The Stanford ENCODE Project

这一建议提出了以识别人类基因组中的功能转录元件为中心的高通量实验和计算方法的组合。这项拟议的研究符合ENCODE对所有目标区域进行全面分析的要求，并将可扩展到整个基因组水平。此外，结果可以很容易地传输到ENCODE数据库和其他公共数据库，以便与该联盟的其他参与者共享。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目标并测试其活动情况。在目标2中，将使用一种强大的选择方法来确定目标区域中的增强子，并对其性质进行表征。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鉴定24个人类细胞系中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。绑定数据段序列，以及 来自AIMS 1和AIMS 2中确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，以确定可能是蛋白质识别位点的基序。在目标4中，将使用先进的计算、进化和统计方法识别在目标区域受到进化限制的所有泛哺乳动物DNA元件，不仅产生完整的部分列表，而且还对每个元件内的进化约束进行高分辨率、定量的估计。在《目标5》中， 人类变异将通过测定48名来自不同背景的人的序列，从最受限制的功能元件的综合样本中识别出来。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对人类遗传学是否像cSNPs一样重要，以及是否可以根据进化比较来预测某个元件对人类遗传学的重要性。这项拟议的工作构成了目标区域中所有功能元件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.