Generation of an In Vivo Human Genome Enhancer Dataset

描述(申请人提供)：尽管远距离作用的转录增强子在人类生物学和疾病中发挥重要作用，但确定它们在基因组中的位置并确定它们在体内的调节活性仍然是一个重大挑战。最近的大量全基因组关联研究有令人信服的证据表明，非编码基因组区间的变异在很大程度上导致了广泛的特征和疾病。然而，缺乏全面的增强子目录在很大程度上排除了对潜在的调控和病因学机制的系统研究。因此，父母资助和这次续签申请的中心目标是确定和定义人类基因组中一组相当大的增强子的体内活动，以作为广泛社区获取的跳板。在资助的第1年到第3年(2006-2009年)，我们建立了极端比较基因组学的力量来确定大量假定的增强子集合，并明确地将体内特定的增强子功能分配给数百个人类保守序列。然而，这种比较方法无法预测哪些保守的非编码片段确实是增强剂，如果是这样的话，它们在体内将在哪里活跃，因此需要大规模的转基因来解决。最近，我们展示了以增强子相关转录辅活化子(P300)为靶点，结合大规模并行下一代测序(CHIP-SEQ)的染色质免疫沉淀的力量，以准确地识别直接在小鼠组织中活性的增强子。这种可扩展的实验方法将假定的增强子功能分配给数千个非编码区，从而极大地扩大了对特定细胞类型或组织中活性的全基因组增强子集的获取。然而，虽然p300代表了体内识别增强子的一个显著的表观基因组标记，但它只是一大类转录辅助激活因子中的一个成员，并且只标记了已知的增强子的子集。基于这些发现，目前的建议包括将这些CHIP-SEQ研究扩展到11个优先的转录辅助激活子，然后通过一系列高通量转基因小鼠检测来验证每个表观基因组标记的增强子预测性。预计公开访问这些数据集将极大地填补我们在人类基因组基因调控注释方面的空白，并破译这些序列的变异如何导致人类疾病。

DESCRIPTION (provided by applicant): Despite the important role of distant-acting transcriptional enhancers in human biology and disease, identifying their location in the genome and determining their in vivo regulatory activities remains a major challenge. There is compelling evidence from a large number of recent genome-wide association studies that variation in noncoding genomic intervals contributes on a substantial scale to a wide range of traits and disorders. However, the paucity of comprehensive enhancer catalogues has largely precluded systematic studies of the underlying regulatory and etiological mechanisms. Accordingly, the central goal of the parent grant and this renewal application is to identify and define the in vivo activities of a sizeable set of enhancers in the human genome to serve as a springboard for broad community access. In funded years 1 through 3 (2006-2009), we established the power of extreme comparative genomics to identify a large collection of putative enhancers and have unambiguously assigned specific in vivo enhancer function to hundreds of human conserved sequences. However, this comparative approach fails to predict a priori which conserved noncoding fragments are indeed enhancers and, if so, where they will be active in vivo, thus requiring massive- scale transgenesis to resolve. More recently, we demonstrated the power of chromatin immuno- precipitation targeting an enhancer-associated transcriptional coactivator (p300) coupled with massively parallel next generation sequencing (ChIP-Seq) to accurately identify enhancers active directly in mouse tissues. This scalable experimental approach has assigned putative enhancer function to thousands of noncoding regions, thereby dramatically expanding access to genome-wide sets of enhancers active in particular cell types or tissues. However, while p300 represents a remarkable epigenomic marker for enhancer identification in vivo, it is just one member of a larger class of transcriptional co-activators and only marks subsets of known enhancers. Based on these findings, the present proposal involves the extension of these ChIP-Seq studies to 11 prioritized transcriptional co-activators followed by validation of enhancer predictivity for each epigenomic mark through a series of high-throughput transgenic mouse assays. It is anticipated that public access to these data sets will significantly fill our void in gene regulatory annotation of the human genome and to decipher how variation in these sequences causes human disease.