The Role of MYST Histone Acetyltransferase in Genome Stability

描述（由申请人提供）：动态蛋白乙酰化对正常细胞生理和发育至关重要。事实上，乙酰转移酶的缺陷与多种人类疾病有关。组蛋白乙酰转移酶的MYST家族是高度保守的，从酵母到人，并且它们作为大的多蛋白质复合物的催化亚基，其结构组成也是保守的。MYST乙酰转移酶是早期哺乳动物胚胎发育所必需的，MYST基因的异常重排或调节与人类癌症相关。我们正在研究MYST家族的两个标志性成员的分子遗传学：芽殖酵母的Esa 1酶和小鼠和人类的Myst 2酶。ESA 1编码芽殖酵母中唯一必需的组蛋白乙酰转移酶。它是两种多蛋白复合物NuA4和picNuA4的催化亚基。我们最近做出了令人惊讶的发现，催化不是Esa 1的基本功能，如以前所认为的那样。相反，我们的数据认为Esa1是一个“分子开关”，它使用辅因子A的结合来控制目前未表征的基本功能。我们将进行旨在了解Esa1的基本功能是做什么的实验，以及它是如何执行的。我们将研究条件突变体的ESA 1，专门暴露其基本功能，并表征表型，基因表达模式，和启动子染色质结构在非许可条件下。我们将专门挑战分子开关模型，使用基因抑制剂和重组picNuA4蛋白质构象的生化测定。这些实验的结果将彻底改变我们对MYST家族蛋白质的看法。Myst 2（Hbo 1）是一种哺乳动物MYST家族酶，作为多蛋白复合物的催化亚基，包括Ing和Jade肿瘤抑制家族的成员。基于我们和其他人的工作，很明显，Myst2是DNA复制许可所必需的，与p53相互作用以介导应激信号，并在转录中发挥作用。我们最近发现，纯合子Myst2基因敲除小鼠胚胎在胚胎日龄E7.5时发育停滞，在这个阶段，快速增殖和广泛的基因重编程即将发生原肠胚形成。Myst2是唯一具有这种敲除表型的MYST基因，我们认为它是DNA复制爆发或基因重编程所必需的。我们将描述野生型、杂合型和纯合型Myst2基因敲除胚胎的基因表达模式，以确定依赖Myst2发育的基因和途径。我们将描述DNA复制许可，S期进展，DNA损伤反应和DNA复制起点的蛋白质占用，以确定Myst2在这个关键发展阶段的参与。最后，我们将利用我们对ESA 1的了解来构建携带突变Myst 2等位基因的条件性敲除小鼠，这将揭示它在发育过程中是否也具有必要的非酶功能。这些实验将大大扩展我们对Myst2在哺乳动物早期发育中调节DNA复制和基因表达的功能的理解。 公共卫生相关性：MYST家族蛋白质复合物执行对于适当的基因表达、DNA复制、DNA损伤修复和胚胎发育至关重要的功能。MYST基因功能的失败与基因组不稳定性和包括人类癌症在内的许多疾病有关。关于它们的功能范围和靶向通路知之甚少。这项申请中提出的研究旨在揭示这些酶如何工作的新原理，它们做什么以及它们如何做到这一点。

DESCRIPTION (provided by applicant): Dynamic protein acetylation is essential for normal cell physiology and development. Indeed, defects in acetyltransferases are associated with a wide variety of human diseases. The MYST family of histone acetyltransferases are highly conserved, from yeast to man, and they serve as the catalytic subunits of large multi-protein complexes whose structural compositions are also conserved. MYST acetyltransferases are required for early mammalian embryonic development and aberrant rearrangements or regulation of MYST genes are associated with human cancers. We are investigating the molecular genetics of two signature members of the MYST family: the Esa1 enzyme of budding yeast, and the Myst2 enzyme of mouse and humans. ESA1 encodes the only essential histone acetyltransferase in budding yeast. It is the catalytic subunit of two multi-protein complexes, NuA4 and picNuA4. We recently made the surprising discovery that catalysis is not the essential function of Esa1, as previously believed. Our data argue, instead, that Esa1 is a "molecular switch" that uses the binding of Cofactor A to control currently uncharacterized essential functions. We will carry out experiments designed to understand what the essential function of Esa1 is doing, and how it is executed. We will study conditional mutants of ESA1 that specifically expose its essential function, and characterize phenotypes, gene expression patterns, and promoter chromatin structure under nonpermissive conditions. We will specifically challenge the molecular switch model using genetic suppressors and biochemical assays of protein conformation in recombinant picNuA4. The results of these experiments are poised to completely change the way we think about MYST family proteins. Myst2 (Hbo1) is a mammalian MYST family enzyme that serves as the catalytic subunit of multi-protein complexes that include members of the Ing and Jade tumor suppressor families. Based on our work and that of others, it is clear that Myst2 is required for DNA replication licensing, interacts with p53 to mediate stress signaling, and has roles in transcription. We recently discovered that homozygous Myst2 knockout mouse embryos arrest development at embryonic day E7.5, a stage at which rapid proliferation and extensive gene reprogramming are about to occur for gastrulation. Myst2 is the only MYST gene with this knockout phenotype and we propose that it is required for the burst of DNA replication, or gene reprogramming at this stage. We will characterize the gene expression pattern of wild type, heterozygous, and homozygous Myst2 knockout embryos to identify the genes and pathways dependent on Myst2 for development. We will characterize DNA replication licensing, S phase progression, DNA damage response, and protein occupancy at DNA replication origins to define the involvement of Myst2 is proliferation at this critical stage of development. Finally, we will use our knowledge of ESA1 to construct conditional knockout mice carrying mutant Myst2 alleles that will reveal if it also has essential non-enzymatic functions during development. These experiments will greatly expand our understanding of Myst2 function in regulating DNA replication and gene expression in early mammalian development. PUBLIC HEALTH RELEVANCE: MYST family protein complexes carry out functions that are essential for proper gene expression, DNA replication, DNA damage repair, and embryonic development. Failures in the function of MYST genes are associated with genome instability and many diseases including human cancers. Little is known about their range of functions and target pathways. The research proposed in this application is designed to uncover new principles in how these enzymes work, what they do, and how they do it.