Highly parallel functional characterization of human regulatory elements

描述（由申请人提供）：人调节元件的高度平行功能表征项目概述鉴定人调节元件的最有效方法之一是通过使用如DNA酶超敏反应或FAIRE的方法发现“开放染色质”。虽然有充分的证据表明开放染色质区域是功能性的，并受到序列特异性调节因子的约束，但我们通常不知道单个元件具有什么功能，或者人类开放染色质区域中的DNA序列变异如何影响该功能。传统上，功能已经在报告基因测定中通过实验测量，一次一个功能元件。然而，使用低通量连续方法来表征存在于每种细胞类型中的约100，000个开放染色质区域是不可行的。我们建议制定两个互补的办法来克服这些障碍。第一个将在一个实验中测试数万个人类调控元件的功能，第二个将在一个实验中测试10，000个这些元件中的天然人类序列变异的影响，这比现有方法提高了1，000到10，000倍。首先，FAIRE分离的假定调控元件将被克隆到基于网关的“进入”载体中，使我们能够容易地将插入片段交换到测试启动子、增强子、绝缘子或沉默子功能的报告子中。含有具有生物活性的插入物的细胞可以通过细胞分选分离，并且相应的插入物可以 可以通过下一代测序来鉴定。我们还将开发一种不需要细胞分选的这种方法的变体。发现人类序列变异对调控元件功能的影响的第二个主要障碍是在高度受控的环境中测量大量设计的DNA序列的影响的能力有限。使用Agilent阵列技术，我们将合成10，000个长度约200 bp的调控序列，其对应于5，000个推定调控区的交替等位基因。将根据其与人类疾病风险的联系选择综合的5，000个区域。在转染到细胞中之后，我们将使用流式细胞仪将所得转染细胞池分选到64个细胞箱中。 报告子水平，使用PCR从每个箱的细胞扩增插入的合成区域，并使用下一代测序测量每个活性箱的DNA含量。对于每个条形码（代表一个测试的元件），其下一代测序读数在表达箱中的分布提供了其表达的平均值和标准偏差的测量。将检测启动子、增强子、绝缘子和沉默子功能。由于所有测试序列都被转染到同一细胞系中，因此反式因子环境保持不变，使我们能够真正测试人类个体之间的遗传变异是否对表达产生因果影响。

DESCRIPTION (provided by applicant): Highly parallel functional characterization of human regulatory elements PROJECT SUMMARY One of the most effective means of identifying human regulatory elements is by discovery of "open chromatin" using methods like DNase hypersensitivity or FAIRE. While there is ample evidence that open chromatin regions are functional and bound by sequence-specific regulatory factors, we typically do not know what function an individual element has, or how DNA sequence variation in human open chromatin regions affects that function. Traditionally, function has been measured experimentally in reporter assays, one functional element at a time. However, it is not feasible to characterize the ~100,000 open chromatin regions that exist in each cell type using low-throughput, serial methods. We propose to develop two complimentary approaches to overcome these obstacles. The first will test the function of tens of thousands of human regulatory elements in a single experiment, and the second will test the effect of natural human sequence variation within 10,000 of those elements in a single experiment, representing 1,000 to 10,000-fold improvements over existing methods. First, putative regulatory elements isolated by FAIRE will be cloned en masse into a Gateway-based "entry" vector, allowing us to easily swap the inserts into reporters that test promoter, enhancer, insulator, or silencer function. Cells containing inserts with biological activity can be isolated by cell sorting, and the corresponding inserts can be identified by next-generation sequencing. We also will develop a variant of this method that does not require cell sorting. A second major obstacle in discovering the effect of human sequence variation on the function of regulatory elements is the limited ability to measure the effect of a large number of designed DNA sequences in a highly controlled setting. Using Agilent array technology, we will synthesize 10,000 regulatory sequences ~200 bp in length that corresponds to alternate alleles of 5,000 putative regulatory regions. The 5,000 regions synthesized will be selected based on their linkage to human disease risk. After transfection into cells, we will use a flow cytometer to sort the resulting pool of transfected cells into 64 bins of reporter levels, amplify the inserted synthesized region from the cells of each bin using PCR, and measure the DNA content of each activity bin using next-generation sequencing. For every barcode (representing one tested element), the distribution of its next-generation sequencing reads across the expression bins provides a measure of both its mean and standard deviation of expression. Promoter, enhancer, insulator, and silencer function will be tested. Since all tested sequences are transfected to the same cell line, the trans-factor environment is held constant, allowing us to truly test whether the genetic variation among human individuals has a causal effect on expression.