Functions of Mammalian Histone Modifiers

哺乳动物组蛋白修饰剂的功能

• 批准号: 6594658
• 负责人: SHARON Y. R. DENT
• 金额: $ 29.18万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6594658
• 关键词: DNA repair; acetylation; acyltransferase; alleles; apoptosis; cell differentiation; developmental genetics; embryonic stem cell; enzyme activity; enzyme mechanism; genetically modified animals; genomic imprinting; laboratory mouse; mammalian embryology; point mutation; polymerase chain reaction; posttranslational modifications; terminal nick end labeling; tissue /cell culture; transcription factor

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.

描述（由申请人提供）：翻译后修饰组蛋白的酶活性对于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抑制相关。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) 活性控制。尽管在过去几年中已经描述了许多具有这些活性的转录共激活子和辅阻遏物，但这些酶在细胞分化转录程序中的作用目前尚未得到充分研究。尽管特定 HAT 和 HDAC 的突变与结肠癌和白血病等人类疾病有关，但这些酶的小鼠基因中几乎没有产生或鉴定出突变。 Gcn5 是第一个被鉴定的核 HAT 活性，它可以作为 HAT 结构和功能的有用范例。 此前，我们证明小鼠 Gcn5 缺失会导致原肠胚形成后胚胎死亡，并导致特定中胚层谱系的缺失。这些谱系正常，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 Gcn5 对正常发育的重要性，但 Gcn5 无效胚胎的死亡妨碍了在后期胚胎时间点对 Gcn5 功能的分析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 Gcn5 在特定发育阶段的重要性 2) 使用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) 确定是否存在发育缺陷嵌合动物中细胞凋亡和凋亡是细胞自主的。 4) 确定 Gcn5 缺失对 Gcn5 缺失胚胎干细胞中细胞生长、转录和 DNA 修复的分子影响。这些研究将提供有关该 HAT 在哺乳动物发育过程中的功能的重要且新颖的信息，并将为长期遗传学研究提供一个跳板，以确定该组蛋白修饰活性和其他组蛋白修饰活性在正常和患病状态下的功能。