Analyses of structure and dynamics with model chromatin

Tachiwana, Hiroaki*; Arimura, Yasuhiro*; Matsumoto, Ryohei*; Takizawa, Yoshimasa*; Hori, Tetsuya*; Matsumoto, Atsushi; Oda, Takashi*; Sato, Mamoru*; Kono, Hidetoshi; Fukagawa, Tatsuo*; Ohi, M. D.*; Kurumizaka, Hitoshi*

Centromeres are specific region of chromosomes, which ensure equal chromosome segregation in mitosis. The histone H3 variant, CENP-A, is a key protein for the establishment and maintenance of centromeres. However, little is known about how the incorporation of CENP-A into the chromatin leads to the centromere formation. We reported that a nucleosome containing human CENP-A (CENP-A nucleosome) has different structural feature, by which the CENP-A nucleosome may contribute to organize a unique chromatin structure in centromeres1. Then, we further analysed whether CENP-A nucleosome affects the poly-nucleosome structure using reconstituted tri-nucleosomes. To enable this, we reconstituted the tri-nucleosome, in which single CENP-A nucleosome is arranged at the centre, and two H3 nucleosomes are arranged on the both sides (H3-CA-H3 nucleosomes), by anucleosome-ligation method. Then, we analyzed structures of H3-CA-H3 nucleosomes and control H3-H3-H3 nucleosomes by dynamic light scattering, small angle X-ray scattering, and electron microscopic analyses. We found that single CENP-A nucleosome significantly alters higher order chromatin configuration. Based on these analyses, we propose a chromatin structure containing CENP-A nucleosomes and discuss the advantage of this structure in centromere formation and maintenance.