Uncovering the biomolecular function of the R-octasome--a nucleosome-like particle with only H3 and H4 histones

揭示 R-octasome 的生物分子功能——一种仅含有 H3 和 H4 组蛋白的核小体样颗粒

• 批准号: 10657120
• 负责人: Edward E Luk
• 金额: $ 31.63万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10657120
• 关键词: Affect; Affinity; Antibodies; Arginine; Binding; Biochemical; Biological; Biological Process; Biology; Biophysics; Cancer Biology; Cell Nucleus; Cell Proliferation; Cell physiology; Cells; Chemicals; Chromatin; Chromatin Fiber; Chromatin Modeling; Chromatin Structure; Collaborations; Cryoelectron Microscopy; Cysteine; DNA; Data; Data Set; Dimerization; Electron Microscopy; Eukaryota; Eukaryotic Cell; Gene Expression; Gene Silencing; Genes; Genetic; Genetic Transcription; Genome; Genomics; Grant; Heterochromatin; Histone H2A; Histone H3; Histone H4; Histones; Human; In Vitro; Left; Length; Link; Maintenance; Malignant Neoplasms; Maps; Methodology; Minor; Modification; Molecular; Molecular Conformation; Mutagenesis; Mutation; N-terminal; Nature; Negative Staining; Normal Cell; Nuclear; Nuclease Protection Assays; Nucleoproteins; Nucleosomes; Outcomes Research; Paper; Positioning Attribute; Productivity; Property; Publishing; Recombinants; Resolution; Role; Saccharomyces cerevisiae; Sedimentation process; Shapes; Site; Structure; Testing; Therapeutic Intervention; Variant; Yeasts; adduct; chromatin remodeling; crosslink; dimer; gene function; in vivo; insight; mutant; new therapeutic target; novel; particle; reconstitution; stoichiometry; telomere; tumor; tumor progression

7. PROJECT SUMMARY/ABSTRACT The chromatin landscape of eukaryotic cells is decorated with landmarks characterized by covalent modifications and variant nucleoprotein structures. Some of these chromatin marks contribute to productive transcription, while others are involved in the establishment and maintenance of silenced chromatin regions. The fact that more than half of human cancers have mutations in genes encoding histones and chromatin regulators underscores the importance of understanding the fundamental mechanism of chromatin remodeling in cells. The canonical nucleosome, which has an inner core consisted of two copies each of histone H2A, H2B, H3, and H4 and an outer DNA coil of 147 bp in length, represents the predominant packaging unit of cellular chromatin. But emerging evidence suggests that nucleoproteins with alternative histone stoichiometries and DNA wrapping configurations are present. These variant packaging units can significantly alter the biophysical and biochemical properties of chromatin, potentially affecting a wide spectrum of nuclear functions. In the proposed studies, we focus on a nucleosome-like particle, called the R-octasome, in which its core is made up of eight subunits of the arginine-rich histone H3 and H4 (without H2A and H2B). Although it has been known for >40 years that R-octasomes can be assembled in vitro, the biological relevance is unknown. Using new structural data of the R-octasome, we strategically placed cysteine probes in yeast H3 and showed by site-directed crosslinking that structures specific to R-octasomes are present in yeast cells, providing the first evidence that R-octasomes exist in vivo. To further study the nature of R-octasomes, in Aim 1, we propose to develop a methodology to purify native R-octasomes from yeast that can be used for biochemical, genomic, and structural studies. To study the biological roles of R-octasomes, in Aim 2, we will interrogate the role of R- octasomes in telomeric gene silencing, as analysis of site-specific chemical mapping data suggest that R- octasomes are linked to telomeres. In parallel, we will investigate how R-octasomes function as substrates of ATP-dependent chromatin remodelers. Finally, in Aim 3, we will explore the role of R-octasomes in higher- order chromatin organization, as our structural data suggest that R-octasomes can nucleate the concatenation of additional H3 and H4 histones. Overall, the outcome of this research will give new insights into how eukaryotes use the highly conserved H3 and H4 as multi-functional substrates to modulate genomic functions.

7. 项目概要/摘要 真核细胞的染色质景观装饰有共价标记 修饰和变异核蛋白结构。其中一些染色质标记有助于提高生产力 转录，而其他参与沉默染色质区域的建立和维持。 超过一半的人类癌症的组蛋白和染色质编码基因发生突变 监管机构强调了解染色质重塑基本机制的重要性 在细胞中。典型的核小体，其内核由组蛋白 H2A 的两个副本组成， H2B、H3 和 H4 以及长度为 147 bp 的外部 DNA 线圈代表了 细胞染色质。但新出现的证据表明，具有替代组蛋白化学计量的核蛋白 并存在 DNA 包裹结构。这些不同的包装单元可以显着改变 染色质的生物物理和生化特性，可能影响广泛的核功能。 在拟议的研究中，我们重点关注一种称为 R-octasome 的核小体样颗粒，其中其核心 由富含精氨酸的组蛋白 H3 和 H4 的八个亚基组成（不含 H2A 和 H2B）。虽然它有 40 年前人们就知道 R-octasomes 可以在体外组装，但其生物学相关性尚不清楚。 利用 R-八体的新结构数据，我们策略性地将半胱氨酸探针放置在酵母 H3 中，并显示 通过定点交联，酵母细胞中存在 R-octasome 特有的结构，从而提供 第一个证据表明 R-octasomes 存在于体内。为了进一步研究 R-octasomes 的性质，在目标 1 中，我们建议 开发一种从酵母中纯化天然 R-octasomes 的方法，可用于生化、基因组、 和结构研究。为了研究 R-octasomes 的生物学作用，在目标 2 中，我们将探讨 R-octasomes 的作用 端粒基因沉默中的八体，对位点特异性化学作图数据的分析表明，R- 八体与端粒相连。同时，我们将研究 R-octasomes 如何作为底物发挥作用 ATP 依赖性染色质重塑剂。最后，在目标 3 中，我们将探讨 R-octasomes 在更高层次中的作用。 有序染色质组织，因为我们的结构数据表明 R-octasomes 可以使串联成核 额外的 H3 和 H4 组蛋白。总体而言，这项研究的结果将为如何 真核生物使用高度保守的 H3 和 H4 作为多功能底物来调节基因组功能。