Functions of Mammalian Histone Modifiers

描述（由申请人提供）：后修饰组蛋白的酶活性对基因表达的调节至关重要。目前研究最多的组蛋白修饰是赖氨酸残基的乙酰化。组蛋白乙酰化增加伴随着基因激活，而乙酰化减少则与基因阻遏有关。乙酰化水平由相反的组蛋白乙酰转移酶（HAT）和组蛋白脱乙酰酶（HDAC）活性决定。虽然在过去的几年中已经描述了一些转录辅激活因子和辅抑制因子，这些酶在细胞分化的转录程序中的作用目前研究不足。这些酶的小鼠基因中很少有突变被创建或鉴定，尽管特定HAT和HDAC中的突变与包括结肠癌和白血病在内的人类疾病相关。Gcn5是第一个被鉴定的核HAT活性，它是HAT结构和功能的有用范例。 以前，我们证明了Gcn 5在小鼠中的损失导致胚胎死亡后不久，原肠胚形成，与特定的中胚层谱系的损失。这些谱系是正常的，但在妊娠7.5天后由于细胞凋亡增加而失败。这些实验证明了Gcn 5对正常发育的重要性，但是Gcn 5无效胚胎的死亡排除了在以后的胚胎时间点分析Gcn 5功能或确定细胞凋亡增加的分子基础的机制研究。这里的实验将利用新开发的遗传工具来解决这些问题。我们的具体目标是1）使用条件（floxed）等位基因确定Gcn 5在特定发育阶段的重要性2）使用在催化中心携带点突变的等位基因确定Gcn 5 HAT活性对发育的重要性3）确定发育缺陷和凋亡在嵌合动物中是否是细胞自主的4）确定Gcn 5缺失对细胞生长、转录、Gcn 5缺失胚胎干细胞中的DNA修复。这些研究将提供重要的和新的信息，这种HAT在哺乳动物发育过程中的功能，并将提供一个跳板，长期的遗传研究，以确定在正常和疾病状态下的功能，这和其他组蛋白修饰活动。

DESCRIPTION (provided by applicant): Enzymatic activities that post-translationally modify the histones are central to the regulation of gene expression. The best studied histone modification at present is acetylation of lysine residues. Increased histone acetylation accompanies gene activation, whereas decreased acetylation is associated with gene repression. Acetylation levels are governed by opposing histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities. Although a number of transcriptional coactivators and corepressors that house these activities have been described in the last few years, the role of these enzymes in transcriptional programs of cellular differentiation is currently understudied. Few mutations in mouse genes for these enzymes have been created or identified, although mutations in specific HATs and HDACs are associated with human diseases including colon cancer and leukemias. Gcn5 was the first nuclear HAT activity to be identified, and it serves as a useful paradigm for HAT structure and function. Previously, we demonstrated that loss of Gcn5 in mouse causes embryonic lethality just after gastrulation, with a loss of particular mesodermal lineages. These lineages are specified normally but failafter 7.5 days of gestation due to increased apoptosis. These experiments demonstrate the importance of Gcn5 to normal development, but the death of Gcn5 null embryos precludes analysis of Gcn5 functions at later embryonic time points or mechanistic studies to determine the molecular basis of the increased apoptosis. Experiments here will make use of newly developed genetic tools to address these questions. Our specific aims are to 1) Determine the importance of Gcn5 at specific developmental stages using a conditional (floxed) allele 2) Determine the importance of Gcn5 HAT activity to development using alleles that carry point mutations in the catalytic center 3) Determine whether developmental defects and apoptosis are cell autonomous in mosaic animals 4) Determine the molecular effects of Gcn5 loss on cell growth, transcription, and DNA repair in Gcn5 null embryonic stem cells. These studies will provide important and novel information about the functions of this HAT during mammalian development and will provide a springboard for long term genetic studies to define the functions of this and other histone modifying activities in normal and diseased states.