Developmental and evolutionary dynamics of tissue-specific mammalian enhancers.

描述（由申请人提供）：增强子是调节基因的组织特异性和发育表达的远程作用非编码DNA元件。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重要作用。两种互补的，强大的方法来确定组织特异性增强子在基因组的规模是使用极端进化保守的非编码序列，最近，映射增强子相关的表观基因组标记染色质免疫沉淀耦合测序（ChIP-seq）。令人惊讶的是，不同胚胎小鼠组织的ChIP-seq研究显示，在某些组织（如胚胎大脑）中有活性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）中有活性的增强子仅受到轻度约束。这些结果提出了一种可能性，即在不同组织中激活的增强子之间，进化约束的模式通常是不同的。或者，在发育过程中，特定组织中增强子约束的程度可能发生变化，这些变化的时间差异解释了组织之间增强子保守性的观察差异。我建议研究这两种可能性，通过结合实验和计算的方法，旨在识别和序列分析的全基因组的增强子集活跃在小鼠从妊娠中期到成年期的三个组织具有不同的发展轨迹。具体目标包括：1）通过在出生前和出生后小鼠发育的八个时间点上取样的脑、肝和心脏组织上进行的ChIP-seq鉴定活性增强子; 2）在所有取样的时间点对与三种组织中的活性增强子相关的进化约束的程度进行分类; 3）使用小鼠转基因增强子测定法对选定的一组增强子通过体内发育来检测活性的变化。这项研究有望阐明在同一发育时间点在不同组织中激活的增强子的进化约束特征，以及在不同发育阶段在同一组织中激活的增强子之间可能的约束差异。重要的是，这些结果将有助于解释 为什么在进化上古老的器官系统（如心脏）中的基因表达似乎是在保守性差的增强子的控制下。预计这将对我们理解增强子在脊椎动物进化中的作用产生直接影响。除了使基本的进化见解，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，因为它们预计将揭示大量目前未被识别的增强子，这些增强子在三种主要生物医学组织中具有短暂的发育活性。

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.