Epigenetic Control of the Cell Cycle During Animal Development

摘要 在细胞增殖期间基因组的准确复制对于正常动物发育是必要的， 体内平衡复制的调节或保真度的破坏有助于许多人类病理， 尤其是癌症。因此，对基因组复制机制的完整理解是 对人类健康至关重要。像人类细胞中发现的大基因组的复制需要启动 在每条染色体上的数千个独立位置进行双向DNA合成。它还需要细胞 大量组蛋白的周期调节合成，将新复制的DNA包装成 染色质这个项目将集中在如何染色质组装和组织影响基因组复制 在动物的发展过程中。染色质的基本组成部分是核小体，它是组蛋白的八聚体 由约147个碱基对的DNA组成的蛋白质。染色质内的大多数组蛋白是合成的 细胞周期的每个S期来自复制依赖性组蛋白基因，其编码唯一的真核生物 以茎环而不是聚腺苷酸尾结束的mRNA。编码所有五种RD-组蛋白的基因是 聚集在后生动物基因组中，以及组蛋白所需的转录和前mRNA加工因子 mRNA生物合成被组织成核体（组蛋白基因座体或HLB），其在 这些基因簇。我们将确定RD-组蛋白的配位合成的要求 在果蝇中使用生物化学和遗传方法的mRNAs，特别关注 HLB在组蛋白转录和前mRNA加工中起作用。每个核心组蛋白的N-末端 从核小体核心突出并经历多种化学修饰的尾部（例如， 甲基化，乙酰化和磷酸化），调节染色质组织，从而影响所有 基因组功能的各个方面，包括DNA复制。我们在果蝇中开发了一种方法， 工程化任何所需的组蛋白尾部突变，为我们提供了一种防止特定组蛋白修饰的方法。 组蛋白残基，从而操纵染色质组织的方式在任何其他动物中都是不可用的。 这种遗传方法将与细胞生物学和下一代DNA测序方法相结合， 确定染色质组织如何调节整个基因组中的DNA复制，从而 解决这个领域的一个重大问题。我们的果蝇实验范式允许在体内 对基因表达和DNA复制中这些基本过程的研究， 在其他实验系统中不可用。

Abstract Accurate replication of the genome during cell proliferation is necessary for normal animal development and homeostasis. Disruption of the regulation or fidelity of replication contributes to many human pathologies, particularly cancer. Thus, a complete understanding of the mechanisms governing genome replication is paramount to human health. Replication of large genomes like that found in human cells requires the initiation of bi-directional DNA synthesis at thousands of individual locations on each chromosome. It also requires cell cycle-regulated synthesis of large amounts of histone proteins to package newly replicated DNA into chromatin. This project will focus on how chromatin assembly and organization influences genome replication during animal development. The basic building block of chromatin is the nucleosome, an octamer of histone proteins encompassed by ~147 base pairs of DNA. Most histone proteins within chromatin are synthesized each S phase of the cell cycle from replication-dependent histone genes, which encode the only eukaryotic mRNAs that end in a stem loop rather than a poly A tail. The genes encoding all five RD-histone proteins are clustered in metazoan genomes, and transcription and pre-mRNA processing factors required for histone mRNA biosynthesis are organized into a nuclear body (the Histone Locus Body or HLB) that assembles at these gene clusters. We will determine the requirements for the coordinate synthesis of the RD-histone mRNAs using both biochemical and genetic approaches in Drosophila, with a particular focus on the role that the HLB plays in histone transcription and pre-mRNA processing. Each core histone protein has an N-terminal tail that protrudes from the nucleosome core and is subject to a variety of chemical modifications (e.g. methylation, acetylation, and phosphorylation) that modulate chromatin organization and thus influence all aspects of genome function, including DNA replication. We have developed a method in Drosophila for engineering any desired histone tail mutation, providing us a means of preventing modification of specific histone residues and thus of manipulating chromatin organization in a way not available in any other animal. This genetic approach will be combined with cell biological and next generation DNA sequencing methods to determine how chromatin organization modulates DNA replication throughout the entire genome, thereby addressing a major question in the field. Our Drosophila experimental paradigm permits the in vivo interrogation of these fundamental processes in gene expression and DNA replication in ways that are unavailable in other experimental systems.