The Role of MYST Histone Acetyltransferase in Genome Stability

描述（由申请人提供）：动态蛋白质乙酰化是正常细胞生理和发育所必需的。事实上，乙酰转移酶的缺陷与多种人类疾病有关。MYST家族的组蛋白乙酰转移酶是高度保守的，从酵母到人类，它们是大型多蛋白复合物的催化亚基，其结构组成也是保守的。MYST乙酰转移酶是哺乳动物早期胚胎发育所必需的，MYST基因的异常重排或调控与人类癌症有关。我们正在研究MYST家族的两个标志性成员：出芽酵母的Esa1酶和小鼠和人类的mys2酶的分子遗传学。ESA1编码出芽酵母中唯一必需的组蛋白乙酰转移酶。它是两个多蛋白复合物NuA4和picNuA4的催化亚基。我们最近有了一个惊人的发现，催化并不是Esa1的基本功能，正如我们之前认为的那样。相反，我们的数据表明，Esa1是一种“分子开关”，它利用辅因子a的结合来控制目前尚未表征的基本功能。我们将进行实验来了解Esa1的基本功能是什么，以及它是如何执行的。我们将研究ESA1的条件突变，具体暴露其基本功能，并在非许可条件下表征表型、基因表达模式和启动子染色质结构。我们将利用基因抑制因子和重组picNuA4蛋白构象的生化分析来挑战分子开关模型。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 （Hbo1）是哺乳动物MYST家族的一种酶，作为包括Ing和Jade肿瘤抑制家族成员在内的多蛋白复合物的催化亚基。根据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敲除小鼠胚胎在胚胎期E7.5停止发育，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，我们认为这是DNA复制爆发或基因重编程所必需的。我们将对野生型、杂合型和纯合型Myst2基因敲除胚胎的基因表达模式进行表征，以确定Myst2依赖于发育的基因和途径。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在这一关键发育阶段的增殖参与。最后，我们将利用ESA1的知识构建携带突变Myst2等位基因的条件敲除小鼠，以揭示其在发育过程中是否也具有必要的非酶功能。这些实验将极大地扩展我们对Myst2在哺乳动物早期发育中调控DNA复制和基因表达的功能的理解。

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.