Highly parallel functional characterization of human regulatory elements

人类调控元件的高度并行功能表征

基本信息

批准号：
8310666
负责人：
JASON D LIEB
金额：
$ 44.63万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-04-23 至 2015-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8310666
关键词：
Affect Alleles Atherosclerosis Bar Codes Binding Biological Biological Assay Cell Culture Techniques Cell Line Cell Separation Cells Chromatin DNA DNA Sequence Data Defect Deoxyribonucleases Diabetes Mellitus Elements Enhancers Environment Flow Cytometry Foundations Functional RNA Genetic Transcription Genetic Variation High Density Lipoprotein Cholesterol Human Hypersensitivity Indium Individual K-562 LDL Cholesterol Lipoproteins Length Libraries Link Malignant Neoplasms Measurement Measures Methods Nucleic Acid Regulatory Sequences Obesity Plasmids Ploidies RNA Reading Regulator Genes Regulatory Element Reporter Sorting - Cell Movement Techniques Technology Testing Time Transcript Transcriptional Regulation Transfection Triglycerides Variant Yeasts age related base cell type design disorder risk drug metabolism human disease next generation programs promoter public health relevance research study trait vector

项目摘要

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. PUBLIC HEALTH RELEVANCE: Highly parallel functional characterization of human regulatory elements PROJECT NARRATIVE Public health relevance Defects in transcriptional regulation are at the foundation of many human diseases, including cancer and many aging-related maladies. Gene transcription programs are determined by regulatory elements encoded in DNA. However, it is currently difficult to test the function of more than a few of these DNA regulatory elements at the same time, and it is difficult to test the effect of sequence variation. We propose to develop two complementary technologies. 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, including variants associated with human disease risk, in 10,000 DNA elements in a single experiment. These experiments may prove valuable in identifying non-coding DNA sequence variants that are causally linked to human disease.

描述（由申请人提供）：人类调节元件的高度平行功能表征摘要摘要识别人类调节元素的最有效方法之一是使用DNase超敏或公平的方法发现“开放染色质”。尽管有足够的证据表明开放的染色质区域是功能性的，并且受序列特异性调节因素的约束，但我们通常不知道单个元素具有什么功能，或者人类开放染色质区域中的DNA序列变化如何影响该功能。传统上，功能是在记者测定中测量的，一次是一个功能元素。但是，使用低通量，串行方法来表征每种单元类型中存在的〜100,000个开放染色质区域是不可行的。我们建议开发两种免费的方法来克服这些障碍。第一个将在一个实验中测试成千上万的人类调节元素的功能，第二个将测试单个实验中10,000个元素内自然人类序列变化的影响，比现有方法的改善1,000至10,000倍。首先，由Faire隔离的推定调节元素将被克隆到基于网关的“输入”矢量中，从而使我们可以轻松地将插入物交换为测试启动子，增强器，绝缘体或消音器功能的记者。可以通过细胞分类分离包含具有生物活性的插入的细胞，相应的插入物可以可以通过下一代测序来识别。我们还将开发该方法的变体，该变体不需要细胞分类。发现人类序列变化对调节元素功能的影响的第二个主要障碍是在高度控制的环境中测量大量设计的DNA序列的影响的有限能力。使用安捷伦阵列技术，我们将综合10,000个调节序列约200 bp的长度，与5,000个推定的监管区域的替代等位基因相对应。 5,000个区域将根据其与人类疾病风险的联系而选择。转染后，我们将使用流式细胞仪将所得的转染细胞池分类为64箱记者水平，使用PCR从每个bin的细胞中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（代表一个测试元素），其下一代测序的分布在表达式箱中读取提供了衡量其表达的平均值和标准偏差的度量。将测试启动子，增强子，绝缘子和消音器功能。由于将所有测试的序列转染至同一细胞系，因此跨因素环境保持恒定，从而使我们能够真正测试人个体之间的遗传变异是否对表达有因果作用。公共卫生相关性：人类监管要素的高度平行功能表征项目叙事公共卫生相关性缺陷是许多人类疾病的基础，包括癌症和许多与衰老有关的疾病。基因转录程序由DNA中编码的调节元素确定。但是，目前很难同时测试这些DNA调节元件中的一些以上的功能，并且很难测试序列变化的效果。我们建议开发两种互补技术。第一个将在一个实验中测试成千上万人类调节元素的功能，第二个将测试自然人类序列变化的效果，包括与人类疾病风险相关的变异，在单个实验中10,000个DNA元素中。这些实验可能证明在识别与人类疾病有因果关系的非编码DNA序列变体方面非常有价值。