Liver “Tick Tock”

Authors

  • Nicholas A. Shackel

    1. Centenary Institute, Central Clinical School, The University of Sydney, and Royal Prince Alfred Hospital, Camperdown, Sydney, Australia
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  • Potential conflict of interest: Nothing to report.

Gatfield D, Le Martelot G, Vejnar CE, Gerlach D, Schaad O, Fleury-Olela F, Ruskeepää AL, et al. Integration of microRNA miR-122 in hepatic circadian gene expression. Genes Dev 2009;23:1313–1326. (Reprinted with permission.)

Abstract

In liver, most metabolic pathways are under circadian control, and hundreds of protein-encoding genes are thus transcribed in a cyclic fashion. Here we show that rhythmic transcription extends to the locus specifying miR-122, a highly abundant, hepatocyte-specific microRNA. Genetic loss-of-function and gain-of-function experiments have identified the orphan nuclear receptor REV-ERBalpha as the major circadian regulator of mir-122 transcription. Although due to its long half-life mature miR-122 accumulates at nearly constant rates throughout the day, this miRNA is tightly associated with control mechanisms governing circadian gene expression. Thus, the knockdown of miR-122 expression via an antisense oligonucleotide (ASO) strategy resulted in the up- and down-regulation of hundreds of mRNAs, of which a disproportionately high fraction accumulates in a circadian fashion. miR-122 has previously been linked to the regulation of cholesterol and lipid metabolism. The transcripts associated with these pathways indeed show the strongest time point-specific changes upon miR-122 depletion. The identification of PPARbeta/delta and the peroxisome proliferator-activated receptor alpha (PPARalpha) coactivator Smarcd1/Baf60a as novel miR-122 targets suggests an involvement of the circadian metabolic regulators of the PPAR family in miR-122-mediated metabolic control.

Comment

The term circadian comes from the Latin circa, (“around”) and dies (“day”) meaning “approximately one day”. The circadian rhythm pervades life and defines the daily cycle of biological processes that is seen in virtually every organism from bacteria through to mammals. The maintenance of a circadian rhythm within a highly metabolically active organ such as the liver is essential for its normal function, because it responds to nutrients and other stimuli in a repetitive manner on a daily basis. The “master” clock located in the suprachiasmatic nuclei of the brain governs the circadian rhythm.1 However, peripheral cellular clocks also maintain circadian rhythm within organs such as the liver by complex molecular mechanisms. Cellular clocks involve two interlocked transcriptional/posttranslational feedback loops, in which “clock” proteins negatively regulate “clock” genes in a rhythmical manner (Fig. 1).1 The clock genes CLOCK, brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like-1 (BMAL1), Period (PER), and Cryptochrome (CRY) are the principal molecular mediators of circadian rhythm within the liver and other tissues.1, 2 The CLOCK/BMAL1 transcription factors mediate activation of PER and CRY genes. Nuclear accumulation of PER and CRY proteins over the circadian day ultimately turns off the transcriptional drive to their encoding genes. Rev-erbα is an orphan receptor, which coordinates the expression of these two feedback loops because it directly controls the expression of the BMAL1 and in turn is regulated by CLOCK/BMAL1 heterodimers.3 Rev-erbα has been shown to be regulated by glucocorticoids as well as ligands of peroxisome proliferator-activated receptor α (PPARα). Importantly, tissue-specific transcripts that change with circadian rhythm can vary greatly, constituting up to 10% of an organ's transcriptome.2 Furthermore, maintenance of circadian rhythm is subject to extensive transcriptional and posttranscriptional regulation as well as posttranslational modification of “clock” proteins. Therefore, in response to organ-specific or tissue-specific gene regulation, there are marked differences in the functional cellular circadian clock.2

Figure 1.

(A) Molecular circadian rhythm is under the control of two interlocked transcriptional/posttranslational feedback loops regulated by Rev-erbα. Clock genes CLOCK, BMAL1, Period (PER), and Cryptochrome (CRY) are the principal molecular mediators of the circadian rhythm. (B) Rev-erbα is regulated by miR-122 and proliferator-activated receptor (PPAR) gene expression. (C) The pre-mir-122, but not miR-122, demonstrates circadian rhythm.

MicroRNAs (miRNAs) are the short, ∼22-nucleotide sequences that regulate ∼30% of all mRNA transcripts. Expression of miRNA is tissue-specific; due to only partial nucleotide complementarity of target sequences, a single miRNA has multiple targets that in some cases can be hundreds of different messenger RNAs (mRNAs). Liver miRNAs have been widely implicated in the development of malignancy, response to infections, as well as the maintenance of normal physiological homeostasis. In the absence of intrahepatic miRNAs, normal hepatocyte function remains, although the livers of such animals are enlarged and display increased hepatocyte apoptosis/proliferation and portal inflammation in the long term.4 Liver miRNA expression has been partially characterized, and it has been established that microRNA-122 (miR-122) constitutes ∼70% of all intrahepatic miRNAs.4 miR-122 is implicated in fatty acid and cholesterol metabolism, acts as a tumor suppressor, and regulates hepatitis C viral replication.

miRNAs are known to be involved in the regulation of circadian gene expression.5, 6 However, little is known about miRNA regulation of the circadian rhythm within the liver. Gatfield and colleagues examined the role of miR-122 in regulating liver circadian rhythm by using a range of approaches including transgenic animals, transcriptome profiling, and bioinformatics.7 Their results are intriguing because they demonstrated circadian variation in the expression of the precursor of miR-122 but not in miR-122 itself, which had constant high-level expression within the liver. Importantly, the results demonstrate that the miR-122 locus gives rise to only a single miRNA. The researchers used antisense oligonucleotides to deplete miR-122 and the resultant mRNA pool was subjected to Affymetrix transcriptome profiling. The ensuing mRNA differential expression was compared to the predicted targets of miR-122, and this set of genes was found to be enriched for circadian rhythm-associated transcripts. Further, in mice with Rev-erbα overexpression, there was reduced miR-122 expression and weaker repression of miR-122 targets. In addition, these researchers demonstrated posttranscriptional regulation of miR-122 targets implicated in circadian rhythm by showing a direct link and that the observed effect is not due to indirect transcriptional regulation. Finally, the work showed cross-talk between miR-122 and PPAR gene expression. In miR-122–deficient mice, the mRNA of Smarcd1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin d1)/Baf60 (a gene that regulates hepatic lipid metabolism) is increased. Smarcd1/Baf60 interacts with PPAR genes, and it was demonstrated that PPARβ/δ protein expression was regulated by miR-122, although there was no change in mRNA expression. Therefore, there is clearly a link between miR-122 expression and intrahepatic circadian gene expression, which determines the rhythmical metabolic phenotype of the liver. However, the results presented raise almost as many questions as they answer. It is unclear how an abundant and constantly expressed miRNA, which is not subject to rhythmical circadian variation in expression, regulates circadian genes. Additionally, are such complex circadian molecular events altered significantly in liver injury? What if any contribution does this have to the changes we observe in progressive fibrosis and cirrhosis? Further, do researchers account for the possibility of circadian variation in liver genes under study when planning experiments? Fundamental research such as that of Gatfield and colleagues helps us understand the normal homeostasis of the liver, and in particular, the importance of the circadian rhythm and miRNA regulation of mRNA expression.