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.
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.
|Chen, Menzies; Licon, Katherine; Otsuka, Rei et al. (2013) Decoupling epigenetic and genetic effects through systematic analysis of gene position. Cell Rep 3:128-37|