Novel histone biotinylation sites and relationships to other epigenetic marks

描述(由申请人提供)： 已知组蛋白是通过维生素生物素的共价结合来修饰的，组蛋白的生物素化在组蛋白修饰中是独一无二的，因为代谢辅助因子也起着染色质标记的作用。我们已经在组蛋白H_2A、H_3和H_4中确定了十个不同的生物素化位点。已知存在其他生物素化位点，但仅初步确定。组蛋白的生物素化是由全羧酶合成酶(HCS)介导的，在抑制反转录转座子、异染色质结构和DNA修复以及基因调控等方面具有重要的生物学功能。目前，由于我们对组蛋白生物素化位点的不完全了解，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，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：一位蛋白质生物化学家同时也是一家质谱学实验室(Chang)的负责人，一位在基因调控的表观遗传机制方面拥有广泛专业知识的遗传学家(Eissenberg)，以及一位发现组蛋白生物素化并开发出该领域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，我们将利用LC/MS/MS鉴定活细胞中所有组蛋白和变异体中所有自然发生的生物素化位点，此外，我们还将产生针对新的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)在目标2中，我们将检验生物素化与人类同一组蛋白分子上的其他修饰标记共存的假设。我们将通过多次修饰来产生针对选定的组蛋白实例的抗体。首先，通过绘制果蝇多线染色体中这些修饰的组蛋白亚型图，将产生对生物功能的洞察。(3)我们实验室正在进行的项目表明，HCS与H3K9-甲基转移酶相互作用，K12-生物素化的组蛋白H4与K9-二甲基化组蛋白H3共定位。在这里，我们将检验一种假设，即在人类细胞和果蝇中敲除HCS会降低选定基因座上K9Me3H3的丰度，并增加这些基因座上K4Me3H3的丰度。我们还将验证组蛋白的生物素化降低的假设，以响应K9-甲基转移酶的敲除、突变和/或过表达。这些突变和转基因生物中的大多数都在我们的实验室中或免费获得。 与公共卫生相关：组蛋白的生物素化是一个独特的表观遗传学标志，因为它取决于膳食中必需的维生素生物素的摄入量。生物素缺乏症在美国人中很普遍，高达50%的孕妇出现了中度生物素缺乏症。以前的研究表明，组蛋白的生物素化在抑制反转录转座子中起着关键作用，从而减少反转录转座、染色体异常的发生率，并可能降低癌症风险。

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.