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

7.项目摘要/摘要 真核细胞的染色质景观上装饰着以共价为特征的标志物 修饰和变异核蛋白结构。其中一些染色质标记有助于多产 转录，而其他参与建立和维持沉默的染色质区域。 事实上，超过一半的人类癌症存在编码组蛋白和染色质的基因突变 监管机构强调了解染色质重塑的基本机制的重要性 在牢房里。典型的核小体，它的内核由两个组蛋白H_2A拷贝组成， H_2B、H_3和H_4以及长度为147个碱基对的外部DNA线圈，代表主要的包装单位 细胞染色质。但新出现的证据表明，具有替代性组蛋白化学计量的核蛋白 并且DNA包装配置已经存在。这些不同的包装单元可以显著改变 染色质的生物物理和生化特性，潜在地影响广泛的核功能。 在所提出的研究中，我们专注于一种类似核小体的粒子，称为R-八酶体，在该粒子中其核心 由富含精氨酸的组蛋白H3和H4的八个亚基组成(不含H_2A和H_2B)。尽管它已经 40年前就知道R-八酶体可以在体外组装，但生物学上的相关性尚不清楚。 利用R-octasome的新结构数据，我们将半胱氨酸探针战略性地放置在酵母H3中，并展示了 通过定点定向交联，酵母细胞中存在R-八酶体特有的结构，提供了 首次有证据表明R-八酶体在体内存在。为了进一步研究R-八酶体的性质，在目标1中，我们提出了 开发一种从酵母中纯化天然R-八聚体的方法学，可用于生化，基因组， 和结构研究。为了研究R-octasome的生物学作用，在目标2中，我们将询问R-octasome的作用 八体在端粒基因沉默中的作用，因为对特定位点的化学图谱数据的分析表明，R- 八体与端粒相连。同时，我们将研究R-八酶体作为底物的作用。 依赖三磷酸腺苷的染色质重构体。最后，在目标3中，我们将探索R-octasome在高等- 由于我们的结构数据表明R-八酶体可以使连接成核，所以染色质组织有序 额外的H3和H4组蛋白。总体而言，这项研究的结果将为我们提供新的见解 真核生物利用高度保守的H3和H4作为多功能底物来调节基因组功能。

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.