the liver. Data are represented as the mean ± S.E.M. (n = 3~6 per group).</p

The effects of C-terminal region of <i>Bmal1</i> on the molecular circadian rhythm.

<p>(A) The structures of BMAL1^wt and Bmal1 mutant constructs, Bmal1^GTΔC, Bmal1^ΔC and Bmal1 ^ΔN. (B) The representative circadian oscillation profiles of the contructs in (A). The constructs were trasfected to WT MEFs. The luciferase activities of <i>Bmal1</i> and <i>Per2</i> promoters were distinguished by the wavelength separation method (n = 3).</p

The progeny genotypes of <i>Bmal1</i> mutant and null mice.

<p>The progeny genotypes of <i>Bmal1</i> mutant and null mice.</p

Identification of a novel circadian clock modulator controlling BMAL1 expression through a ROR/REV-ERB-response element-dependent mechanism

Circadian rhythms, biological oscillations with a period of about 24 h, are maintained by an innate genetically determined time-keeping system called the molecular circadian clockwork. Despite the physiological and clinical importance of the circadian clock, development of small molecule modulators targeting the core clock machinery has only recently been initiated. BMAL1, a core clock gene, is controlled by a ROR/REV-ERB-response element (RORE)-dependent mechanism, which plays an important role in stabilizing the period of the molecular circadian clock. Therefore, we aimed to identify a novel small molecule modulator that regulates Bmal1 gene expression in RORE-dependency, thereby influencing the molecular feedback loop of the circadian clock. For this purpose, we carried out a cell-based screen of more than 1000 drug-like compounds, using a luciferase reporter driven by the proximal region of the mouse Bmal1 promoter. One compound, designated KK-S6, repressed the RORE-dependent transcriptional activity of the mBmal1 promoter and reduced endogenous BMAL1 protein expression. More importantly, KK-S6 significantly altered the amplitude of circadian oscillations of Bmal1 and Per2 promoter activities in a dose-dependent manner, but barely affected the period length. KK-S6 effectively decreased mRNA expression of metabolic genes acting downstream of REV-ERBα, Pai-1 and Citrate synthase, that contain RORE cis-element in their promoter. KK-S6 likely acts in a RORE-dependent manner by reinforcing the REV-ERBα activity, though not by the same mechanism as known REV-ERB agonists. In conclusion, the present study demonstrates that KK-S6 functions as a novel modulator of the amplitude of molecular circadian rhythms by influencing RORE-mediated BMAL1 expression. © 2015 The Authors. Published by Elsevier Inc.