Evidence for an Overlapping Role of CLOCK and NPAS2 Transcription Factors in Liver Circadian Oscillators

Abstract

The mechanisms underlying the circadian control of gene expression in peripheral tissues and influencing many biological pathways are poorly defined. Factor VII (FVII), the protease triggering blood coagulation, represents a valuable model to address this issue in liver since its plasma levels oscillate in a circadian manner and its promoter contains E-boxes, which are putative DNA-binding sites for CLOCK-BMAL1 and NPAS2-BMAL1 heterodimers and hallmarks of circadian regulation. The peaks of FVII mRNA levels in livers of wild-type mice preceded those in plasma, indicating a transcriptional regulation, and were abolished in Clock ؊/؊ ; Npas2 ؊/؊ mice, thus demonstrating a role for CLOCK and NPAS2 circadian transcription factors. The investigation of Npas2؊/؊ and Clock ⌬19/⌬19 mice, which express functionally defective heterodimers, revealed robust rhythms of FVII expression in both animal models, suggesting a redundant role for NPAS2 and CLOCK. The molecular bases of these observations were established through reporter gene assays. FVII transactivation activities of the NPAS2-BMAL1 and CLOCK-BMAL1 heterodimers were (i) comparable (a fourfold increase), (ii) dampened by the negative circadian regulators PER2 and CRY1, and (iii) abolished upon E-box mutagenesis. Our data provide the first evidence in peripheral oscillators for an overlapping role of CLOCK and NPAS2 in the regulation of circadianly controlled genes.Circadian clocks are endogenous oscillators that drive biochemical, physiological, and behavioral processes in organisms (36), and the alteration of which is associated with human diseases (17,26,42). It is well documented that the circadian rhythms of mammals are controlled by a master circadian pacemaker located in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus (38) as well as by peripheral oscillators located in most tissues (43).The basic circadian molecular clockworks consist of interacting positive and negative transcriptional/translational feedback loops (11). The positive loop involves CLOCK-BMAL1 and NPAS2-BMAL1 heterodimers, three basic helix-loop-helix transcriptional activators that bind to E-box elements (CA CGTG) located in the regulatory regions of the Period (Per1, Per2, and Per3) and Cryptochrome (Cry1 and Cry2) genes (3, 13, 16, 24). CRY and PER proteins form oligomers that, once transported into the nucleus, repress their own transcription by inhibiting CLOCK-BMAL1 or NPAS2-BMAL1 (25). Furthermore, other transcription factors (CIPC, DEC1, DEC2, and Fbxl3) play important roles in the negative-feedback loop (15,19,44). The positive and negative limbs are interlaced: CLOCK-BMAL1 heterodimers indirectly regulate a rhythm in Bmal1 transcription; the nuclear orphan receptor genes Reverb␣ and Rora are coordinately activated by CLOCK-BMAL1 to produce proteins that compete for the same promoter element but have opposing actions on Bmal1 transcription (1, 32). Oscillations in gene expression resulting from this feedback loop have period lengths of approximately 24 h, thus giving ri...

Impact of the human circadian system, exercise, and their interaction on cardiovascular function

“…33). The circadian influence on platelet aggregability is consistent with animal experiments, showing effects of core molecular clock components on coagulation (34).…”

Mutations in the Circadian Gene CLOCK in Colorectal Cancer

“…CLOCK acts as a heterodimer with another transcription factor, BMAL1, to activate transcription of a large number of target genes (1). Recent data suggest that Npas2 (neuronal PAS domain protein 2) may play a redundant role in the circadian clock (3). Several of the oscillating circadian clock target genes, such as c-MYC, MDM2, p53, caspases, and cyclins, are involved in the regulation of the cell cycle, cellular homeostasis, and metabolism (2).…”

“…Some of the circadian genes also directly modulate cell proliferation. Members of the casein kinase 1 (CK1) family, which are key kinases in establishing the circadian oscillations of the core clock (4), also phosphorylate and destabilize β-catenin (3,5), a component in the WNT signaling pathway that plays a key role in colorectal cell proliferation and tumorigenesis (2). PER1 has been found to interact with ATM, a kinase involved in DNA double-strand breakinduced events, thus playing a role in DNA damage control (6).…”

Characterization of the Core Mammalian Clock Component, NPAS2, as a REV-ERBα/RORα Target Gene

“…The expression of arrays of genes is a function of the molecular clock in the liver and regulate functions, including carbohydrate, fatty acid, cholesterol and bile acid metabolism. HepG2 hepatocellular carcinoma cells have been used as a model to study circadian rhythms in the liver, including the function of CLOCK and BMAL1 as well as the circadian expression of a wide number of genes, including a number of cytochrome p450s (12)(13)(14)(15)(16)(17)(18)(19).…”