Novel histone biotinylation sites and relationships to other epigenetic marks

新型组蛋白生物素化位点及其与其他表观遗传标记的关系

• 批准号: 7895893
• 负责人: YIE-HWA CHANG
• 金额: $ 25.64万
• 依托单位: UNIVERSITY OF NEBRASKA LINCOLN
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-20 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7895893
• 关键词: Acetylation; Affect; American; Antibodies; Area; Binding; Biological; Biological Process; Biotin; Biotinylation; Cataloging; Catalogs; Cells; Chromatin; Chromosome abnormality; Chromosomes; Chromosomes, Human, Pair 3; DNA Repair; Dietary intake; Drosophila genus; Enzymes; Epigenetic Process; Event; Frequencies; Future; Gene Expression Regulation; Genes; Genome Stability; Goals; Head; Heterochromatin; Histone H1; Histone H1(s); Histone H2A; Histone H3; Histone H4; Histones; Human; Incidence; Incubated; K-18 conjugate; Laboratories; Life; Ligase; Lysine; Maps; Mediating; Metabolic; Methylation; Modification; Mutation; Nutritionist; Organism; Orthologous Gene; Pathway interactions; Peptides; Play; Pregnant Women; Protein Isoforms; Proteins; Public Health; Repression; Research; Research Personnel; Retrotransposition; Retrotransposon; Role; SECTM1 gene; SU(VAR)3-9; Site; Structure; Testing; Transferase; Transgenic Organisms; Variant; Vitamins; base; biotin 1; cancer risk; fly; histone modification; holocarboxylase synthetases; insight; mutant; novel; overexpression; polyclonal antibody; public health relevance; response; tool

DESCRIPTION (provided by applicant): Histones are known to be modified by covalent binding of the vitamin biotin Histone biotinylation is unique among histone modifications in that a metabolic co-factor also functions as a chromatin mark. We have identified ten distinct biotinylation sites in histones H2A, H3, and H4. Additional biotinylation sites are known to exist but have only been tentatively identified. Biotinylation of histones is mediated by holocarboxylase synthetase (HCS) and has important biological functions, e.g., in the repression of retrotransposons, in heterochromatin structures and DNA repair, and in gene regulation. The elucidation of the full spectrum of biological functions of histone biotinylation is currently slowed by our incomplete understanding of histone biotinylation sites, the lack of availability of antibodies to some biotinylation sites, and our lack of understanding of the crosstalk between histone biotinylation and other epigenetic marks. Here, we propose to overcome these obstacles by taking advantage of a unique combination of investigators with expertise in diverse areas of research: a protein biochemist who also is the head of a mass spec laboratory (Chang), a geneticist with extensive expertise in epigenetic mechanisms of gene regulation (Eissenberg), and a nutritionist who discovered histone biotinylation and developed many unique tools in this field (Zempleni). The long-term objective of this project is to identify pathways by which histone biotinylation maintains genome stability. Specific aims: (1) In aim 1, we will identify all naturally occurring biotinylation sites in all histones and variants in living cells by using LC/MS/MS. In addition, we will generate antibodies to novel biotinylation sites. These antibodies will be essential for use in future studies to determine the biological importance of histone biotinylation. (2) In aim 2 we will test the hypothesis that biotinylation co-exists with other modification marks on the same histone molecule in humans. We will generate antibodies to selected examples of histones with multiple modifications. First insights into biological functions will be generated by mapping these modified histone isoforms in Drosophila polytene chromosomes. (3) Ongoing projects in our laboratories suggest that HCS interacts with H3 K9-methyl transferases and that K12-biotinylated histone H4 co-localizes with K9- dimethylated histone H3. Here we will test the hypothesis that HCS knockdown in human cells and flies decreases the abundance of K9Me3H3 at selected loci, and increases the abundance of K4Me3H3 at these loci. We will also test the hypothesis that biotinylation of histones decreases in response to knockdown, mutation, and/or overexpression of K9-methyl transferases. Most of these mutant and transgenic organisms are in our laboratories or freely available. PUBLIC HEALTH RELEVANCE: Biotinylation of histones is a unique epigenetic mark because it depends on the dietary intake of the essential vitamin biotin. Biotin deficiency is prevalent among Americans, and moderate biotin deficiency has been observed in up to 50% of pregnant women. Previous studies suggest that biotinylation of histones plays a critical role in the repression of retrotransposons, thereby decreasing the incidence of retrotranspositions, chromosomal abnormalities, and probably cancer risk.

描述（由申请人提供）： 已知组蛋白通过维生素生物素组蛋白生物素化的共价结合在组蛋白修饰中是独一无二的，因为代谢辅助因子也可以用作染色质标记。我们已经确定了组蛋白H2a，H3和H4中的十个不同的生物素化位点。已知其他生物素化位点存在，但仅暂时鉴定。组蛋白的生物素化是由全羧化酶合成酶（HCS）介导的，并且具有重要的生物学功能，例如，在逆转录转座子的抑制中，在异染色质结构和DNA修复中以及基因调节中。目前，由于我们对组蛋白生物素化位点的不完全了解，缺乏对某些生物素化位点的抗体的可用性，以及我们对组蛋白生物素化和其他表观遗传学分子之间的串扰缺乏理解，阐明了组蛋白生物素化的全部生物学功能的阐明速度减慢了。在这里，我们提议通过利用在不同研究领域具有专业知识的研究人员的独特组合来克服这些障碍：蛋白质生物化学家也是群众特定实验室（CHANG）的负责人（CHANG），这是一位具有广泛的基因调节表观遗传机制专业知识（Eissenberg）（Eissenberg）的专业知识（Eissenberg）的专业知识（Eissenberg），并且是一种自来具型的构造，并发现了许多统一的历史培训。该项目的长期目标是识别组蛋白生物素化维持基因组稳定性的途径。具体目的：（1）在AIM 1中，我们将使用LC/MS/MS确定活细胞中所有组蛋白和变体中所有天然存在的生物素化位点。此外，我们将生成与新型生物素化位点的抗体。这些抗体将在未来的研究中使用，以确定组蛋白生物素化的生物学重要性。 （2）在AIM 2中，我们将测试以下假设：生物素化与人类同一组蛋白分子的其他修饰标记并存。我们将生成与具有多个修饰的组蛋白示例的选定示例的抗体。通过映射果蝇多丹尼染色体中的这些改良的组蛋白同工型，将产生对生物学功能的首先见解。 （3）我们实验室中正在进行的项目表明，HCS与H3 K9-甲基转移酶相互作用，并且K12-Biotinylated组蛋白H4与K9-二甲基化组蛋白H3共定位。在这里，我们将测试以下假设：人类细胞中的HCS敲低和苍蝇降低了选定基因座的K9ME3H3的丰度，并增加了这些基因座的K4ME3H3的丰度。我们还将检验以下假设：组蛋白的生物素化响应于K9-甲基转移酶的敲低，突变和/或过表达而降低。这些突变体和转基因生物大多数都在我们的实验室中或自由使用。 公共卫生相关性：组蛋白的生物素化是一个独特的表观遗传标记，因为它取决于必需维生素生物素的饮食摄入。在美国人中，生物素缺乏症普遍存在，并且在多达50％的孕妇中观察到了中度的生物素缺乏症。先前的研究表明，组蛋白的生物素化在抑制逆转座子中起着至关重要的作用，从而降低了逆转录倾角的发生率，染色体异常和可能的癌症风险。