Functions of Mammalian Histone Modifiers

描述（由申请人提供）：翻译后修饰组蛋白的酶活性是基因表达调控的核心。目前研究得最好的组蛋白修饰是赖氨酸残基的乙酰化。增加的组蛋白乙酰化伴随着基因激活，而降低的乙酰化与基因抑制有关。乙酰化水平受组蛋白乙酰转移酶（HAT）和组蛋白去乙酰化酶（HDAC）活性的影响。尽管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，但这些酶在细胞分化转录程序中的作用目前还没有得到充分的研究。尽管特定的hat和hdac的突变与包括结肠癌和白血病在内的人类疾病有关，但这些酶的小鼠基因中很少有突变被创造或鉴定出来。Gcn5是第一个被确定的核HAT活性，它可以作为HAT结构和功能的有用范例。先前，我们证明了小鼠Gcn5的缺失会导致原肠胚形成后的胚胎死亡，并导致特定中胚层谱系的缺失。这些谱系通常是指定的，但在妊娠7.5天后由于细胞凋亡增加而失效。这些实验证明了Gcn5对正常发育的重要性，但Gcn5缺失胚胎的死亡阻碍了Gcn5在胚胎后期时间点的功能分析或机制研究，以确定细胞凋亡增加的分子基础。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)利用一个条件等位基因确定Gcn5在特定发育阶段的重要性；2)利用在催化中心携带点突变的等位基因确定Gcn5 HAT活性对发育的重要性；3)确定嵌合体动物的发育缺陷和细胞凋亡是否自主发生；4)确定Gcn5缺失对Gcn5无源胚胎干细胞的细胞生长、转录和DNA修复的分子效应。这些研究将提供关于该HAT在哺乳动物发育过程中的功能的重要和新颖的信息，并将为长期遗传学研究提供跳板，以确定该和其他组蛋白修饰活性在正常和患病状态下的功能。

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.