in-vivo-monitoring-circadian-clock-gene-expression-mouse

<em>In Vivo</em> Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene ...

National Institute of Biological Sciences, Beijing

Although a deficiency in CRY1 or CRY2 correlates with a shorter or longer circadian period, the regulation of CRY proteins in the circadian period has not been well studied. In this study, we found that both CRY1 and CRY2 were able to rescue oscillation in CRY null cells and that they displayed different periods. Furthermore, we demonstrated that protein nuclear import rates, not protein stability, regulate the period-length at the cellular level. Co-transfection of CRY1 and CRY2 in various ratios in the same cells gives rise to the predicted period length in a dose-dependent manner. Given the distinct characteristics of the C-terminal tails of the CRY1 and CRY2 proteins, our study addresses a long-standing hypothesis that the ratio of these two CRY molecules affects the clock period. 

The ratio of intracellular CRY proteins determines the clock period length.

Circadian regulation is critically important in maintaining metabolic and physiological homeostasis. However, little is known about the possible influence of the clock on physiological abnormalities occurring under pathological conditions. Here, we report the discovery that hypoxia, a condition that causes catastrophic bodily damage, is gated by the circadian clock in vivo. Hypoxia signals conversely regulate the clock by slowing the circadian cycle and dampening the amplitude of oscillations in a dose-dependent manner. ChIP-seq analyses of hypoxia-inducible factor HIF1A and the core clock component BMAL1 revealed crosstalk between hypoxia and the clock at the genome level. Further, severe consequences caused by acute hypoxia, such as those that occur with heart attacks, were correlated with defects in circadian rhythms. We propose that the clock plays functions in fine-tuning hypoxic responses under pathophysiological conditions. We argue that the clock can, and likely should, be exploited therapeutically to reduce the severity of fatal hypoxia-related diseases.

Reciprocal Regulation between the Circadian Clock and Hypoxia Signaling at the Genome Level in Mammals.

Circadian disruption has been implicated in tumour development, but the underlying mechanism remains unclear. Here, we show that the molecular clockwork within malignant human pancreatic epithelium is disrupted and that this disruption is mediated by miR-135b-induced BMAL1 repression. miR-135b directly targets the BMAL1 3'-UTR and thereby disturbs the pancreatic oscillator, and the downregulation of miR-135b is essential for the realignment of the cellular clock. Asynchrony between miR-135b and BMAL1 expression impairs the local circadian gating control of tumour suppression and significantly promotes tumourigenesis and resistance to gemcitabine in pancreatic cancer (PC) cells, as demonstrated by bioinformatics analyses of public PC data sets and in vitro and in vivo functional studies. Moreover, we found that YY1 transcriptionally activated miR-135b and formed a 'miR-135b-BMAL1-YY1' loop, which holds significant predictive and prognostic value for patients with PC. Thus, our work has identified a novel signalling loop that mediates pancreatic clock disruption as an important mechanism of PC progression and chemoresistance.

The MiR-135b-BMAL1-YY1 loop disturbs pancreatic clockwork to promote tumourigenesis and chemoresistance.

Endogenous circadian clocks control 24-h physiological and behavioral rhythms in mammals. Here, we report a real-time in vivo fluorescence recording system that enables long-term monitoring of circadian rhythms in the brains of freely moving mice. With a designed reporter of circadian clock gene expression, we tracked robust  transcription reporter rhythms in the suprachiasmatic nucleus (SCN) of WT,  , and  mice in LD (12 h light, 12 h dark) and DD (constant darkness) conditions and verified that signals remained stable for over 6 mo. Further, we recorded  transcriptional rhythms in the subparaventricular zone (SPZ) and hippocampal CA1/2 regions of WT mice housed under LD and DD conditions. By using a Cre-loxP system, we recorded  and  transcription rhythms specifically in vasoactive intestinal peptide (VIP) neurons of the SCN. Finally, we demonstrated the dynamics of  and  transcriptional rhythms in SCN VIP neurons following an 8-h phase advance in the light/dark cycle.

Article	Year
The ratio of intracellular CRY proteins determines the clock period length. Biochemical and biophysical research communications\| PubMed ID: 26966073	2016
Reciprocal Regulation between the Circadian Clock and Hypoxia Signaling at the Genome Level in Mammals. Cell metabolism\| PubMed ID: 27773697	2017
The MiR-135b-BMAL1-YY1 loop disturbs pancreatic clockwork to promote tumourigenesis and chemoresistance. Cell death & disease\| PubMed ID: 29396463	2018
Long-term in vivo recording of circadian rhythms in brains of freely moving mice. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 29610316	2018

Eric Erquan Zhang

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