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.

演化研究常受限于关键数据的缺失，而此类数据正是解析选择作用演化历史的核心要件。在受控条件下复刻快速演化过程的选择实验，是研究基因组如何在选择压力下演化的绝佳工具。本研究针对长胫（Longshanks）选择实验开展基因组剖析：该实验以小鼠为对象，针对体质量相对比例下的更长胫骨进行了20代选择性繁育，最终在两个重复实验组中实现了胫骨长度13%的增长。我们整合演化理论、基因组序列与分子遗传学手段解析选择响应机制，发现该响应同时涉及多基因适应性演化与主效离散位点，其中效应最强的位点大概率在两个重复实验组中受到平行选择。研究表明，选择压力可能通过使抑制因子Nkx3-2的两个肢体特异性增强子失活，从而促进骨生长的去抑制过程。综上，我们的整合基因组分析证实，可将单个碱基对突变与整体选择响应建立关联。

整体实验设计：对三个小鼠品系的奠基种群及第17代种群进行全基因组测序（共6个群体样本）。为鉴定驱动胫骨长度快速增长的候选区域，我们开展了选择响应的统计建模。在小鼠基因组5号染色体上发现了一个呈现平行选择响应的关键位点Nkx3-2。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4C测序（4C-Seq）等功能基因组学技术，以辅助鉴定相关功能元件用于后续研究与功能剖析。我们以ENCODE项目的染色质修饰标记与开放染色质ATAC-Seq信号联合鉴定出的三个增强子（标记为"锚定位点"），分别在前肢、后肢组织与肝脏对照样本中开展4C测序（4C-Seq）实验。针对BglII酶切位点开展染色质邻近连接反应，随后对选定锚定位点的连接扩增子进行扩增并测序。嵌合连接产物被视为潜在染色质相互作用的指示信号。