Single cell analysis of chromatin changes at individual genes

描述（由申请人提供）：在这些研究中，我们提出开发用于对感兴趣的基因组位点的表观遗传变化进行定量单细胞分析的新技术。 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，这些基因表达状态决定了分化、生物体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，能够捕获驻留在异质组织内的单细胞中的染色质变化对于更好地理解生物体发育、衰老和疾病的机制是非常重要的。为体外表观遗传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时应用有限。需要新的有效技术来以单细胞分辨率探测染色质结构。在单个细胞的特定基因组位点上可视化表观遗传标记的方法的发展将促进表观遗传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，并用它来检查动物组织。我们的方法是基于原位生化反应，如果感兴趣的蛋白质与选择用于研究的单个基因或一组基因结合，则在显微镜下产生可见的荧光信号。在该方法中，与链霉亲和素缀合的荧光底物通过生物素化的DNA探针连接到感兴趣的基因组基因座;并且同源酶作为抗体缀合物募集到DNA结合的蛋白质（即修饰的组蛋白）。由于试剂非常接近，酶促反应原位将底物转化为荧光团。通过显微镜检测的荧光信号的强度预期与基因座处的蛋白质密度成比例。 首先，我们将在哺乳动物细胞培养中开发和验证这种方法，在那里我们将研究由有丝分裂原诱导的Egr 1基因组蛋白修饰的变化。 其次，我们将使用这种方法来检查细胞类型特异性和管家基因的表观遗传标记在大鼠肾脏中的细胞分布。该技术将以单细胞分辨率提供肾脏表观遗传景观的3D视图。 我们预计，这种方法将对基础和医学研究的许多领域产生广泛的影响，表观遗传机制在这些领域发挥作用。 公共卫生相关性：表观遗传机制对生物体发育和衰老的调节至关重要，表观遗传网络的改变有助于各种疾病。由于人体器官的复杂性和缺乏有效的方法，体内表观遗传过程的分析是一个挑战。在这项工作中，我们建议开发新的技术，用于定量分析单个细胞中特定基因组位点的表观遗传标记，这将推进人类疾病的表观遗传学研究，并有助于确定新的治疗目标。

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. PUBLIC HEALTH RELEVANCE: Epigenetic mechanisms are crucial for regulation of organism development and aging, and alterations in epigenetic networks contribute to various diseases. Analysis of epigenetic processes in vivo is a challenge due to the complexity of human organs and lack of efficient approaches. In this work we propose to develop novel technology for quantitative analysis of epigenetic marks at specific genomic sites in individual cells that will advance epigenetic research of human diseases, and help to define novel targets for development of treatment.