Rhythmic gene expression is central to the circadian control of physiology in mammals. Transcriptional activation of Per and Cry genes by heterodimeric bHLH-PAS proteins is a key event in the feedback loop that drives rhythmicity; however, the mechanism is not clearly understood. Here we show the transcriptional coactivators and histone acetyltransferases, p300/CBP, PCAF, and ACTR associate with the bHLH-PAS proteins, CLOCK and NPAS2, to regulate positively clock gene expression. Furthermore, Cry2 mediated repression of NPAS2:BMAL1 is overcome by overexpression of p300 in transactivation assays. Accordingly, p300 exhibits a circadian time-dependent association with NPAS2 in the vasculature, which precedes peak expression of target genes. In addition, a rhythm in core histone H3 acetylation on the mPer1 promoter in vivo correlates with the cyclical expression of their mRNAs. Temporal coactivator recruitment and HAT-dependent chromatin remodeling on the promoter of clock controlled genes in the vasculature permits the mammalian clock to orchestrate circadian gene expression.Circadian rhythms, which are generated by cell autonomous biological clocks, allow for the appropriate temporal synchronization of physiology and behavior, optimizing the efficiency of biological systems (1, 2). In mammals, the circadian timing system is hierarchical, with the master clock located in the hypothalamic suprachiasmatic nuclei (SCN) 1 (3). Circadian oscillators have been uncovered in both central and peripheral tissues, with the suprachiasmatic nucleus (SCN) coordinating temporal physiology by synchronizing peripheral oscillators through both neural and humoral outputs (4 -8).Transcriptional regulation is central to clock function. Pacemaker rhythms are generated and sustained by positive and negative transcriptional/translational feedback loops (9). Positive components include the bHLH-PAS proteins CLOCK and BMAL1 (also known as MOP3) driving transcription as functional heterodimers through E-box enhancer elements (CACGTG) (10). NPAS2 (also known as MOP4) is a paralogue of CLOCK and behaves similarly in cell and biochemical assays (8,(11)(12)(13)(14). We have observed a robust rhythm in NPAS2 mRNA expression in the aorta, kidney, and heart. Furthermore, NPAS2 can operate in the core feedback loop in the vasculature and forebrain (8,13,14). Known targets for these effector molecules are the Period (Per1-3) and Cryptochrome (Cry1-2) genes (2). As PER and CRY cytoplasmic levels rise, they can translocate back to the nucleus, and negatively regulate their own transcription by directly interacting with CLOCK:BMAL1 or NPAS2:BMAL1 heterodimers (9, 15). Thereafter, nuclear levels of the PER/CRY complex decline, relieving repression on the bHLH-PAS heterodimer and restarting the cycle with a period of ϳ24 h. The positive limb of the feedback loop also involves the regulation of bmal1, which cycles robustly antiphase to Per and Cry (9). CLOCK and BMAL1 also drive the expression of the orphan nuclear receptor Rev-erb␣ in the SCN and liver (1...

Background-Myocardial infarction, stroke, and sudden death undergo diurnal variation. Although genes relevant to hemostasis and vascular integrity undergo circadian oscillation, the role of the molecular clock in thrombotic events remains to be established. Methods and Results-A diurnal variation in the time to thrombotic vascular occlusion (TTVO) subsequent to a photochemical injury was observed in wild-type mice: TTVO varied from 24.6Ϯ2.7 minutes at zeitgeber time (ZT) 2 to 40.3Ϯ4.3 minutes at ZT8, 24.3Ϯ2.3 minutes at ZT14, and 31.0Ϯ4.4 minutes at ZT20. This pattern was disrupted or altered when core clock genes-BMAL1, CLOCK, and NPAS2-were mutated or deleted. Mutation of CLOCK abolished the diurnal variation in TTVO, whereas deletion of NPAS2 altered its temporal pattern. NPAS2 deletion prolonged TTVO and reduced blood pressure irrespective of clock time. Global BMAL1 deletion shortened TTVO at ZT8, and the diurnal variation in TTVO, but not in systemic blood pressure, was disrupted in mice in which BMAL1 had been selectively deleted in endothelium. Conclusions-Key components of the molecular clock regulate the response to a thrombogenic stimulus in vivo. Such a phenomenon may interact with environmental variables, and together with the influence of these genes on blood pressure may contribute to the diurnal variation in cardiovascular events observed in humans.

Abstract-Living organisms have adapted to the daily rotation of the earth and regular changes in the light environment.Life forms anticipate environmental transitions, adapt their own physiology, and perform activities at behaviorally advantageous times during the day. This is achieved by means of endogenous circadian clocks that can be synchronized to the daily changes in external cues, most notably light and temperature. For many years it was thought that neurons of the suprachiasmatic nucleus (SCN) uniquely controlled circadian rhythmicity of peripheral tissues via neural and humoral signals. The cloning and characterization of mammalian clock genes revealed that they are expressed in a circadian manner throughout the body. It is now accepted that peripheral cells, including those of the cardiovascular system, contain a circadian clock similar to that in the SCN. Many aspects of cardiovascular physiology are subject to diurnal variation, and serious adverse cardiovascular events including myocardial infarction, sudden cardiac death, and stroke occur with a frequency conditioned by time of day. This has raised the possibility that biological responses under the control of the molecular clock might interact with environmental cues to influence the phenotype of human cardiovascular disease. (Arterioscler Thromb Vasc Biol. 2007;27:1694-1705.)ircadian rhythms are daily cycles of physiology and behavior that are driven by an endogenous oscillator with a period of approximately (circa-) one day (dies). 1 The most obvious circadian rhythm in humans is the cycle of sleep and wakefulness. 2 These cycles are not simply consequences of light perception, but are generated by endogenous circadian clocks that can adapt the physiology of an organism to its needs in an anticipatory manner. Their expression continues (free-runs) when subjects are isolated from the light cycle, with the oscillator defining predicted day and night, organizing our behavior and physiology appropriately to adapt to the contrasting demands encountered throughout the 24-hour period.Cardiovascular or hemodynamic parameters such as heart rate, blood pressure, endothelial function, and fibrinolytic activity exhibit variations consistent with circadian rhythm. Additionally, several types of acute pathological cardiac events exhibit diurnal patterns. The incidence of acute myocardial infarction, myocardial ischemia, cardiac arrest, ventricular tachycardia, post myocardial infarction, and sudden death in heart failure all vary according to the time of day. 3,4 Social and commercial pressures such as shift work, which oppose the temporal circadian order, may be underlying factors contributing to the incidence of chronic illnesses such as cardiovascular disease and cancer. 5,6 Understanding this molecular clock and its mechanisms may ultimately allow treatment of conditions where either the severity of the illness or therapeutic efficacy exhibit circadian rhythmicity. Molecular Basis of Circadian ClocksCircadian rhythms are regulated by three components: (1)...

Objective-The incidence of heart attack and stroke undergo diurnal variation. Molecular clocks have been described in the heart and the vasculature; however it is largely unknown how the suprachiasmatic nucleus (SCN) entrains these peripheral oscillators. Methods and Results-Norepinephrine and epinephrine, added to aortic smooth muscle cells (ASMCs) in vitro, altered Per1, E4bp4, and dbp expression and altered the observed oscillations in clock gene expression. However, oscillations of Per1, E4bp4, dbp, and Per2 were preserved ex vivo in the aorta, heart, and liver harvested from dopamine ␤-hydroxylase knockout mice (Dbh Ϫ/Ϫ ) that cannot synthesize either norepinephrine or epinephrine. Furthermore, clock gene oscillations in heart, liver, and white adipose tissue phase shifted identically in Dbh Ϫ/Ϫ mice and in Dbh ϩ/Ϫ controls in response to daytime restriction of feeding. Oscillation of clock genes was similarly preserved ex vivo in tissues from