Abstract
ABSTRACTAlthough it is generally accepted that chromatin containing the histone H3 variant CENP-A is an epigenetic mark maintaining centromere identity, the pathways leading to the formation and maintenance of centromere chromatin remain unclear. We previously generated human artificial chromosomes (HACs) whose centromeres contain a synthetic alpha-satellite (alphoid) DNA array containing the tetracycline operator (alphoidtetO). We also obtained cell lines bearing the alphoidtetO array at ectopic integration sites on chromosomal arms. Here, we have examined the regulation of CENP-A assembly at centromeres as well as de novo assembly on the ectopic arrays by tethering tetracycline repressor (tetR) fusions of substantial centromeric factors and chromatin modifiers. This analysis revealed four classes of factors that influence CENP-A assembly. Interestingly, many kinetochore structural components induced de novo CENP-A assembly at the ectopic site. We showed that these components work by recruiting CENP-C and subsequently recruiting M18BP1. Furthermore, we found that CENP-I can also recruit M18BP1 and, as a consequence, enhances M18BP1 assembly on centromeres in the downstream of CENP-C. Thus, we suggest that CENP-C and CENP-I are key factors connecting kinetochore to CENP-A assembly.
Highlights
The kinetochore directs accurate chromosome segregation by controlling chromosome movements through interactions with spindle microtubules, and by serving as a platform for various regulatory pathways
Tethering tetracycline repressor (tetR)-EYFP–HJURP significantly increased the CENP-A signal on the human artificial chromosomes (HACs), whereas tethering tetR-EYFP–Suv39h1 caused a corresponding decrease (Fig. 1D)
The CENPA recruitment caused by CENP-C domain III might not depend only on the interaction with M18BP1. These results suggest that CENP-C induces de novo CENP-A assembly through at least two M18BP1 recruitment pathways: CENP-I recruitment by domain II and directly through its C terminus (Fig. 7I)
Summary
The kinetochore directs accurate chromosome segregation by controlling chromosome movements through interactions with spindle microtubules, and by serving as a platform for various regulatory pathways. The interphase centromere complex (ICEN) associates with the CENP-A nucleosome (Izuta et al, 2006; Obuse et al, 2004), and the. The CCAN factors CENP-C (Saitoh et al, 1992) and CENP-T act as a crucial platform for the kinetochore during mitosis (Gascoigne et al, 2011; Hori et al, 2008, 2013; Nishino et al, 2013; Przewloka et al, 2011; Rago et al, 2015). The KMN network, composed of KNL1, the Mis complex and the NDC80 complex is the main microtubule-binding component of the outer kinetochore (Cheeseman et al, 2006; DeLuca et al, 2006)
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