Scm3 Is a Centromeric Nucleosome Assembly Factor

Abstract

The Cse4 nucleosome at each budding yeast centromere must be faithfully assembled each cell cycle to specify the site of kinetochore assembly and microtubule attachment for chromosome segregation. Although Scm3 is required for the localization of the centromeric H3 histone variant Cse4 to centromeres, its role in nucleosome assembly has not been tested. We demonstrate that Scm3 is able to mediate the assembly of Cse4 nucleosomes in vitro, but not H3 nucleosomes, as measured by a supercoiling assay. Localization of Cse4 to centromeres and the assembly activity depend on an evolutionarily conserved core motif in Scm3, but localization of the CBF3 subunit Ndc10 to centromeres does not depend on this motif. The centromere targeting domain of Cse4 is sufficient for Scm3 nucleosome assembly activity. Assembly does not depend on centromeric sequence. We propose that Scm3 plays an active role in centromeric nucleosome assembly.

The CENP-A chaperone Scm3 becomes enriched at kinetochores in anaphase independently of CENP-A incorporation

“…[13][14][15] In an alternative model, however, it was proposed that the Scm3-Cse4 interaction is merely important for the incorporation of Cse4 into centromeric chromatin and that Scm3 is not a structural component of centromeres making Scm3 a bona fide loading factor for Cse4. 16,17 More consistent with the latter model, overexpression of CSE4 is able to bypass the essential function of SCM3. 17 Recent data showed that the non-essential E3 ubiquitin ligase Psh1 regulates the stability of excess Cse4.…”

The Composition, Functions, and Regulation of the Budding Yeast Kinetochore

“…One posited that centromeric nucleosomes completely lack H2A and H2B and instead contain two copies of Scm3 (Mizuguchi et al 2007). The demonstration that Scm3 is a chaperone for Cse4/H4 and that Cse4/ H4 cannot simultaneously bind to DNA and Scm3 eliminated this model (Cho and Harrison 2011;Dechassa et al 2011;Shivaraju et al 2011;Xiao et al 2011;Zhou et al 2011). In a revised model, the centromeric nucleosome was proposed to be a tetramer containing two copies of Cse4 and H4 and completely lacking H2A and H2B (Xiao et al 2011).…”

“…Scm3 recognizes Cse4 through the centromere-targeting domain (CATD) in the histone fold and mediates its incorporation into chromatin in vivo and in vitro (Camahort et al 2007;Shivaraju et al 2011). Scm3 also interacts with the Ndc10 component of the CBF3 complex, which can explain the specific deposition of Cse4 at centromeres (Camahort et al 2007).…”

Evolutionary insights into the role of the essential centromere protein CAL1 in Drosophila

“…HJURP selectively recognizes pre-nucleosomal CENP-A from canonical histone H3 and targets it to centromeres, starting in telophase and continuing through G1 Lagana et al 2010). Both HJURP and Scm3 have been shown to possess CENP-A assembly activities in vitro (Dechassa et al 2011;Barnhart et al 2011;Shivaraju et al 2011). In humans, HJURP is recruited to the centromere by M18BP1 (Barnhart et al 2011), a subunit of the Mis18 complex, which is essential for CENP-A incorporation (Fujita et al 2007;Hayashi et al 2004).…”