The genomic landscape of human cellular circadian variation points to a novel role for the signalosome
- University of Zurich, Switzerland
- University of Geneva, Switzerland
- Laboratory of Chronobiology, Germany
- University of Lausanne, Switzerland
- Institute of Physiology, Charité-Universitätsmedizin Berlin, Working Group Sleep Research & Clinical Chronobiology, Germany
Figures
Figure 1 with 3 supplements
eQTLs influencing circadian function.
(a) Manhattan plot of identified polymorphisms for all measured traits. Blue line, threshold for suggestive candidates, p<10−5, approx. FDR ≤ 0.1. Genes likely associated with the most significant …
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Figure 1—source data 1
SNPs associated with (a) PER1 and (b) PER2 expression, (c) Amplitude, (d) Phase, and (e) Period.
The closest annotated gene is indicated for each SNP, as well as the unadjusted GWAS p-value.
- https://doi.org/10.7554/eLife.24994.006
Figure 1—figure supplement 1
Umbilical cord fibroblasts from different individuals show large variations in circadian parameters.
(a) Circadian period length (blue), circadian phase (red) and circadian amplitude (black) measured in 159 umbilical cord fibroblast lines of the Genecord II library. Left y-axis, circadian period …
Figure 1—figure supplement 2
Quantile-quantile plots for GWAS of each clock parameter.
https://doi.org/10.7554/eLife.24994.004
Figure 1—figure supplement 3
‘Larks’ and ‘owls’ show skewed distribution of GWAS-positive alleles.
(a) For all SNPs at the p-value indicated in the legend, the relative density distribution of differences in GWAS-positive allele frequency between self-declared ‘larks’ and ‘owls’. Y-axis, density …
Figure 2 with 1 supplement
Assessment of genes associated with identified eQTLs.
(a) Genome-wide high-throughput chromosome conformation capture (Hi-C) for probes spanning rs920400 (blue) and rs10195385 (black). Y-axis, read counts. X-axis, genomic location of reads. Gene …
Figure 2—figure supplement 1
Clock properties of U2OS cells after RNAi depletion of GWAS-identified transcripts.
(a) Z-scored circadian period and amplitude values (left) and RSA scores (right, reflecting the reproducibility-weighted average of all RNAi hairpins; number of different hairpins indicated) for …
Figure 3 with 2 supplements
Positive SNPs highlight protein catabolism as essential for human circadian variation.
(a) Abbreviated Gene Ontology diagram from the Gene SeT Analysis Toolkit (WebGestalt), showing biological process categories containing genes (Supplementary file 1) associated with positive SNPs. …
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Figure 3—source data 1
Multiple-comparison-adjusted p-values and significant associated genes for each pathway identified in Figure 3a (and in more detail in Figure 3—figure supplement 1).
- https://doi.org/10.7554/eLife.24994.012
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Figure 3—source data 2
Multiple-comparison-adjusted p-values, false discovery rates, and significant associated genes for each pathway identified in Figure 3—figure supplement 2a.
Significantly associated genes, as well as all genes in the pathway not significantly associated, can be perused via the ‘view detail’ links for each pathway.
- https://doi.org/10.7554/eLife.24994.013
Figure 3—figure supplement 1
Full Gene SeT Analysis Toolkit (WebGestalt) biological process category diagram containing genes associated with positive SNPs.
Nodes with red labels represent enriched categories and nodes with black labels represents non-enriched parents. Numbers of genes are indicated in enriched categories, as well as multiple …
Figure 3—figure supplement 2
Clock properties of U2OS cells after RNAi depletion of GSEA-identified transcripts.
(a) GSEA4GWAS annotated significant pathways associated with circadian traits. A genelist associated with these pathways is available in Supplementary file 3, and a clickable list of pathways, …
Figure 4 with 1 supplement
COPS7B influences PER2 expression and cellular clock function.
(a) Expression levels of PER2 stratified by rs920400 genotype. (AA vs. GG genotype, T-test p=0.00026). Insets - Correlation between PER2 and COPS7B gene expression within each genotype. (b) …
Figure 4—figure supplement 1
Effects of COPS4 depletion upon circadian period and amplitude.
(a) Raw bioluminescence profiles of U2OS cells cotransfected with a Bmal-luciferase circadian reporter and siRNAs targeting COPS4 (dotted lines) or scrambled siRNA (solid lines). (d) Reduction in …
Figure 5 with 2 supplements
COPS7B stabilizes the essential clock protein BMAL1.
(a–b) Coimmunoprecipitation assay of HEK293T cells transfected with plasmids expressing FLAG epitope-tagged clock proteins (BMAL1-flag, PER2-flag and CRY1-flag). *Unspecific background bands. (a) …
Figure 5—figure supplement 1
Negative control immunoprecipitation experiments identical to those described in Figure 5a,b, but using untransfected HEK293T cells as a substrate.
Coimmunoprecipitation assay of untransfected HEK293T cells. (left) immunoprecipitated with antibodies against flag and probed with anti COPS7B and (right) immunoprecipitated with antibodies against …
Figure 5—figure supplement 2
Pulse-chase analysis of PER2 (a) and CRY1 (b) protein half-lives in absence (left-control lanes; black diamonds in graph below) or presence (right lanes; grey squares in graph below) of siRNAs targeting COPS7B.
HEK293T cells were transfected either with FLAG epitope-tagged clock proteins (PER2-flag and CRY1-flag) or FLAG epitope-tagged clock proteins plus siRNA targeting COPS7B, incubated with 35S-labelled …
Figure 6 with 1 supplement
The signalosome is rhythmically imported into the nucleus.
(a) BMAL1, PER2 and COPS7B RNA levels in U2OS cells at different times of day, as determined via qPCR. Inset - Western blot analysis of nuclear COPS7B protein under identical conditions. Top panel, …
Figure 6—figure supplement 1
Relative expression of signalosome and proteasome subunits.
(a) Nuclear protein expression levels of signalosome subunits, measured relative to each other, by mass spectrometry in mouse liver nuclei. Y axis, arbitrary units . (b) Nuclear protein expression …
Additional files
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Supplementary file 1
Spearman correlations between the sets of positive alleles (p<10−5) identified for all phenotypes.
- https://doi.org/10.7554/eLife.24994.021
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Supplementary file 2
List of genes associated with positive SNPs, determined via GWAS, Hi-C and RTC analysis.
Right columns, unadjusted p-values and –logP values.
- https://doi.org/10.7554/eLife.24994.022
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Supplementary file 3
List of genes implicated in the pathways determined via Gsea4GWAS analysis.
Middle columns, unadjusted p-values and –logP values. Right column, lowest-level GO term associated with each pathway.
- https://doi.org/10.7554/eLife.24994.023
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Transparent reporting form
- https://doi.org/10.7554/eLife.24994.024
