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.项目总结/摘要 真核细胞的染色质景观是由以共价键为特征的地标装饰的。 修饰和变异核蛋白结构。这些染色质标记中的一些有助于生产 转录，而其他参与沉默的染色质区域的建立和维持。 事实上，超过一半的人类癌症在编码组蛋白和染色质的基因中存在突变， 调节剂强调了理解染色质重塑的基本机制的重要性 在细胞中。典型的核小体具有由两个组蛋白H2 A拷贝组成的内核， H2 B、H3和H4以及长度为147 bp的外部DNA螺旋，代表了 细胞染色质但新的证据表明，具有不同组蛋白化学计量的核蛋白 并且存在DNA缠绕构型。这些变体包装单元可以显著改变 染色质的生物物理和生物化学性质，可能影响广泛的核功能。 在拟议的研究中，我们专注于核小体样粒子，称为R-八体，其中其核心 由富含丝氨酸的组蛋白H3和H4的八个亚基组成（不含H2 A和H2 B）。虽然 已知R-八体可以在体外组装已有>40年，但生物学相关性尚不清楚。 使用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，酵母细胞中存在对R-八体特异的结构， 第一个证明R-八体存在于体内的证据。为了进一步研究R-八体的性质，在目的1中，我们提出了 开发一种从酵母中纯化天然R-八体的方法，该方法可用于生物化学，基因组学， 结构研究。为了研究R-octasomes的生物学作用，在目的2中，我们将询问R-octasomes的作用。 八体在端粒基因沉默中的作用，因为位点特异性化学作图数据的分析表明， 八体与端粒相连。与此同时，我们将研究R-八体如何作为底物发挥作用， ATP依赖性染色质重塑。最后，在目标3中，我们将探讨R-八体在高等生物学中的作用。 有序染色质组织，因为我们的结构数据表明，R-八体可以使连接成核 额外的H3和H4组蛋白。总的来说，这项研究的结果将提供新的见解，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。