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: 7568716
• 负责人: YIE-HWA CHANG
• 金额: $ 27.91万
• 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/7568716
• 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的共价结合而被修饰。组蛋白生物素化在组蛋白修饰中是独特的，因为代谢辅因子也起染色质标记的作用。我们已经确定了10个不同的生物素化位点在组蛋白H2 A，H3和H4。已知存在其他生物素化位点，但只是暂时确定。组蛋白的生物素化由全羧化酶合成酶（HCS）介导，并且具有重要的生物学功能，例如，在逆转录转座子的抑制、异染色质结构和DNA修复以及基因调控中。由于我们对组蛋白生物素化位点的不完全理解，缺乏针对某些生物素化位点的抗体，以及我们对组蛋白生物素化与其他表观遗传标记之间的串扰缺乏了解，目前对组蛋白生物素化的全谱生物学功能的阐明进展缓慢。在这里，我们建议通过利用具有不同研究领域专业知识的调查人员的独特组合来克服这些障碍：一位蛋白质生物化学家，他也是质谱实验室的负责人（Chang），一位在基因调控的表观遗传机制方面具有广泛专业知识的遗传学家（Zemsenberg），以及一位发现组蛋白生物素化并在该领域开发了许多独特工具的营养学家（Zempleni）。该项目的长期目标是确定组蛋白生物素化维持基因组稳定性的途径。具体目标：（1）在目标1中，我们将通过使用LC/MS/MS鉴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，我们将产生针对新生物素化位点的抗体。这些抗体将是必不可少的，用于在未来的研究，以确定组蛋白生物素化的生物学意义。(2)在目标2中，我们将测试生物素化与人类相同组蛋白分子上的其他修饰标记共存的假设。我们将产生抗体的组蛋白与多种修饰的选择的例子。通过在果蝇多线染色体中绘制这些修饰的组蛋白亚型，将产生对生物学功能的初步认识。(3)我们实验室正在进行的项目表明，HCS与H3 K9-甲基转移酶相互作用，K12-生物素化组蛋白H4与K9-二甲基化组蛋白H3共定位。在这里，我们将测试的假设，在人类细胞和苍蝇的HCS敲低减少K9 Me 3 H3在选定的基因座的丰度，并增加在这些基因座的K4 Me 3 H3的丰度。我们还将测试组蛋白的生物素化在K9-甲基转移酶的敲低、突变和/或过表达时降低的假设。大多数这些突变和转基因生物都在我们的实验室或免费提供。 公共卫生关系：组蛋白的生物素化是一种独特的表观遗传标记，因为它依赖于膳食中必需维生素生物素的摄入。生物素缺乏症在美国人中很普遍，在高达50%的孕妇中观察到中度生物素缺乏症。先前的研究表明，组蛋白的生物素化在逆转录转座子的抑制中起着关键作用，从而降低逆转录转座子、染色体异常和可能的癌症风险的发生率。