Project description:Copy-number variants (CNVs) are large-scale amplifications or deletions of DNA that can drive rapid adaptive evolution and result in large-scale changes in gene expression. Whereas alterations in the copy number of one or more genes within a CNV can confer a selective advantage, other genes within a CNV can decrease fitness when their dosage is changed. Dosage compensation - in which the gene expression output from multiple gene copies is less than expected - is one means by which an organism can mitigate the fitness costs of deleterious gene amplification. Previous research has shown evidence for dosage compensation at both the transcriptional level and at the level of protein expression; however, the extent of compensation differs substantially between genes, strains, and studies. Here, we investigated sources of dosage compensation at multiple levels of gene expression regulation by defining the transcriptome, translatome and proteome of experimentally evolved yeast (Saccharomyces cerevisiae) strains containing adaptive CNVs. We quantified the gene expression output at each step and found evidence of widespread dosage compensation at the protein abundance (~47%) level. By contrast we find only limited evidence for dosage compensation at the transcriptional (~8%) and translational (~3%) level. We also find substantial divergence in the expression of unamplified genes in evolved strains that could be due to either the presence of a CNV or adaptation to the environment. Detailed analysis of 82 amplified and 411 unamplified genes with significantly discrepant relationships between RNA and protein abundances identified enrichment for upstream open reading frames (uORFs). These uORFs are enriched for binding site motifs for SSD1, an RNA binding protein that has previously been associated with tolerance of aneuploidy. Our findings suggest that, in the presence of CNVs, SSD1 may act to alter the expression of specific genes by potentiating uORF mediated translational regulation.

Project description:The glutathione S-transferase (GST) gene family codes for enzymes that detoxify xenobiotics by catalyzing the conjugation of xenobiotics to glutathione. Based on reports that inherited copy number variations (CNV) in the genome modulate some GST expression levels and with the knowledge that cigarette smoke contains >3000 xenobiotics, and that the small airway epithelium and alveolar macrophages are involved early in the pathogensis of smoking-induced lung disease, we asked: do germline CNVs modulate GST expression level in the small airway epithelium and alveolar macrophages? Affymetrix HG U133 Plus 2.0 microarrays were used to survey GST gene expression in the small airway epithelium and alveolar macrophages obtained by bronchoscopic brushings from current smokers (n=35) and nonsmokers (n=35). The CNV genotypes of these 70 subjects were determined by Affymetrix Human SNP array 5.0 chips. Sixteen % of subjects had deletions of both GSTM1 alleles. These deletions were associated with reduced GSTM1 mRNA levels in both the small airway epithelium (p<10-7) and alveolar macrophages (p<0.05). Thirty % of subjects had homozygous deletions of GSTT1 with concomitant reduced mRNA levels in both small airway epithelium and alveolar macrophages (p<10-7). In contrast, genes flanking the CNV regions of both GST genes showed no difference in expression level among subjects with and without the GST deletions (p>0.3). Interestingly, GSTT2B, a duplicate gene of GSTT2, exhibited homozygous deletion in blood in 27% of subjects and was not expressed in small airway epithelium in the remainder of subjects but was expressed in alveolar macrophages of heterozygotes and wild type subjects, proportionate to genotype (p<10-3). These data demonstrate that highly prevalent CNV deletions of genes critical to ameliorating smoking-associated xenobiotic-induced damage in the lung can result in significant modulation of the gene expression levels, with the linear relationship of genotype to expression level suggesting minimal compensation of gene expression levels in heterozygotes consistent with GST polymorphisms playing a role in the risk for development of smoking-induced lung disease.

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Copy number variation: the balance between gene dosage and expression in Drosophila melanogaster

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