Abstract: The chromatin landscape of eukaryotic cells is decorated with landmarks characterized by covalent modifications and variant nucleoprotein structures. In this proposed project, we aim to investigate the R-octasome, a nucleosome-like particles but with a core made up of four H3 and H4 histones. We recently solved the cryo-EM structure of the R-octasome and detected its presence in yeast using in vivo crosslinking techniques. However, the biological function of R-octasomes remains unknown. Preliminary data suggest that R-octasomes are associated with the telomeric repeats. Using a mutant that disrupts R-octasome formation, our data suggest that its integrity is crucial for sub-telomeric gene silencing. We hypothesize that R-octasomes are integral components of telomeric heterochromatin. We proposed three aims to further study the biological roles of the R-octasome. Aim 1: Develop a methodology to purify native R-octasomes from yeast for biochemical, genomic, and structural studies. Aim 2: Investigate the role of R-octasomes in telomeric gene silencing and their function as substrates of ATP-dependent chromatin remodelers. Aim 3: Explore the role of R-octasomes in higher-order chromatin organization. Our structural data suggest that R-octasomes can nucleate the concatenation of additional H3 and H4 histones. Overall, the outcome of this research will provide new insights into how eukaryotes use the highly conserved H3 and H4 as multi-functional substrates to modulate genomic functions.