The Stanford ENCODE Project

该提案提出了一个高通量的实验和计算方法的结合，集中在人类基因组中的功能转录元件的识别。拟议的研究符合ENCODE对所有目标区域进行综合分析的要求，并将扩展到全基因组水平。此外，研究结果可以很容易地转移到ENCODE数据库和其他公共数据库，以便与联合体的其他参与者分享。在目标1中，所有基因的启动子 在ENCODE中，将通过筛选计划和选择计划全面确定和测试目标的活性。在目标2中，将使用强大的选择方法来识别靶区域中的增强子，并表征其特性。在目标3中，染色质免疫沉淀和微阵列杂交的组合将被用来确定在24个人类细胞系中被12个一般转录因子和染色质蛋白占据的顺式作用结合位点。结合片段的序列，沿着 目的1和2中鉴定的启动子和增强子的序列，将用统计计算方法进行深入研究，以鉴定作为蛋白质的可能识别位点的基序。在目标4中，将通过使用先进的计算、进化和统计方法来识别在目标区域中进化上受到约束的所有泛哺乳动物DNA元件，不仅产生完整的部件列表，而且还产生每个元件内进化约束的高分辨率定量估计。在目标5中， 通过确定来自不同背景的48个人的元件序列，将在最受约束的功能元件的综合样本中识别人类变异。这些序列数据将有助于回答以下问题：非编码功能元件中的SNPs是否与cSNPs一样对人类遗传学重要，以及是否可以根据进化比较预测元件对人类遗传学的重要性。这项拟议的工作构成了对目标区域所有功能要素的一个重要子集的全面表征。

This proposal presents a combination of high-throughput experimental and computational approaches centered on the identification of functional transcriptional elements in the human genome. The proposed research is consistent with ENCODE's requirement for comprehensive analyses of all target regions, and will be scalable to the whole genome level. In addition, the results can easily be transferred to the ENCODE database and other public databases for sharing with the other participants of the consortium. In Aim 1, promoters of all the genes in the ENCODE targets will be comprehensively identified and tested for activity by a screening scheme and a selection scheme. In Aim 2, a powerful selection method will be used to identify enhancers in the target regions, and their properties will be characterized. In Aim 3, a combination of chromatin immunoprecipitation and microarray hybridization will be used to identify cis-acting binding sites that are occupied by 12 general transcription factors and chromatin proteins in 24 human cell lines. Sequences of bound segments, along with sequences from promoters and enhancers identified in Aims 1 and 2, will be examined intensively with statistical computational methods to identify motifs that are the likely recognition sites for the proteins. In Aim 4, all the pan-mammalian DNA elements that are evolutionarily constrained in the target regions will be identified by using advanced computational, evolutionary, and statistical methodology, producing not only a complete parts list, but also high-resolution, quantitative estimates of evolutionary constraint within each element. In Aim 5, human variation will be identified m a comprehensive sample of the most constrained functional elements by determining the sequence of the element in 48 humans from diverse backgrounds. This sequence data will help answer the question of whether SNPs in noncoding functional elements are as important for human genetics as are cSNPs, and whether the importance of an element for human genetics can be predicted on the basis of evolutionary comparisons. This proposed work constitutes a comprehensive characterization of an important subset of all functional elements in the target regions.