An integrative genomic analysis of the Longshanks selection experiment for longer limbs in mice (ATAC-seq)

Evolutionary studies are often limited by missing data that are critical to understanding the history of selection. Selection experiments, which reproduce rapid evolution under controlled conditions, are excellent tools to study how genomes evolve under selection. Here we present a genomic dissection of the Longshanks selection experiment, in which mice were selectively bred over 20 generations for longer tibiae relative to body mass, resulting in 13% longer tibiae in two replicates. We synthesized evolutionary theory, genome sequences and molecular genetics to understand the selection response and found that it involved both polygenic adaptation and discrete loci of major effect, with the strongest loci likely to be selected in parallel between replicates. We show that selection may favor de-repression of bone growth through inactivating two limb enhancers of an inhibitor, Nkx3-2. Our integrative genomic analyses thus show that it is possible to connect individual base-pair changes to the overall selection response. Whole genome sequencing of the founding and generation 17 of three lines of mice (6 population samples in total). Statistical modelling of the selection response was performed in order to identify candidate regions responsible for the rapid increase in tibia length. One major locus found to show parallel selection response was Nkx3-2 on Chr5 of the mouse genome. Since no coding mutations were found that could be attributable as the target of selection, functional genomic techniques such as ATAC-Seq and 4C-Seq were performed to help identify relevant functional elements for further investigation and functional dissection. 4C-Seq was performed from fore- and hindlimb tissues as well as liver controls from the "viewpoint" of three enhancers (labelled as N1-N3) as identified by a combination of chromatin modification marks from the ENCODE project and ATAC-Seq signals of open chromatin. Proxmity ligation was performed on BglII sites, and religated amplicons from the chosen viewpoints were amplified for sequencing. Chimeric ligation products were interpreted as indicative of potential chromosome contacts.