Beyond the reference: gene expression variation and transcriptional response to RNAi in C. elegans

Abstract

A universal feature of living systems is that natural variation in genotype underpins variation in phenotype. Yet, research in model organisms is often constrained to a single genetic background, the reference strain. Further, genomic studies that do evaluate wild strains typically rely on the reference strain genome for read alignment, leading to the possibility of biased inferences based on incomplete or inaccurate mapping; the extent of reference bias can be difficult to quantify. As an intermediary between genome and organismal traits, gene expression is well positioned to describe natural variability across genotypes generally and in the context of environmental responses, which can represent complex adaptive phenotypes. C. elegans sits at the forefront of investigation into small-RNA gene regulatory mechanisms, or RNA interference (RNAi), and wild strains exhibit natural variation in RNAi competency following environmental triggers. Here, we examine how genetic differences among five wild strains affect the C. elegans transcriptome in general and after inducing RNAi responses to two germline target genes. Approximately 34% of genes were differentially expressed across strains; 411 genes were not expressed at all in at least one strain despite robust expression in others, including 49 genes not expressed in reference strain N2. Despite the presence of hyper-diverse hotspots throughout the C. elegans genome, reference mapping bias was of limited concern: over 92% of variably expressed genes were robust to mapping issues. Overall, the transcriptional response to RNAi was strongly strain-specific and highly specific to the target gene, and the laboratory strain N2 was not representative of the other strains. Moreover, the transcriptional response to RNAi was not correlated with RNAi phenotypic penetrance; the two germline RNAi incompetent strains exhibited substantial differential gene expression following RNAi treatment, indicating an RNAi response despite failure to reduce expression of the target gene. We conclude that gene expression, both generally and in response to RNAi, differs across C. elegans strains such that choice of strain may meaningfully influence scientific conclusions. To provide a public, easily accessible resource for querying gene expression variation in this dataset, we introduce an interactive website at https://wildworm.biosci.gatech.edu/rnai/ .

Figure 1.

Genotype (strain) dominates expression variation across five C. elegans strains treated with RNAi targeting the genes par-1 and pos-1 or an empty vector control. A) Principal components analysis (PCA) of gene expression. PCs 1 vs. 2 (left) and 2 vs. 3 (right) of PCA of the 500 most variably expressed genes are plotted; the proportion of variance explained is noted on the axes. B) In the control condition, 34.2% of 15,654 nominally expressed genes are differentially expressed across strains (genome-wide adjusted p < 0.1 in a likelihood-ratio test between models including and excluding the strain term); a subset of these (approximately 2.6% overall) are not expressed at all in at least one strain (in any condition, see text for details). Related Supplementary Material: File S1 contains the genes differentially expressed based on strain File S2 contains the ‘off’ genes identified as potentially unexpressed in one strain but expressed in others

Figure 2.

Improving confidence in differential expression calls by integrating DNA alignment data. A) The number of genes with low (<25% of the median) and missing (zero raw coverage) DNA alignment coverage (from CeNDR sequencing (Cook et al., 2017)) in each strain, of the 18,589 genes included in the expression analysis. Strain note: CeNDR assessed DNA coverage in EG4349, the genetically identical isotype to EG4348. B) The total number of genes differentially expressed based on strain (likelihood-ratio test of models including and excluding strain term, genome-wide adjusted p < 0.1) and their overlap with genes classified as missing or low DNA coverage in any strain (417 are both differentially expressed across strains and low DNA coverage, hypergeometric enrichment test p = 9.8 × 10⁻⁴⁶). Areas are proportional to number of observations. C) The number of unexpressed ‘off’ genes per strain, subset into three categories: called as turned off at the RNA level with high confidence; missing in the strain genome (zero raw coverage); called with uncertainty, given low DNA sequence coverage (<25% but >0 median DNA coverage). Related Supplementary Material: Figure S2 shows DNA coverage distributions and cutoffs File S2 contains details on each ‘off’ gene File S3 contains raw per-gene DNA sequence coverage estimates File S4 contains median-normalized per-gene DNA sequence coverage estimates Files S5 contains the list of genes flagged as low DNA coverage Files S6–7 provide numerical summaries of ‘off’ genes

Figure 3.

The transcriptional response to dsRNA is highly strain- and target-specific. A) The number of genes up- and down-regulated in each strain upon par-1 and pos-1 dsRNA ingestion/RNAi induction. Genes were called differentially expressed if their shrunken absolute fold change was > 1.5 and genome-wide adjusted p-value/FDR < 0.1. B) Gene set enrichment analysis results for genes upregulated on par-1 dsRNA in each strain. Gene ontology (GO) categories that were significantly enriched (false discovery rate Q < 0.1) in any strain are included. GO terms are ranked and colored by median significance across strains. Related Supplementary Material: Figure S5 shows volcano plots for RNAi treatments for each strain Figure S6 contains Venn diagrams of overlap among strains in specific DE genes Figure S7 shows results from the same gene set enrichment analysis of genes downregulated under par-1 RNAi and up- and down-regulated under pos-1 RNAi Table S1 gives number of up and downregulated genes in each strain and included in each analysis File S8 contains the genes differentially expressed based on strain-treatment interaction Files S9a-j contain the genes differentially expressed in each strain in each RNAi treatment vs. control File S10 gives all enriched GO categories.