Identification of Functional DNA Elements by HSqPCR

描述（由申请人提供）：本研究提案的总体目标是应用功能方法（最初在UW开发，并由Regulome Corporation适应高通量）进行定量染色质分析，以描绘ENCODE工作组选择的30兆碱基人类基因组序列（“ENCODE区域”）中的功能DNA元件。 首先，我们将在人淋巴母细胞系中产生ENCODE区域的定量染色质谱。 该概况将使得能够精确定位所有主要的Dnasel-超敏位点（下文中的“HS”）。 序列特异性Dnasel敏感性的定量将通过应用一种新的、经测试的高通量定量实时PCR测定（“超敏qPCR”或“HSqPCR”）来完成。 将设计和合成以高分辨率跨越ENCODE区域的专用引物组。 该引物组将用于所有实验中。 第二，我们将在来自3个主要胚胎谱系（内胚层、中胚层、外胚层）的另外3种细胞类型中产生ENCODE区域的定量染色质谱。 这些图谱将能够定位每种组织类型中存在的所有主要HS。 与淋巴样细胞的图谱相结合，将进一步能够鉴定组织或谱系限制性HS以及存在于多个谱系中的位点。 第三，我们将使用高分辨率超敏Southern对已识别的HS进行确证性验证实验。 南方超敏反应代表了一种广泛应用的“金标准”方法来识别HS。 每年将从高通量方法确定的研究中心中选择250个研究中心，并使用该独立方法进行验证。 第四，产生的定量染色质谱将进行详细的计算分析，目的是确定HS和进化保守的非编码序列之间的对应关系。平行分析将集中于与组织特异性、谱系特异性或多谱系HS相关的DNA序列基序的鉴定。 将开发和测试人类基因组中HS的识别和分类算法。 还将评价用于鉴定活性调控序列的选定已发表算法的性能。预计这项研究工作将导致鉴定ENCODE区域的大多数（如果不是全部）调控元件，并将提供有关应用这种功能方法鉴定人类基因组所有调控元件的可行性的信息。

DESCRIPTION (provided by applicant): The overall aim of this research proposal is to apply functional methodology (initially developed at UW and adapted to high throughput by Regulome Corporation) for quantitative chromatin profiling to delineate the functional DNA elements across 30 megabases of human genome sequence selected by the ENCODE working group (the 'ENCODE regions'). First we will produce a quantitative chromatin profile of the ENCODE regions in a human lymphoblastoid cell line. This profile will enable precise localization of all major Dnasel-hypersensitive sites ('HSs' hereafter). Quantification of sequence-specific Dnasel sensitivity will be accomplished through application of a novel, tested high-throughput quantitative real-time PCR assay ('Hypersensitivity qPCR' or 'HSqPCR'). A specialized primer set that spans the ENCODE regions at high resolution will be designed and synthesized. This primer set will be employed in all the experiments. Second, we will produce quantitative chromatin profiles of the ENCODE regions in 3 additional cell types derived from the 3 major embryonic lineages (endoderm, mesoderm, ectoderm). These profiles will enable localization of all major HSs present in each tissue type. The profiles, in combination with that of lymphoid cells, will further enable identification of tissue or lineage-restricted HSs and also of sites present in multiple lineages. Third, we will perform confirmatory validation experiments on identified HSs using high-resolution hypersensitivity Southerns. Hypersensitivity Southerns represent an extensively applied 'gold-standard' methodology for identification of HSs. 250 sites per year will be selected from those identified with the high throughput method and validated with this independent methodology. Fourth, the quantitative chromatin profiles produced will be subjected to detailed computational analysis with the aim of determining the correspondence between HSs and evolutionarily-conserved non-coding sequences. Parallel analyses will focus on identification of DNA sequence motifs associated with tissue-specific, lineage-specific, or multilineage HSs. Algorithms for identification and classification of HSs in the human genome will be developed and tested. The performance of selected published algorithms for identification of active regulatory sequences will also be evaluated. It is expected that this research effort will result in the identification of the majority (if not all) of the regulatory elements of the ENCODE regions, and will provide information on the feasibility of the application of this functional approach to the identification of all regulatory elements of the human genome.