Hybrid mice reveal parent-of-origin and cis- and trans-regulatory effects in the retina

A fundamental challenge in genomics is to map DNA sequence variants onto changes in gene expression. Gene expression is regulated by cis-regulatory elements (CREs, i.e., enhancers, promoters, and silencers) and the trans factors (e.g., transcription factors) that act upon them. A powerful approach to dissecting cis and trans effects is to compare F1 hybrids with F0 homozygotes. Using this approach and taking advantage of the high frequency of polymorphisms in wild-derived inbred Cast/EiJ mice relative to the reference strain C57BL/6J, we conducted allele-specific mRNA-seq analysis in the adult mouse retina, a disease-relevant neural tissue. We found that cis effects account for the bulk of gene regulatory divergence in the retina. Many CREs contained functional (i.e., activating or silencing) cis-regulatory variants mapping onto altered expression of genes, including genes associated with retinal disease. By comparing our retinal data with previously published liver data, we found that most of the cis effects identified were tissue-specific. Lastly, by comparing reciprocal F1 hybrids, we identified evidence of imprinting in the retina for the first time. Our study provides a framework and resource for mapping cis-regulatory variants onto changes in gene expression, and underscores the importance of studying cis-regulatory variants in the context of retinal disease. Retinas from four classes of 8 week old male mice were collected: F0 C57BL/6J (B6), F0 Cast/EiJ (Cast), F1 B6xCast, and F1 CastxB6. Three replicates per class were generated. Each replicate consisted of a pool of 6-8 retinas. The mRNA-seq was conducted with paired-end 2x101 sequencing on the Illumina HiSeq 2000 platform. One lane of sequencing was run for all twelve samples. An additional lane of sequencing was run for the six F1 samples.基因组学领域的一项核心挑战，在于将DNA序列变异与基因表达变化建立对应关联。基因表达受顺式调控元件（cis-regulatory elements, CREs，即增强子、启动子与沉默子）以及作用于这些元件的反式作用因子（例如转录因子）调控。解析顺式与反式调控效应的一种高效方法，是将F1杂交个体与F0纯合子进行对比分析。本研究采用该方法，并利用野生来源近交系Cast/EiJ小鼠相较于参考品系C57BL/6J具有更高频率多态性的优势，在与疾病相关的神经组织——成年小鼠视网膜中开展了等位基因特异性mRNA测序（mRNA-seq）分析。本研究发现，顺式调控效应构成了视网膜中基因调控差异的主体部分。诸多CREs携带有功能性（即激活或沉默型）顺式调控变异，这些变异与基因表达改变存在对应关联，其中包括与视网膜疾病相关的基因。通过将本研究的视网膜数据集与已发表的肝脏数据集进行对比，我们发现所鉴定出的绝大多数顺式调控效应具有组织特异性。最后，通过对比互交F1杂交个体，我们首次在视网膜中发现了基因组印记的相关证据。本研究为建立顺式调控变异与基因表达变化的对应关联提供了研究框架与数据资源，并强调了在视网膜疾病背景下研究顺式调控变异的重要性。本研究收集了4组8周龄雄性小鼠的视网膜样本：F0代C57BL/6J（B6）、F0代Cast/EiJ（Cast）、F1代B6xCast以及F1代CastxB6。每组设置3次生物学重复，每次重复由6~8个视网膜混合而成。本研究采用Illumina HiSeq 2000测序平台，进行双端2×101bp的mRNA-seq测序。所有12个样本共用一个测序泳道，6个F1样本额外增设一个测序泳道。