Single cell analysis of chromatin changes at individual genes

描述(由申请人提供)：在这些研究中，我们建议开发新的技术，用于对感兴趣的基因组位置的表观遗传变化进行定量的单细胞分析。 表观遗传机制，包括组蛋白和DNA修饰，对于建立和维持基因表达状态至关重要，基因表达状态决定了分化、生物发育和衰老过程中不同的细胞表型。在疾病中，染色质结构的异常变化发生在一小群细胞、特定细胞类型或整个器官中。因此，能够捕捉异质组织中单个细胞的染色质变化对于更好地了解生物发育、衰老和疾病的机制是非常重要的。为体外表观遗传学分析开发的强大技术，如染色质免疫沉淀(CHIP)，在研究由多种不同细胞类型组成的组织方面的应用有限。在新的有效技术中需要在单细胞分辨率下探测染色质结构。在单个细胞的特定基因组位置可视化表观遗传标记的方法的发展将促进表观遗传学研究的许多领域的研究，包括发育生物学、衰老和人类疾病。在这些研究中，我们将开发这样一种方法，并将其用于检查动物组织。我们的方法是基于原位生化反应，如果感兴趣的蛋白质I与选定用于研究的单个基因或一组基因结合，就会产生在显微镜下可见的荧光信号。在这种方法中，通过生物素化的DNA探针将与链霉亲和素结合的荧光底物连接到感兴趣的基因组位置，并将同源酶招募到DNA结合的蛋白质(即修饰的组蛋白)上作为抗体结合物。在试剂非常接近的情况下，酶反应在原位将底物转化为荧光团。显微镜检测到的荧光信号强度预计与基因座上蛋白质的密度成正比。首先，我们将在哺乳动物细胞培养中开发和验证这种方法，在那里我们将检测由有丝分裂原诱导的Egr1基因组蛋白修饰的变化。其次，我们将使用这种方法来检测表观遗传标记在细胞类型特定和看家基因上在大鼠肾脏中的细胞分布。这项技术将在单细胞分辨率下提供肾脏表观遗传景观的3D视图。我们预计，这种方法将对许多基础和医学研究领域产生广泛影响，在这些领域，表观遗传机制发挥着作用。

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.