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While it is generally known that metabolic disorders and circadian dysfunction are intertwined, how the two systems affect each other is not well understood, nor are the genetic factors that might exacerbate this pathological interaction. Blood chemistry is profoundly changed in metabolic disorders, and we have previously shown that serum factors change cellular clock properties. To investigate if circulating factors altered in metabolic disorders have circadian modifying effects, and whether these effects are of genetic origin, we measured circadian rhythms in U2OS cell in the presence of serum collected from diabetic, obese or control subjects. We observed that circadian period lengthening in U2OS cells was associated with serum chemistry that is characteristic of insulin resistance. Characterizing the genetic variants that altered circadian period length by genome-wide association analysis, we found that one of the top variants mapped to the E3 ubiquitin ligase MARCH1 involved in insulin sensitivity. Confirming our data, the serum circadian modifying variants were also enriched in type 2 diabetes and chronotype variants identified in the UK Biobank cohort. Finally, to identify serum factors that might be involved in period lengthening, we performed detailed metabolomics and found that the circadian modifying variants are particularly associated with branched chain amino acids, whose levels are known to correlate with diabetes and insulin resistance. Overall, our multi-omics data showed comprehensively that systemic factors serve as a path through which metabolic disorders influence circadian system, and these can be examined in human populations directly by simple cellular assays in common cultured cells.
RESUMEN
Human cells, especially primary fibroblasts from skin punch biopsy, have emerged over the last decade as powerful, unlimited, and easily accessible resources that bridge the gap between animal models and human subjects in basic as well as clinical research. The cells also retain molecular circadian clocks that reflect subject-specific differences in circadian physiology, and the cellular rhythms can be measured easily in large scale. This is a series of protocols that describes the procedure to measure circadian rhythms in these cells, starting from deriving fibroblasts from skin punch biopsy, to generation of stable cells expressing a circadian reporter, and finally measurement of cellular rhythms in large scale.
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Relojes Circadianos , Cultivo Primario de Células/métodos , Línea Celular , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Piel/citologíaRESUMEN
MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression post-transcriptionally. MiRNAs are implicated in various biological processes associated with obesity, including adipocyte differentiation and lipid metabolism. We used a neuronal-specific inhibition of miRNA maturation in adult mice to study the consequences of miRNA loss on obesity development. Camk2a-CreERT2 (Cre+) and floxed Dicer (Dicerlox/lox) mice were crossed to generate tamoxifen-inducible conditional Dicer knockouts (cKO). Vehicle- and/or tamoxifen-injected Cre+;Dicerlox/lox and Cre+;Dicer+/+ served as controls. Four cohorts were used to a) measure body composition, b) follow food intake and body weight dynamics, c) evaluate basal metabolism and effects of food deprivation, and d) assess the brain transcriptome consequences of miRNA loss. cKO mice developed severe obesity and gained 18 g extra weight over the 5 weeks following tamoxifen injection, mainly due to increased fat mass. This phenotype was highly reproducible and observed in all 38 cKO mice recorded and in none of the controls, excluding possible effects of tamoxifen or the non-induced transgene. Development of obesity was concomitant with hyperphagia, increased food efficiency, and decreased activity. Surprisingly, after reaching maximum body weight, obese cKO mice spontaneously started losing weight as rapidly as it was gained. Weight loss was accompanied by lowered O2-consumption and respiratory-exchange ratio. Brain transcriptome analyses in obese mice identified several obesity-related pathways (e.g. leptin, somatostatin, and nemo-like kinase signaling), as well as genes involved in feeding and appetite (e.g. Pmch, Neurotensin) and in metabolism (e.g. Bmp4, Bmp7, Ptger1, Cox7a1). A gene cluster with anti-correlated expression in the cerebral cortex of post-obese compared to obese mice was enriched for synaptic plasticity pathways. While other studies have identified a role for miRNAs in obesity, we here present a unique model that allows for the study of processes involved in reversing obesity. Moreover, our study identified the cortex as a brain area important for body weight homeostasis.
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Eliminación de Gen , MicroARNs/genética , MicroARNs/metabolismo , Neuronas/metabolismo , Obesidad/genética , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Animales , Metabolismo Basal/genética , Encéfalo/metabolismo , Exones , Ayuno , Técnicas de Silenciamiento del Gen , Estudios de Asociación Genética , Genotipo , Hiperfagia/genética , Locomoción , Ratones , Ratones Noqueados , Obesidad/metabolismo , Especificidad de Órganos/genética , Fenotipo , Tamoxifeno/administración & dosificación , TranscriptomaRESUMEN
A considerable proportion of mammalian gene expression undergoes circadian oscillations. Post-transcriptional mechanisms likely make important contributions to mRNA abundance rhythms. We have investigated how microRNAs (miRNAs) contribute to core clock and clock-controlled gene expression using mice in which miRNA biogenesis can be inactivated in the liver. While the hepatic core clock was surprisingly resilient to miRNA loss, whole transcriptome sequencing uncovered widespread effects on clock output gene expression. Cyclic transcription paired with miRNA-mediated regulation was thus identified as a frequent phenomenon that affected up to 30% of the rhythmic transcriptome and served to post-transcriptionally adjust the phases and amplitudes of rhythmic mRNA accumulation. However, only few mRNA rhythms were actually generated by miRNAs. Overall, our study suggests that miRNAs function to adapt clock-driven gene expression to tissue-specific requirements. Finally, we pinpoint several miRNAs predicted to act as modulators of rhythmic transcripts, and identify rhythmic pathways particularly prone to miRNA regulation.DOI: http://dx.doi.org/10.7554/eLife.02510.001.
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Ritmo Circadiano/genética , Regulación de la Expresión Génica , Hígado/metabolismo , MicroARNs/metabolismo , Transcriptoma/genética , Regiones no Traducidas 3'/genética , Animales , Relojes Biológicos/genética , Biomarcadores/metabolismo , Células Cultivadas , ARN Helicasas DEAD-box/metabolismo , Genes Reporteros , Genoma , Hepatocitos/metabolismo , Ratones , Ratones Noqueados , MicroARNs/genética , Modelos Biológicos , Proteínas Circadianas Period/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Ribonucleasa III/metabolismo , Análisis de Secuencia de ARN , Transcripción GenéticaRESUMEN
In mammalian circadian clockwork, the CLOCK-BMAL1 complex binds to DNA enhancers of target genes and drives circadian oscillation of transcription. Here we identified 7,978 CLOCK-binding sites in mouse liver by chromatin immunoprecipitation-sequencing (ChIP-Seq), and a newly developed bioinformatics method, motif centrality analysis of ChIP-Seq (MOCCS), revealed a genome-wide distribution of previously unappreciated noncanonical E-boxes targeted by CLOCK. In vitro promoter assays showed that CACGNG, CACGTT, and CATG(T/C)G are functional CLOCK-binding motifs. Furthermore, we extensively revealed rhythmically expressed genes by poly(A)-tailed RNA-Seq and identified 1,629 CLOCK target genes within 11,926 genes expressed in the liver. Our analysis also revealed rhythmically expressed genes that have no apparent CLOCK-binding site, indicating the importance of indirect transcriptional and posttranscriptional regulations. Indirect transcriptional regulation is represented by rhythmic expression of CLOCK-regulated transcription factors, such as Krüppel-like factors (KLFs). Indirect posttranscriptional regulation involves rhythmic microRNAs that were identified by small-RNA-Seq. Collectively, CLOCK-dependent direct transactivation through multiple E-boxes and indirect regulations polyphonically orchestrate dynamic circadian outputs.
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Proteínas CLOCK/fisiología , Ritmo Circadiano , Elementos E-Box , Interferencia de ARN , Animales , Secuencia de Bases , Sitios de Unión , Secuencia de Consenso , Células HEK293 , Humanos , Factores de Transcripción de Tipo Kruppel/metabolismo , Hígado , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/genética , MicroARNs/metabolismo , Unión Proteica , TranscriptomaRESUMEN
In the molecular oscillatory mechanism governing circadian rhythms, positive regulators, including CLOCK and BMAL1, transactivate Per and Cry genes through E-box elements, and translated PER and CRY proteins negatively regulate their own transactivation. Like BMAL1, its paralog BMAL2 dimerizes with CLOCK to activate the E-box-dependent transcription, but the role of BMAL2 in the circadian clockwork is still elusive. Here we characterized BMAL2 function in NIH3T3 cells and found that the cellular rhythms monitored by Bmal1 promoter-driven bioluminescence signals were blunted by RNA interference-mediated suppression of Bmal2 as well as that of Bmal1. Transcription assays with a 2.1-kb mPer1 promoter revealed that CRY2 inhibited the transactivation mediated by BMAL1-CLOCK more strongly than that by BMAL2-CLOCK. In contrast, PER2 showed a stronger inhibitory effect on BMAL2-CLOCK than on BMAL1-CLOCK. The molecular link between BMAL2 and PER2 was further strengthened by the fact that PER2 exhibited a greater affinity for BMAL2 than for BMAL1 in co-immunoprecipitation experiments. These results indicate a functional partnership between BMAL2 and PER2 and reemphasize the negative role of PER2 in the circadian transcription. As a broad spectrum function, BMAL2-CLOCK activated transcription from a variety of SV40-driven reporters harboring various E/E'-box-containing sequences present in the upstream regions of clock and clock-controlled genes. Importantly, the efficiencies of BMAL2-CLOCK-mediated transactivation relative to that achieved by BMAL1-CLOCK were dependent heavily on the E-box-containing sequences, supporting distinguishable roles of the two BMALs. Collectively, it is strongly suggested that BMAL2 plays an active role in the circadian transcription.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Proteínas de Ciclo Celular/fisiología , Regulación de la Expresión Génica , Proteínas Nucleares/fisiología , Transactivadores/fisiología , Factores de Transcripción/fisiología , Transcripción Genética , Factores de Transcripción ARNTL , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas CLOCK , Proteínas de Ciclo Celular/metabolismo , Ritmo Circadiano , Humanos , Ratones , Modelos Biológicos , Células 3T3 NIH , Proteínas Nucleares/metabolismo , Proteínas Circadianas Period , Transactivadores/metabolismo , Factores de Transcripción/metabolismoRESUMEN
In mammalian circadian clockwork, the CLOCK-BMAL1 heterodimer activates E-box-dependent transcription, while its activity is suppressed by circadian binding with negative regulators, such as CRYs. Here, we found that the CLOCK protein is kept mostly in the phosphorylated form throughout the day and is partly hyperphosphorylated in the suppression phase of E-box-dependent transcription in the mouse liver and NIH 3T3 cells. Coexpression of CRY2 in NIH 3T3 cells inhibited the phosphorylation of CLOCK, whereas CIPC coexpression markedly stimulated phosphorylation, indicating that CLOCK phosphorylation is regulated by a combination of the negative regulators in the suppression phase. CLOCK-BMAL1 purified from the mouse liver was subjected to tandem mass spectrometry analysis, which identified Ser38, Ser42, and Ser427 as in vivo phosphorylation sites of CLOCK. Ser38Asp and Ser42Asp mutations of CLOCK additively and markedly weakened the transactivation activity of CLOCK-BMAL1, with downregulation of the nuclear amount of CLOCK and the DNA-binding activity. On the other hand, CLOCK Delta 19, lacking the CIPC-binding domain, was far less phosphorylated and much more stabilized than wild-type CLOCK in vivo. Calyculin A treatment of cultured NIH 3T3 cells promoted CLOCK phosphorylation and facilitated its proteasomal degradation. Together, these results show that CLOCK phosphorylation contributes to the suppression of CLOCK-BMAL1-mediated transactivation through dual regulation: inhibition of CLOCK activity and promotion of its degradation.