A rapidly reversible mutation generates subclonal genetic diversity and unstable drug resistance

Most genetic changes have negligible reversion rates. As most mutations that confer resistance to an adverse condition (e.g., drug treatment) also confer a growth defect in its absence, it is challenging for cells to genetically adapt to transient environmental changes. Here, we identify a set of rapidly reversible drug-resistance mutations in Schizosaccharomyces pombe that are caused by microhomology-mediated tandem duplication (MTD) and reversion back to the wild-type sequence. Using 10,000× coverage whole-genome sequencing, we identify nearly 6,000 subclonal MTDs in a single clonal population and determine, using machine learning, how MTD frequency is encoded in the genome. We find that sequences with the highest-predicted MTD rates tend to generate insertions that maintain the correct reading frame, suggesting that MTD formation has shaped the evolution of coding sequences. Our study reveals a common mechanism of reversible genetic variation that is beneficial for adaptation to environmental fluctuations and facilitates evolutionary divergence.

Different mechanisms of adaptation have different timescales. Epigenetic changes are often rapid and reversible, while most genetic changes have nearly negligible rates of reversion (1). This poses a challenge for genetic adaptation to transient conditions such as drug treatment; mutations that confer drug resistance are often deleterious in the absence of drug, and the second-site suppressor mutations are required to restore fitness (2, 3). Preexisting tandem repeats (satellite DNA) undergo frequent expansion and contraction (4–6). While repeats are rare inside of most coding sequences and functional elements, there is some evidence for conserved repetitive regions that undergo expansion and contraction to regulate protein functions or expression (6–8). RNA interference– or Chromatin-based epigenetic states have been associated with transient drug resistance in fungi (9) and cancer cells (10, 11), and transient resistant states have been characterized by differences in organelle state, growth rate, and gene expression in budding yeast (12, 13). In bacteria and in fungi, copy-number gain and subsequent loss can result in reversible drug resistance (14–18). However, all genetic systems developed so far for studying unstable genotypes rely on reporter genes and thus investigate only one genetic locus and only one type of genetic change.Unbiased, next-generation sequencing-based approaches could give a more global view, allowing us to understand the rules that govern unstable genotypes at a genome-wide scale. However, genetic changes with high rates of reversion tend to remain subclonal (19–21), and it is challenging to distinguish most types of low-frequency mutations from sequencing errors (22), especially in complex genomes with large amount of repetitive DNA or de novo duplicated genes. Thus, fast-growing organisms with relatively small and simple genomes are particularly well suited for determining whether transient mutations exist, for the genome-wide characterization of such mutations, and for identification of the underlying mechanisms.