Mapping Sites of Transcription and Regulation

描述：(申请人提供)人类基因组的工作草稿已经完成。RNA转录是允许编码信息从DNA序列转移到功能的功能过程之一。最近的证据表明，为人类基因组制作的转录本目录比目前的注释所表明的更加复杂。基因组中控制这些转录本表达的调控元件的位置和特征更是知之甚少。这项建议的目标是描述一组通用的和无偏见的策略，这些策略可以用于在全基因组范围内定位转录位点和调节RNA表达的功能元件。这些战略拥有高密度寡核苷酸阵列和染色质免疫沉淀(芯片)作为核心技术，将使这些地点的测绘成为可能。在我们提出的ENCODE计划的第一年，从44个大小从500kb到2Mb的不同染色体位置选择的30Mb的分布基因组序列(约占基因组的1%)将作为目标序列。将合成一个具有大约810,000个探针对的单个阵列，平均每37bp询问30Mb的序列，并用作一个公共平台来定位转录和功能调控元件的位置。将要监测的功能元件是15个转录因子和4个抑制子的结合位点，以及与RNA调控相关的7种组蛋白修饰的位置。三个特性良好的佛波酯或维甲酸反应细胞系(Jurkat，NCCIT和HL-60)将被这些分子激活时进行时间监测，并将为这些研究提供生物学背景。为了证明这套策略的可扩展性，我们提议的第二年和第三年的研究将专注于构建类似的图谱集合，平均分辨率为35BP，但覆盖整个人类基因组。

DESCRIPTION: (provided by applicant) A working draft of the human genome has been completed. Transcription of RNA is one of the functional processes which permit the transfer of encoded information from the DNA sequence into function. Recent evidence suggests that the catalogue of transcripts that are made for the human genome is more complex than indicated by current annotations. The locations and characteristics of regulatory elements encoded in the genome which control the expression of these transcripts are even less well understood. The goal of this proposal is to describe a collection of generic and unbiased strategies which can be used, on a genome-wide scale, to locate the sites of transcription and the functional elements which regulate RNA expression. These strategies have high density oligonucleotide arrays and chromatin immunoprecipation (CHIP) as core technologies that will enable the mapping of these sites. For the first year of our proposed ENCODE project, 30 Mb of distributed genomic sequence (approximately 1% of genome) selected from 44 different chromosomal locations ranging in size from 500 kb to 2 Mb will serve as target sequences. A single array with approximately 810,000 probe pairs that interrogate the 30 Mb of sequence at every 37 bp, on average, will be synthesized and used as a common platform to map the locations of both transcription and functional regulatory elements. The functional elements which will be monitored are the binding sites for 15 transcription factors and 4 repressors as well as the locations for 7 types of histone modifications which have been correlated with RNA regulation. Three well-characterized phorbol ester or retinoic acid- responsive cell lines (Jurkat, NCCIT and HL-60) will be temporally monitored when activated by these molecules and will provide the biological context for these studies. To demonstrate the scalability of this collection of strategies, the second and third years of our proposed studies will be focused on constructing a similar collection of maps, at a resolution of 35 bp on average, but across the entire human genome.