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. 2016 Mar 17;11(3):e0149418.
doi: 10.1371/journal.pone.0149418. eCollection 2016.

Natural Genetic Variation Influences Protein Abundances in C. elegans Developmental Signalling Pathways

Kapil Dev Singh  1 , Bernd Roschitzki  2 , L Basten Snoek  3 , Jonas Grossmann  2 , Xue Zheng  1 , Mark Elvin  4 , Polina Kamkina  1 , Sabine P Schrimpf  1 , Gino B Poulin  4 , Jan E Kammenga  3 , Michael O Hengartner  1
Affiliations

Natural Genetic Variation Influences Protein Abundances in C. elegans Developmental Signalling Pathways

Kapil Dev Singh et al. PLoS One. .

Abstract

Complex traits, including common disease-related traits, are affected by many different genes that function in multiple pathways and networks. The apoptosis, MAPK, Notch, and Wnt signalling pathways play important roles in development and disease progression. At the moment we have a poor understanding of how allelic variation affects gene expression in these pathways at the level of translation. Here we report the effect of natural genetic variation on transcript and protein abundance involved in developmental signalling pathways in Caenorhabditis elegans. We used selected reaction monitoring to analyse proteins from the abovementioned four pathways in a set of recombinant inbred lines (RILs) generated from the wild-type strains N2 (Bristol) and CB4856 (Hawaii) to enable quantitative trait locus (QTL) mapping. About half of the cases from the 44 genes tested showed a statistically significant change in protein abundance between various strains, most of these were however very weak (below 1.3-fold change). We detected a distant QTL on the left arm of chromosome II that affected protein abundance of the phosphatidylserine receptor protein PSR-1, and two separate QTLs that influenced embryonic and ionizing radiation-induced apoptosis on chromosome IV. Our results demonstrate that natural variation in C. elegans is sufficient to cause significant changes in signalling pathways both at the gene expression (transcript and protein abundance) and phenotypic levels.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Signalling pathway proteins show a similar variation in abundance between N2 and CB4856 as a C. elegans shotgun proteome dataset.
Histogram with Tukey-style box plot [36] on the top for protein abundances measured in CB4856 relative to N2. Vertical dashed lines represent the fold change cut-off of 1.3 (~ 0.38 on log2 scale). (A) The C. elegans shotgun proteome dataset was quantified using SILAC (data from [35]). (B) Signalling pathway proteins were quantified using SRM.
Fig 2
Fig 2. Many RILs have a higher transcript expression level variation than the parental strains.
Gene expression at the transcript level was quantified in N2, CB4856, and 47 RILs by two-colour microarray analysis. (A) Tukey-style box plot representing log2 scaled deviations of the gene expression value from the mean across all samples. Four genetically different RILs (WN31, WN71, WN105, and WN186) that showed large variation in transcript abundance were selected for proteome quantification. (B) Hierarchical clustering of the 47 RILs and the two parental strains based on their genotype (see “Additional file 1” of [25] for details). Clustering was done with R functions “dist” and “hclust” from the package stats (version 3.2.2) using “euclidean” distance and “ward.D2” method. Four RILs (black arrows) were chosen from different clusters to ensure maximum genetic diversity; red arrows indicate parental strains. (C) Genotype of the four selected RILs and the two parental strains. Vertical lines separate chromosomes I to V and X from left to right.
Fig 3
Fig 3. Signalling pathway proteins tend to be up-regulated in CB4856 and in RILs compared to N2.
Protein abundance was quantified by SRM. Identification of the true peak group was performed using the mProphet software, followed by protein significance analysis using intensity-based linear mixed-effects model implemented in MSstats. (A) Heat map showing differential abundance of 44 proteins in CB4856 and four empirically selected RILs relative to N2. Blue and red shades represent log2 scaled fold changes, grey colour shows the fold change cut-off of 1.3 (~ 0.38 on log2 scale) and number of asterisks represent BH corrected P-values. Black bands on the left side indicate proteins selected for subsequent pQTL mapping. Number of asterisks on top of each column represent BH corrected P-values from one sample t-test (H0: μ = 0 and H1: μ ≠ 0) on protein fold changes relative to N2; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. (B) Tukey-style box plot of broad-sense heritability for the 44 proteins shown in panel A. Scatter points overlaid on the box plot represent the broad-sense heritability values for the individual proteins (solid black circles correspond to selected proteins). (C) Bar graph of selected proteins from panel A. Horizontal dashed lines represent the fold change cut-off of 1.3 (~ 0.38 on log2 scale). Error bars represent SEM between three biological replicates.
Fig 4
Fig 4. RILs show similar protein and transcript abundance variation for the tested 44 genes as the parental strains.
Comparison of protein and transcript abundance (log2 scaled fold changes relative to N2) for 44 signalling pathway proteins in CB4856 and four selected RILs. Horizontal and vertical dashed lines represent the fold change cut-off of 1.3 (~ 0.38 on log2 scale). Tukey-style box plot on top and right side represents variability in protein and transcript log2 fold changes respectively. Pearson correlation coefficient is denoted by r. Table on bottom right represents the P-values from the Fligner-Killeen test for homogeneity of variances between protein (column 1) and transcript (column 2) data for RILs compared with CB4856.
Fig 5
Fig 5. pQTL profiles of seven selected signalling pathway proteins.
Blue curves show the significance of the pQTLs multiplied by the sign of the effect of the N2 allele (positive values of blue curve indicate higher protein abundance when the N2 allele is present, whereas negative values indicate higher protein abundance when the CB4856 allele is present). Horizontal orange and red dashed lines show 0.1 and 0.05 FDR thresholds respectively. Vertical dotted grey lines separate chromosomes I to V and X from left to right. Vertical magenta bands indicate the position of the gene in the genome. PSR-1 shows a significant pQTL on the left arm of chromosome II.
Fig 6
Fig 6. Protein and transcript abundance variation of the signalling pathway proteins used in pQTL mapping in CB4856 and 45 RILs.
Comparison of protein and transcript abundance (log2 scaled fold changes relative to N2) in CB4856 (solid orange circle) and RILs for the seven proteins used in pQTL mapping. Horizontal and vertical dashed lines represent the fold change cut-off of 1.3 (~ 0.38 on log2 scale). Tukey-style box plot on top and right side represents variability in protein and transcript log2 fold changes respectively. P-value from Fligner-Killeen test for homogeneity of variances between protein and transcript data is denoted by PFK.
Fig 7
Fig 7. Analysis of variation in embryonic and germ line apoptosis in parental strains and RILs.
(A-C) Quantification of apoptotic cell corpse numbers in embryos (A), germ line without ionizing radiation (IR; B), and with 60 Gy IR (C). Error bars represent SEM between numbers of biological replicates indicated at the bottom of each bar. (D-F) Natural variation in apoptosis levels of parental strains and RILs does not correlate with the PSR-1 protein abundance (relative to N2). Scatter plots (CB4856 in orange and N2 in green) of PSR-1 protein abundance and apoptotic levels in embryos (D), germ line without IR (E), and with 60 Gy IR (F). Pearson correlation coefficient is denoted by r. (G-I) Embryonic and IR-induced apoptosis shows a significant QTL on chromosome IV. QTL profiles of apoptotic phenotype in embryos (G), adult germ line without IR (H) and with 60 Gy IR (I). Blue curves show the significance of the QTLs multiplied by the sign of the effect of the N2 allele (positive values of blue curve indicate higher apoptosis level when the N2 allele is present, whereas negative values indicate higher apoptosis level when the CB4856 allele is present). Horizontal orange and red dashed lines show 0.1 and 0.05 FDR thresholds respectively. Vertical dotted grey lines separate chromosomes I to V and X from left to right. Vertical magenta band indicates the position of psr-1 gene in the genome.
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This work was supported by the Swiss National Science Foundation (grant No. 31003A_143932; http://www.snf.ch) to MOH and the European Community's Health Seventh Framework Programme under project PANACEA (project No. 222936; http://www.panaceaproject.eu) to JEK. LBS was funded by the Netherlands Organisation for Scientific Research (project No. 823.01.001; http://www.nwo.nl). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.