The daily timing of gene expression and physiology in mammals

Abstract

Mammalian behavior and physiology undergo daily rhythms that are coordinated by an endogenous circadian timing system. This system has a hierarchical structure, in that a master pacemaker, residing in the suprachiasmatic nucleus of the ventral hypothalamus, synchronizes peripheral oscillators in virtually all body cells. While the basic molecular mechanisms generating the daily rhythms are similar in all cells, most clock outputs are cell-specific. This conclusion is based on genome-wide transcriptome profiling studies in several tissues that have revealed hundreds of rhythmically expressed genes. Cyclic gene expression in the various organs governs overt rhythms in behavior and physiology, encompassing sleep-wake cycles, metabolism, xenobiotic detoxification, and cellular proliferation. As a consequence, chronic perturbation of this temporal organization may lead to increased morbidity and reduced lifespan.

Figure 1.. Feedback loop model for mammalian circadian oscillator. The transcription of Per and Cry genes is activated by heterodimers between BMAL1 (B) and either of the two related proteins CLOCK (C) or NPAS2 (N). The polycomb protein EZH2 interacts with these heterodimers and thereby facilitates their action. The accumulation and activity of PER and CRY proteins are also influenced by phosphorylation by protein kinases (CK1δ, ε), by ubiquitination via a complex containing the F-box protein FBXL3 (specific for CRYs), by the histone methyl transferase binding protein WDR5, and by MONO, an RNAand DNA binding protein. DEC1 and DEC2 (D1, 2) compete with BMAL1-CLOCK/NPAS2 heterodimers for E-box binding and thereby reduce E-box-mediated transactivation. A accessory feedback loop, employing the nuclear orphan receptors RORα, RORβ, and RORγ (RORα,β, γ) as activators, and REV-ERBα and REV-ERβ (REV-ERBα, β) as repressors, regulates the circadian transcription of Bmal1 See text for further explanations.

Figure 2.. A mouse with conditionally active hepatocyte clocks. A transgenic mouse was generated in which the function of hepatocyte circadian oscillators requires the tetracycline derivative doxycycline (Dox). Hepatocyte-specific, Dox-dependent overexpression of REV-ERBα (rREV= rat REV-ERBα) was achieved by placing a rat REV-ERBa (rREV-ERBα) cDNA transgene under the control of seven tetracycline operators. In the liver of transgenic mice expressing the Tet activator from the hepatocyte-specific Clebpβ locus control region (LCR), rREV-ERBα transcription is constitutively high in the absence of the tetracycline analog Dox. This leads to a constitutive repression of Bmal1 transcription and thereby to an attenuation of circadian oscillator function, since BMAL1 is required for circadian rhythm generation. The addition of Dox to the food or drinking water abolishes the binding of Tet activators to their operators of the rREV-ERBα transgene promoter, and thereby provokes the reactivation of Bmail transcription. Under these conditions, the circadian regulation of Bmal1 transcription is accomplished by a rhythmic exchange of ROR activators and endogenous REV-ERB repressors (mREV= murine REV-ERBα), as in wild-type mice (see Figure 1). Hence, circadian hepatocyte oscillators are operative in the presence of Dox.

Figure 3.. Systemically and oscillator-driven circadian liver genes. Circadian liver transcripts were identified in the transgenic mice presented in Figure 2, using genome-wide Affymetrix microarray hybridization with liver RNAs harvested at 4-hour intervals over 2 days from doxycycline-treated and untreated animals (see ref 84). This technique enables the simultaneous quantification of mRNA levels for virtually all of the 15 000 genes that are active in the liver. In doxycycline-treated mice, core clock and clock-controlled genes (CCGs) as well as systemically controlled genes, are expressed in a circadian manner. In mice not receiving doxycycline in the food, only systemically controlled genes are rhythmically expressed. The heat maps below the livers are phase maps of cyclically accumulating transcripts (red for high expression, green for low expression). S stands for mRNAs whose rhythmic transcription is controlled by systemic cues, and S+O stands for transcripts whose rhythmic transcription can be controlled by either systemic cues or hepatocyte oscillators. Comparison of the S+O heat maps of untreated and doxycycline-treated mice reveals that the circadian expression of most genes requires functional hepatocyte clocks. RNA, ribonucleic acid; mRNA, messenger ribonucleic acid

Figure 4.. A clock output pathway regulating circadian xenobiotic detoxification. The SCN master pacemaker synchronizes circadian oscillators in peripheral organs, such as liver, kidney and small intestine. The molecular signaling pathways involved in this process are still poorly understood, but they are related to feeding fasting cycles, body temperature rhythms, cyclic hormone secretion (eg, glucocorticoids), and possibly the rhythmic accumulation of metabolites. The molecular clocks in liver, kidney and small intestine govern the circadian expression of the PAR bZip transcription factors DBP, HLF, and TEF, which in turn modulate the rhythmic expression of regulators and enzymes involved in detoxification (see ref 122 for details). SCN, suprachiasmatic nuclei