Massively parallel epigenomics: building new value with current resources

大规模并行表观基因组学：利用现有资源创造新价值

• 批准号: 7774587
• 负责人: Trey Ideker
• 金额: $ 21.84万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7774587
• 关键词: Address; Affect; Aging; Antibodies; Behavior; Binding; Biological Assay; Biomedical Research; Birds; Cell Differentiation process; Cell physiology; Cells; Chromosomes; Custom; DNA Binding; DNA Methylation; DNA Repair; DNA-Binding Proteins; Data; Defect; Development; Diploidy; Disease; Elements; Epigenetic Process; Event; Frequencies; Funding; Future; Gene Activation; Gene Expression; Gene Expression Regulation; Gene Proteins; Gene Silencing; Genes; Genetic; Genetic Markers; Genetic Transcription; Genomics; Goals; Grouping; Health; Histones; Human; Immunoprecipitation; Individual; Instruction; International; Knock-out; Libraries; Location; Malignant Neoplasms; Maps; Measurement; Measures; Methods; Microarray Analysis; Modification; Molecular; Pathology; Pattern; Positioning Attribute; Regulation; Research; Research Personnel; Resources; Reverse Transcriptase Polymerase Chain Reaction; Reverse Transcription; Syndrome; Technology; Testing; Tissue-Specific Gene Expression; United States National Institutes of Health; Variant; Yeasts; base; carcinogenesis; deletion library; disease phenotype; epigenomics; functional genomics; genome-wide; histone modification; homologous recombination; interest; microchip; programs; promoter; public health relevance; research study; stem; technology development; transcription factor

DESCRIPTION (provided by applicant): The goal of this project is to develop platform technology that measures how the epigenome influences transcription factor binding and gene activation at a controlled promoter sequence as the chromosome location is varied. This platform is built around the idea that the value of the existing yeast deletion libraries (Yeast Knock Outs, YKO) can be used for position-effect experiments to measure epigenetic regulation on a genome- wide scale. The availability of thousands of isogenic strains, each of which differs only in the position of a single gene cassette at a distinct chromosomal locus, provides a controlled genetic marker that can be assayed to understand how epigenetic features at different positions influence transcription. YKO libraries will be used in conjunction with molecular barcode microarrays, ChIP-chip, and pooled transformation technologies to measure position effects on transcription factor binding, gene expression, and homologous recombination. The NIH has previously funded each of these individual technologies for functional genomics research. Here, we propose to give them new value by leveraging them for epigenetic studies. Three technologies are proposed: 1) A molecular barcode immunoprecipitation on microchip (BIP-chip) assay that measures genome-wide position effects on binding between a DNA-binding protein and a controlled promoter sequence. Development will involve combining elements of traditional and histone ChIP-chip with elements of quantitative barcode microarrays. 2) Quantitative RT-PCR of the expression level of the kanMX gene as its chromosomal location is varied. Reverse transcription and quantitative PCR will be used to quantify the expression level of kanMX in individual yeast strains in the YKO library covering all of yeast chromosome I. 3) En masse integrative transformation of custom promoter sequences into pooled yeast cultures. Methods for performing pooled transformations developed for diploid-based Synthetic Lethal Analysis by Microarray (dSLAM) will be adapted to investigate position effects on homologous recombination and to enable the use of BIP-chip for evaluating epigenetic effects on any transcription factor (TF). Together, the development of these technologies will enable future research to directly measure the effects of epigenetic regulation on DNA binding and gene activation for any TF-promoter combination of interest. PUBLIC HEALTH RELEVANCE: Epigenetics contribute to critical cellular functions and pathologies ranging from gene silencing and DNA repair to tissue-specific gene expression, cell differentiation, carcinogenesis, and aging. Throughout development, differentiating cells accumulate epigenetic instructions that ultimately determine fully differentiated patterns of expression. Many developmental syndromes, and specific disease phenotypes, including cancer, stem from fundamental epigenetic changes that inactivate critical genes or activate disruptive genes. Identifying disease- causing epigenetic changes and finding ways to mitigate, alter, or reverse deleterious ones will be the subject of biomedical research for the foreseeable future. Combining reference epigenome maps with TF-epigenome interaction measurements, as proposed in this study, will enable researchers to specifically pinpoint epigenetic changes that induce altered TF binding behavior and contribute to developmental defects and disease.

描述(申请人提供)：这个项目的目标是开发平台技术，测量当染色体位置不同时，表观基因组如何影响受控启动子序列上的转录因子结合和基因激活。这个平台是基于这样一个想法，即现有酵母删除文库(酵母敲除，YKO)的价值可以用于位置效应实验，以在全基因组范围内测量表观遗传调节。数以千计的等基因菌株的可用性，其中每个不同的只是在一个不同的染色体位置的单个基因盒的位置不同，提供了一个受控的遗传标记，可以分析，以了解不同位置的表观遗传特征如何影响转录。YKO文库将与分子条形码微阵列、芯片和混合转化技术结合使用，以测量转录因子结合、基因表达和同源重组的位置效应。美国国立卫生研究院此前曾为这些单独的功能基因组研究技术提供资金。在这里，我们建议通过利用它们进行表观遗传学研究来赋予它们新的价值。提出了三种技术：1)基于微芯片的分子条码免疫沉淀法(BIP-CHIP)，它测量基因组范围内DNA结合蛋白与受控启动子序列之间结合的位置效应。开发将涉及将传统芯片和组蛋白芯片的元素与定量条形码微阵列的元素相结合。2)由于kanMX基因的染色体位置不同，定量RT-PCR检测其表达水平。KanMX基因在YKO文库中的表达水平将被用反转录和定量聚合酶链式反应来定量。3)将定制启动子序列整体整合到酵母混合培养物中。为基于二倍体的微阵列合成致死分析(DSLAM)开发的混合转化方法将适用于研究同源重组的位置效应，并使BIP-CHIP能够用于评估对任何转录因子(Tf)的表观遗传效应。总之，这些技术的发展将使未来的研究能够直接测量表观遗传调控对感兴趣的任何TF-启动子组合的DNA结合和基因激活的影响。 公共卫生相关性：表观遗传学有助于关键的细胞功能和病理，从基因沉默和DNA修复到组织特异性基因表达、细胞分化、癌症发生和衰老。在整个发育过程中，分化细胞积累表观遗传指令，最终决定完全分化的表达模式。许多发育综合征和特定的疾病表型，包括癌症，源于根本的表观遗传变化，即使关键基因失活或激活破坏性基因。在可预见的未来，识别致病的表观遗传变化并找到减轻、改变或逆转有害变化的方法将是生物医学研究的主题。将参考表观基因组图谱与TF-表观基因组相互作用测量相结合，将使研究人员能够特别准确地定位导致TF结合行为改变并导致发育缺陷和疾病的表观遗传学变化。