Abstract
Lead (Pb) poisoning has been a major public health issue globally and the recent Flint water crisis has drawn nation-wide attention to its effects. To better understand how lead plays a role as a neurotoxin, we utilized the Drosophila melanogaster model to study the genetic effects of lead exposure during development and identified lead-responsive genes. In our previous studies, we have successfully identified hundreds of lead-responsive expression QTLs (eQTLs) by using RNA-seq analysis on heads collected from the Drosophila Synthetic Population Resource. Cis-eQTLs, also known as allele-specific expression (ASE) polymorphisms, are generally single-nucleotide polymorphisms in the promoter regions of genes that affect expression of the gene, such as by inhibiting the binding of transcription factors. Trans-eQTLs are genes that regulate mRNA levels for many genes, and are generally thought to be SNPs in trans-acting transcription or translation factors. In this study, we focused our attention on alternative splicing events that are affected by lead exposure. Splicing QTLs (sQTLs), which can be caused by SNPs that alter splicing or alternative splicing (AS), such as by changing the sequence-specific binding affinity of splicing factors to the pre-mRNA. We applied two methods in search for sQTLs by using RNA-seq data from control and lead-exposed w1118 Drosophila heads. First, we used the fraction of reads in a gene that falls in each exon as the phenotype. Second, we directly compared the transcript counts among the various splicing isoforms as the phenotype. Among the 1,236 potential Pb-responsive sQTLs (p < 0.0001, FDR < 0.39), mostly cis-sQTLs, one of the most distinct genes is Dscam1 (Down Syndrome Cell Adhesion Molecule), which has over 30,000 potential alternative splicing isoforms. We have also identified a candidate Pb-responsive trans-sQTL hotspot that appears to regulate 129 genes that are enriched in the “cation channel” gene ontology category, suggesting a model in which alternative splicing of these channels might lead to an increase in the elimination of Pb2+ from the neurons encoding these channels. To our knowledge, this is the first paper that uses sQTL analyses to understand the neurotoxicology of an environmental toxin in any organism, and the first reported discovery of a candidate trans-sQTL hotspot.
Highlights
Lead Burdensthe phase-out of leaded paint and gasoline has substantially reduced mean blood lead levels in the United States (White et al, 2007), lead contamination in the city of Detroit and the neighboring city of Flint have been of extreme concern in the past two years due to the Flint water crisis (Hanna-Attisha et al, 2015)
In order to search for Splicing quantitative trait locus (QTL) (sQTLs), we collected RNA-seq data from 79 fly lines from The Drosophila Synthetic Population Resource (DSPR) (King et al, 2012)
We note that it is not the variable exons that are alternatively spliced by the sQTLs we identified in Dscam1, but rather the invariant exons that are downstream of the variant exons (Figure 6B)
Summary
Lead Burdensthe phase-out of leaded paint and gasoline has substantially reduced mean blood lead levels in the United States (White et al, 2007), lead contamination in the city of Detroit and the neighboring city of Flint have been of extreme concern in the past two years due to the Flint water crisis (Hanna-Attisha et al, 2015). In our genetic and physiology studies, 250 μM lead acetate in the standard fly food causes adult soft-tissue lead levels to be 50–100 μg/dL (Hirsch et al, 2009). This is in the high range of human lead exposure, and the currently CDC level of concern for lead exposure is 5 μg/dL (Bellinger, 2013). We use 250 μM developmental exposure to lead in the food because it consistently affects synaptic (He et al, 2009a), behavioral (Hirsch et al, 2009), and gene expression changes (Ruden et al, 2009). We are confident that Drosophila is a useful model to understand some of the mechanisms for how developmental lead exposure to lead affects gene expression and splicing in neurons, some of which are likely to be conserved in humans
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