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

7. 项目总结/文摘

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.