Developmental and evolutionary dynamics of tissue-specific mammalian enhancers.

描述(申请人提供)：增强子是远程作用的非编码DNA元件，调节基因的组织特异性和发育表达。增强剂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。在基因组水平上识别组织特异性增强子的两种互补的、强大的方法是使用非编码序列的极端进化保守，以及最近通过染色质免疫沉淀与测序相结合的增强子相关表观基因组标记的映射(CHIP-SEQ)。令人惊讶的是，对不同胚胎小鼠组织的芯片序列研究显示，在某些组织中活跃的增强子，如胚胎大脑，受到严格的进化序列限制，而在其他组织中活跃的增强子，如胚胎心脏，只受到轻微的限制。这些结果提出了这样一种可能性，即不同组织中活性的增强剂之间的进化限制模式通常不同。或者，可能是特定组织中增强子的限制程度在发育过程中发生了变化，这些变化的时间上的差异解释了观察到的不同组织之间增强子保守性的差异。我建议通过实验和计算相结合的方法来研究这两种可能性，这些方法旨在识别和分析小鼠从妊娠中期到成年期三个不同发育轨迹的组织中活跃的基因组范围的增强子集。具体目标包括：1)通过对小鼠出生前和出生后八个时间点的大脑、肝脏和心脏组织进行芯片序列分析来确定活性增强剂；2)对所有采样时间点的三种组织中与活性增强剂相关的进化约束程度进行分类；3)通过对选定的一组增强剂进行小鼠转基因增强子分析，验证通过体内发育过程中活性的变化。这项研究有望阐明在同一发育时间点不同组织中活性的增强子的进化约束特征，以及同一组织中不同发育阶段活性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 为什么像心脏这样的进化古老的器官系统中的基因表达似乎受到保守程度不高的增强子的控制。这有望对我们理解增强剂在脊椎动物进化中的作用产生直接的影响。除了实现基本的进化洞察外，通过这一提议产生的数据集也将为生物医学研究提供宝贵的资源，因为它们有望揭示大量目前未被识别的增强剂，它们在三个主要生物医学感兴趣的组织中具有短暂的发育活动。

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.