Engineering histone genes to interrogate the epigenetic code in space and time

描述(申请人提供)：染色质包含DNA序列以外的信息，这是调节基因组和允许多细胞生物体正常发育所必需的。组蛋白的翻译后修饰(PTM)调节染色质的组织，以复制、修复和转录遗传信息，并 被认为是信息的携带者，这些信息是通过表观遗传在多代细胞中遗传的。重要的是，在多细胞真核生物中，组蛋白PTM功能的研究一直受到药物和遗传操作产生的多效性效应的限制，表观遗传调节蛋白产生、移除或响应组蛋白PTM。由于这一限制，关于单个组蛋白残基作为信息载体的作用的知识存在差距。这项建议的目的是在果蝇身上建立一个全面的实验平台，用于在动物发育过程中对翻译后修饰的组蛋白残基进行空间和时间操作。为了研究特定组蛋白PTM的生物学功能，必须将受体残基改变为不能适当修饰的氨基酸。然后，组蛋白基因的所有野生型拷贝都必须被突变拷贝所取代。我们将使用转基因策略，用表达特定组蛋白突变的工程基因簇取代整个果蝇组蛋白多基因家族，并为这些突变的时间和空间限制创造实验工具。这些研究将直接测试翻译后修饰的组蛋白残基在动物发育中的作用，并将为后生动物表观遗传现象的研究创造一个可持续和可扩展的资源。由于进化上保守的组蛋白PTM的破坏被认为是包括癌症在内的许多人类疾病的基础，这项研究将直接影响人类健康。

DESCRIPTION (provided by applicant): Chromatin contains information beyond the DNA sequence that is needed to regulate the genome and allow the proper development of multi-cellular organisms. Posttranslational modifications (PTMs) of histones modulate the organization of chromatin for replication, repair and transcription of genetic information, and are hypothesized to be the carriers of information that is inherited 'epigenetically' over multiple cel generations. Importantly, investigations into histone PTM function in multicellular eukaryotes have been limited by pleiotropic effects resulting from pharmacological and genetic manipulation of epigenetic regulatory proteins that create, remove, or respond to histone PTMs. Because of this limitation, there is a gap in knowledge regarding the role of individual histone residues as carriers of information. The objective of this proposal is to generate a comprehensive experimental platform in Drosophila melanogaster for spatial and temporal manipulation of post-translationally modified histone residues during animal development. To study the biological function of a specific histone PTM, the acceptor residue must be changed to an amino acid that cannot be appropriately modified. Then, all wild-type copies of that histone gene must be replaced with the mutant copy. We will use a transgenic strategy for replacement of the entire Drosophila histone multigene family with engineered gene clusters that express specific histone mutants, and create experimental tools for the temporal and spatial restriction of these mutations. These studies will directly test the roles of post- translationally modified histone residues during animal development, and will create a sustainable and expandable resource for the study of epigenetic phenomena in metazoans. Because disruption of evolutionarily conserved histone PTMs is thought to underlie many human diseases, including cancer, this research will directly impact human health.