Single cell analysis of chromatin changes at individual genes

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DESCRIPTION (provided by applicant): In these studies we propose to develop novel technology for quantitative single cell analysis of epigenetic changes at genomic loci of interest. Epigenetic mechanisms, including histone and DNA modifications, are critical for establishing and maintenance of gene expression states that determine distinct cell phenotypes during differentiation, organism development, and aging. In disease, aberrant changes in chromatin structure occur in a small group of cells, specific cell types or entire organs. Therefore capabiliy to capture chromatin changes in single cells residing within heterogeneous tissues is very important to better understand mechanisms of organism development, ageing and disease. Powerful techniques developed for epigenetic analysis in vitro, such as chromatin immunoprecipitation (ChIP), have limited application to study tissues composed of multiple distinct cell types. There is need in novel efficient technologies to probe chromatin structure at single cell resolution. Development of approaches for visualizing epigenetic marks at specific genomic sites in individual cells will facilitate studies in many areas of epigenetic research, including developmental biology, aging, and human diseases. In these studies we will develop such a method and use it to examine animal tissues. Our approach is based on biochemical reaction in situ that produces fluorescent signal visible under microscope if protein of interest i bound to an individual gene or a group of genes selected for the study. In this method, fluorogenic substrate conjugated to streptavidin is tethered to a genomic locus of interest via biotinylated DNA probe; and the cognate enzyme is recruited to a DNA-bound protein (i.e. modified histone) as an antibody conjugate. Given close proximity of the reagents, enzymatic reaction in situ convert substrate to a fluorophore. Intensity of fluorescent signal detected by microscopy is expected to be proportional to the density of protein at the locus. First, we will develop and validate this method in mammalian cell culture, where we will examine changes in histone modification at Egr1 gene induced by a mitogen. Second, we will use this method to examine cellular distribution of epigenetic marks at cell-type specific and house-keeping genes in rat kidneys. This technology will provide a 3D view at epigenetic landscape in kidneys at single cell resolution. We anticipate that this method will have a broad impact on many areas of basic and medical research, where epigenetic mechanisms play a role.