Developmental and evolutionary dynamics of tissue-specific mammalian enhancers.

描述（由申请人提供）：增强子是远程作用的非编码DNA元件，可调节基因的组织特异性和发育表达。增强子被认为是脊椎动物进化的主要驱动力，越来越多的证据表明它们在人类疾病中发挥着重要作用。在基因组尺度上鉴定组织特异性增强子的两种互补的、强大的方法是利用非编码序列的极端进化守恒，以及最近通过染色质免疫沉淀耦合测序（ChIP-seq）绘制增强子相关表观基因组标记。令人惊讶的是，对不同小鼠胚胎组织的ChIP-seq研究显示，在某些组织（如胚胎脑）中活跃的增强子受到严重的进化序列约束，而在其他组织（如胚胎心脏）中活跃的增强子仅受到轻度约束。这些结果提出了一种可能性，即在不同组织中活跃的增强子之间，进化约束模式通常是不同的。另一种可能是，特定组织中的增强子约束程度在发育过程中发生了变化，这些变化的时间差异解释了组织之间观察到的增强子保护差异。我建议通过实验和计算方法的结合来检验这两种可能性，旨在鉴定和序列分析全基因组增强子，这些增强子在具有不同发育轨迹的小鼠中从妊娠中期到成年期的三种组织中都有活性。具体目标包括：1)通过在小鼠产前和产后发育的八个时间点取样的大脑、肝脏和心脏组织进行ChIP-seq鉴定活性增强子；2)在采样的所有时间点对三种组织中与活性增强子相关的进化约束程度进行分类；3)通过对一组选定的增强子进行小鼠转基因增强子试验，在体内开发来验证活性的变化。本研究旨在阐明在同一发育时间点活跃于不同组织的增强子的进化约束特征，以及在不同发育阶段活跃于同一组织的增强子之间可能存在的约束差异。重要的是，研究结果将有助于解释

DESCRIPTION (provided by applicant): Enhancers are remotely acting non-coding DNA elements that regulate the tissue-specific and developmental expression of genes. Enhancers are thought to be a major driver of vertebrate evolution, and there is increasing evidence that they play important roles in human disorders. Two complementary, powerful approaches to identify tissue-specific enhancers at a genomic scale are the use of extreme evolutionary conservation of non-coding sequences and, more recently, the mapping of enhancer-associated epigenomic marks by chromatin immunoprecipitation coupled to sequencing (ChIP-seq). Surprisingly, ChIP-seq studies of different embryonic mouse tissues revealed that enhancers active in some tissues, such as the embryonic brain, are under severe evolutionary sequence constraint whereas enhancers active in other tissues, such as the embryonic heart, are only mildly constrained. These results raise the possibility that patterns of evolutionary constraint generally differ between enhancers active in different tissues. Alternatively, it is possible that he degree of enhancer constraint in a specific tissue changes during development, and those differences in timing of these changes account for the observed differences in enhancer conservation between tissues. I propose to examine these two possibilities through a combination of experimental and computational approaches aimed at the identification and sequence analysis of genome-wide sets of enhancers active in the mouse from mid-gestation through adulthood in three tissues with different developmental trajectories. The specific aims include: 1) Identify active enhancers through ChIP-seq performed on brain, liver and heart tissue sampled across eight time points of pre- and postnatal mouse development; 2) Classify the degree of evolutionary constraint associated with active enhancers in the three tissues at all time points sampled; 3) Validate changes in activity through development in vivo using mouse transgenic enhancer assays for a selected set of enhancers. This study is expected to elucidate the evolutionary constraint signatures of enhancers active in different tissues at the same developmental time point, as well as possible constraint differences between enhancers active in the same tissue at different developmental stages. Importantly, the results will help to explain why gene expression in evolutionarily old organ systems like the heart appears to be under the control of poorly conserved enhancers. This is expected to have direct implications for our understanding of the role of enhancers in vertebrate evolution. In addition to enabling fundamental evolutionary insights, the data sets generated through this proposal will also provide a valuable resource for biomedical studies, since they are expected to reveal large numbers of currently unrecognized enhancers with transient developmental activities in three tissues of major biomedical interest.