An integrative genomic analysis of the Longshanks selection experiment for longer limbs in mice (4C-seq). An integrative genomic analysis of the Longshanks selection experiment for longer limbs in mice (4C-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. Overall design: 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.

进化研究往往受限于关键数据的缺失——这类数据正是解析选择历程的核心依据。选择实验（selection experiment）可在可控条件下重现快速进化过程，是探究基因组在选择压力下演化规律的优质工具。本研究针对Longshanks选择实验（Longshanks selection experiment）开展基因组解析：该实验通过选择性繁育小鼠历经20代，以体质量校正后的胫骨长度为选育指标，最终两个重复组的胫骨长度均较基线提升13%。我们整合进化理论、基因组序列与分子遗传学手段解析该选择响应机制，发现其同时涉及多基因适应性（polygenic adaptation）与效应显著的离散基因座（discrete major-effect loci），且效应最强的基因座大概率在两个重复组中发生平行选择。研究证实，选择压力可通过使抑因子Nkx3-2的两个肢体增强子失活，解除对骨骼生长的抑制。综上，本研究的整合基因组分析表明，可将单个碱基对突变与整体选择响应直接关联起来。

实验整体设计：对3个小鼠品系的奠基种群及第17代个体开展全基因组测序（whole genome sequencing，WGS），共计6个群体样本。通过对选择响应开展统计建模，筛选出与胫骨长度快速增加相关的候选基因组区域。研究发现，位于小鼠基因组5号染色体（Chr5）上的Nkx3-2是呈现平行选择响应的核心基因座之一。由于未发现可作为选择靶标的编码突变，我们采用转座酶可及性测序（ATAC-Seq）与环状染色体构象捕获测序（4C-Seq）等功能基因组学技术，以筛选相关功能元件用于后续研究与功能解析。我们以ENCODE项目的染色质修饰标记与ATAC-Seq检测到的开放染色质信号联合筛选出的3个增强子（标记为N1-N3）为锚定位点，分别对前肢、后肢组织及肝脏对照样本开展4C-Seq实验。实验采用BglII限制性内切酶酶切位点进行邻近连接，对选定锚定位点的环化扩增子进行扩增并测序。嵌合连接产物被视为染色体潜在相互作用的指示信号。