Generation of an In Vivo Human Genome Enhancer Dataset

描述（由申请人提供）：尽管远距离作用转录增强子在人类生物学和疾病中具有重要作用，但鉴定其在基因组中的位置并确定其体内调节活性仍然是一个重大挑战。最近大量的全基因组关联研究有令人信服的证据表明，非编码基因组间隔的变异在很大程度上导致了广泛的性状和疾病。然而，缺乏全面的增强子目录在很大程度上排除了潜在的监管和病因机制的系统研究。因此，母基金和本更新申请的中心目标是鉴定和定义人类基因组中相当大的一组增强子的体内活性，以作为广泛社区访问的跳板。在资助的第1年至第3年（2006-2009年），我们建立了极端比较基因组学的力量，以确定大量的推定增强子，并明确指定特定的体内增强子功能的数百人保守序列。然而，这种比较方法无法先验地预测哪些保守的非编码片段确实是增强子，如果是的话，它们将在体内何处具有活性，因此需要大规模的转基因来解决.最近，我们证明了染色质免疫沉淀靶向增强子相关转录共激活因子（p300）与大规模并行下一代测序（ChIP-Seq）结合的能力，以准确鉴定直接在小鼠组织中有活性的增强子。这种可扩展的实验方法已经将推定的增强子功能分配给了数千个非编码区，从而极大地扩展了对特定细胞类型或组织中活跃的全基因组增强子的访问。然而，虽然p300代表了一个显着的表观基因组标记增强子识别体内，它只是一个更大的转录共激活因子类的成员之一，只标记已知的增强子的子集。基于这些发现，本提案涉及将这些ChIP-Seq研究扩展到11个优先的转录共激活因子，然后通过一系列高通量转基因小鼠试验验证每个表观基因组标记的增强子预测性。预计公众对这些数据集的访问将大大填补我们在人类基因组基因调控注释方面的空白，并破译这些序列中的变异如何导致人类疾病。 公共卫生相关性：整个人类基因组序列的产生是一个庞大的研究基础的常规起点，并有助于确定我们基因组中的大多数基因。然而，我们对调节这些基因的序列的理解是贫乏的，尽管它们在人类疾病中被假定为改变。在这里，我们建议识别和测试DNA的表观基因组标记，以确定它们在转基因小鼠中作为基因调控序列的能力。增强子在体内的阳性特征的鉴定有望显著填补我们在人类基因组的基因调控注释方面的空白，并将其突变解释为人类疾病的原因。

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. PUBLIC HEALTH RELEVANCE: The generation of the entire human genome sequence serves as a routine starting point for a huge investigator base and has aided in defining the majority of genes in our genome. However, our understanding of the sequences that regulate these genes is meager, despite their presumed alterations in human disease. Here, we propose to identify and test epigenomic marks of DNA for their ability to act as gene regulatory sequences in transgenic mice. The identification of positive signatures of enhancers in vivo is expected to significantly fill our void in gene regulatory annotation of the human genome and to decipher their mutation as a cause of human disease.