Feeding Cues and Injected Nutrients Induce Acute Expression of Multiple Clock Genes in the Mouse Liver

The circadian clock is closely associated with energy metabolism. The liver clock can rapidly adapt to a new feeding cycle within a few days, whereas the lung clock is gradually entrained over one week. However, the mechanism underlying tissue-specific clock resetting is not fully understood. To characterize the rapid response to feeding cues in the liver clock, we examined the effects of a single time-delayed feeding on circadian rhythms in the liver and lungs of Per2::Luc reporter knockin mice. After adapting to a night-time restricted feeding schedule, the mice were fed according to a 4, 8, or 13 h delayed schedule on the last day. The phase of the liver clock was delayed in all groups with delayed feeding, whereas the lung clock remained unaffected. We then examined the acute response of clock and metabolism-related genes in the liver using focused DNA-microarrays. Clock mutant mice were bred under constant light to attenuate the endogenous circadian rhythm, and gene expression profiles were determined during 24 h of fasting followed by 8 h of feeding. Per2 and Dec1 were significantly increased within 1 h of feeding. Real-time RT-PCR analysis revealed a similarly acute response in hepatic clock gene expression caused by feeding wild type mice after an overnight fast. In addition to Per2 and Dec1, the expression of Per1 increased, and that of Rev-erbα decreased in the liver within 1 h of feeding after fasting, whereas none of these clock genes were affected in the lung. Moreover, an intraperitoneal injection of glucose combined with amino acids, but not either alone, reproduced a similar hepatic response. Our findings show that multiple clock genes respond to nutritional cues within 1 h in the liver but not in the lung

<i>E2F1</i> and miRNA expression in a set of patient samples.

<p>A) <i>E2F1</i> expression in tumor relative to normal tissue was evaluated by qPCR; <i>PUM1</i> and <i>HMBS</i> expression were used for normalization. B) MIR205-5p and C) MIR136-5p tumor expression relative to normal expression was evaluated by qPCR; <i>RNU6b</i> expression was used for normalization. Red: patient #2 carries a somatic mutation at <i>E2F1</i>:MIR136-5p target site. Blue: patient #11 carries a germline alteration at <i>E2F1</i>:MIR205-5p target site. Dashed line represents the same expression value between normal and tumor tissues. Error bars indicate the SEM of experiments in triplicate.</p

List of genes screened by next-generation sequencing for genetic alterations at miRNA predicted target sites.

<p>List of genes screened by next-generation sequencing for genetic alterations at miRNA predicted target sites.</p

<i>E2F1</i> somatic mutation within miRNA target site impairs gene regulation in colorectal cancer

<div><p>Background</p><p>Genetic studies have largely concentrated on the impact of somatic mutations found in coding regions, and have neglected mutations outside of these. However, 3’ untranslated regions (3' UTR) mutations can also disrupt or create miRNA target sites, and trigger oncogene activation or tumor suppressor inactivation.</p><p>Methods</p><p>We used next-generation sequencing to widely screen for genetic alterations within predicted miRNA target sites of oncogenes associated with colorectal cancer, and evaluated the functional impact of a new somatic mutation. Target sequencing of 47 genes was performed for 29 primary colorectal tumor samples. For 71 independent samples, Sanger methodology was used to screen for <i>E2F1</i> mutations in miRNA predicted target sites, and the functional impact of these mutations was evaluated by luciferase reporter assays.</p><p>Results</p><p>We identified germline and somatic alterations in <i>E2F1</i>. Of the 100 samples evaluated, 3 had germline alterations at the MIR205-5p target site, while one had a somatic mutation at MIR136-5p target site. <i>E2F1</i> gene expression was similar between normal and tumor tissues bearing the germline alteration; however, expression was increased 4-fold in tumor tissue that harbored a somatic mutation compared to that in normal tissue. Luciferase reporter assays revealed both germline and somatic alterations increased <i>E2F1</i> activity relative to wild-type <i>E2F1</i>.</p><p>Conclusions</p><p>We demonstrated that somatic mutation within <i>E2F1</i>:MIR136-5p target site impairs miRNA-mediated regulation and leads to increased gene activity. We conclude that somatic mutations that disrupt miRNA target sites have the potential to impact gene regulation, highlighting an important mechanism of oncogene activation.</p></div

Position and sequencing characteristics of mutations found in miRNA predicted target sites.

<p>Position and sequencing characteristics of mutations found in miRNA predicted target sites.</p

Study overview.

<p>BWA: Burrows-Wheeler Aligner.</p

<i>E2F1</i> and miRNA expression in a set of patient samples.

<p>A) <i>E2F1</i> expression in tumor relative to normal tissue was evaluated by qPCR; <i>PUM1</i> and <i>HMBS</i> expression were used for normalization. B) MIR205-5p and C) MIR136-5p tumor expression relative to normal expression was evaluated by qPCR; <i>RNU6b</i> expression was used for normalization. Red: patient #2 carries a somatic mutation at <i>E2F1</i>:MIR136-5p target site. Blue: patient #11 carries a germline alteration at <i>E2F1</i>:MIR205-5p target site. Dashed line represents the same expression value between normal and tumor tissues. Error bars indicate the SEM of experiments in triplicate.</p