Mapping Sites of Transcription and Regulation

描述：（申请人提供）人类基因组工作草图已经完成。 RNA的转录是允许将编码信息从DNA序列转移到功能中的功能过程之一。 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要复杂得多。 在基因组中编码的控制这些转录物表达的调控元件的位置和特征甚至更不清楚。 这个建议的目的是描述一个通用的和无偏见的策略，可以使用，在全基因组范围内，定位的转录和调节RNA表达的功能元件的网站的集合。 这些策略以高密度寡核苷酸阵列和染色质免疫沉淀（CHIP）为核心技术，将使这些位点的定位成为可能。 对于我们提出的ENCODE项目的第一年，从大小从500 kb到2 Mb的44个不同染色体位置选择的30 Mb分布的基因组序列（约占基因组的1%）将作为靶序列。 将合成具有约810，000个探针对的单个阵列，其平均每37 bp询问30 Mb的序列，并将其用作通用平台以映射转录和功能调节元件的位置。 将被监测的功能元件是15个转录因子和4个阻遏物的结合位点，以及7种与RNA调控相关的组蛋白修饰的位置。 三种充分表征的佛波酯或视黄酸响应细胞系（Jurkat、NCCIT和HL-60）将在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。 为了证明这些策略的可扩展性，我们提出的研究的第二年和第三年将集中在构建一个类似的图谱集合上，平均分辨率为35 bp，但跨越整个人类基因组。

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.