Histone Modifications During Embryogenesis

描述（由申请人提供）：拟议研究的长期目标是确定胚胎发生过程中染色质如何调节基因表达。重点是与发育控制基因相关的两种组蛋白修饰的作用。组蛋白H3 （H3K27me3）赖氨酸27的三甲基化通常与沉默基因相关，而组蛋白H3 （H3K4me3）赖氨酸4的三甲基化通常与活跃或允许被激活的基因相关。对多能胚胎细胞的研究表明，在这些基因表达之前，这两个标记都与发育控制基因相关。这些二价H3K4me3/H3K27me3标记被认为有助于发育基因表达的定量、时间或空间精度，但该领域缺乏能够直接测试这些假设的技术。本项目旨在(1)开发一种在特定基因上诱导特异性组蛋白修饰的方法，(2)应用该技术确定二价组蛋白标记在斑马鱼胚胎发生过程中的作用。该方法涉及设计具有组蛋白甲基转移酶或去甲基化酶活性的锌指融合蛋白。这些蛋白将针对特定基因改变H3K4或H3K27甲基化。然后，这项技术将用于分析在特定基因上破坏或引入H3K4me3或H3K27me3标记的后果。由于特定的组蛋白修饰伴随着所有真核生物的发育和生理状态，开发一种基因特异性调节组蛋白标记的技术将有助于阐明这种修饰在许多情况下的作用。提出的技术还可以对研究和调节疾病过程产生直接影响。例如，组蛋白异常修饰与几种癌症有关。组蛋白修饰的局部操作可能有助于确定许多失调基因中的哪一个负责肿瘤的形成。甚至有可能使用所提出的技术专门激活疾病保护基因或抑制疾病诱导基因。

DESCRIPTION (provided by applicant): The long-range goal of the proposed studies is to determine how chromatin modulates gene expression during embryogenesis. The focus is on the role of two histone modifications that are associated with developmental control genes. Trimethylation of lysine 27 of histone H3 (H3K27me3) is commonly associated with silenced genes, whereas trimethylation of lysine 4 of histone H3 (H3K4me3) is commonly associated with genes that are active or permissive to be activated. Studies in pluripotent embryonic cells have revealed that both these marks are associated with developmental control genes before these genes are expressed. These bivalent H3K4me3/H3K27me3 marks have been proposed to contribute to the quantitative, temporal or spatial precision of developmental gene expression, but the field lacks a technology that would allow a direct test of these hypotheses. This project aims to (1) develop a method to induce specific histone modifications at defined genes and (2) apply this technology to determine the role of bivalent histone marks during zebrafish embryogenesis. The method involves the design of zinc finger fusion proteins that have histone methyltransferase or demethylase activities. These proteins will be targeted to defined genes to change H3K4 or H3K27 methylation. The technology will then be used to analyze the consequences of disrupting or introducing H3K4me3 or H3K27me3 marks at specific genes. Since specific histone modifications accompany developmental and physiological states in all eukaryotes, developing a technology for the gene-specific modulation of histone marks would help elucidate the role of such modifications in numerous settings. The proposed technology could also have direct impact in studying and modulating disease processes. For example, abnormal histone modifications have been implicated in several cancers. Local manipulation of histone modifications might help to determine which of the many misregulated genes are responsible for tumor formation. It might even become possible to use the proposed technology to specifically activate disease-protective genes or repress disease-inducing genes.