Massively parallel epigenomics: building new value with current resources

描述（由申请人提供）：该项目的目标是开发平台技术，该技术测量当染色体位置变化时，表观基因组如何影响受控启动子序列处的转录因子结合和基因激活。该平台是围绕现有酵母缺失文库（Yeast Knock Outs，YKO）的价值可用于位置效应实验以在全基因组范围内测量表观遗传调控的想法构建的。数以千计的等基因菌株的可用性，其中每一个仅在不同染色体基因座处的单个基因盒的位置上不同，提供了受控的遗传标记，其可以被测定以了解不同位置处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、ChIP芯片和合并转化技术结合使用，以测量位置对转录因子结合、基因表达和同源重组的影响。美国国立卫生研究院以前曾为功能基因组学研究资助过这些单独的技术。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）微芯片上的分子条形码免疫沉淀（BIP-芯片）测定，其测量对DNA结合蛋白和受控启动子序列之间的结合的全基因组位置效应。 开发将涉及传统和组蛋白ChIP芯片的元素与定量条形码微阵列的元素相结合。2)kanMX基因的表达水平的定量RT-PCR，因为其染色体位置是变化的。 逆转录和定量PCR将用于定量覆盖所有酵母染色体I的YKO文库中单个酵母菌株中kanMX的表达水平。3)将定制启动子序列整合转化到合并的酵母培养物中。 用于执行针对基于二倍体的通过微阵列的合成致死分析（dSLAM）开发的合并转化的方法将适于研究位置对同源重组的影响，并且使得能够使用BIP芯片来评估对任何转录因子（TF）的表观遗传影响。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任何感兴趣的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、致癌和衰老。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，都源于基本的表观遗传变化，这些变化会破坏关键基因或激活破坏性基因。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。结合参考表观基因组图谱与TF-表观基因组相互作用的测量，如本研究所提出的，将使研究人员能够特异性地确定诱导TF结合行为改变并导致发育缺陷和疾病的表观遗传变化。

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.