CENP-T-W-S-X Forms a Unique Centromeric Chromatin Structure with a Histone-like Fold

Abstract

Summary The multi-protein kinetochore complex must assemble at a specific site on each chromosome to achieve accurate chromosome segregation. Defining the nature of the DNA-protein interactions that specify the position of the kinetochore and provide a scaffold for kinetochore formation remain key goals. Here, we demonstrate that the centromeric histone-fold containing CENP-T-W and CENP-S-X complexes co-assemble to form a stable CENP-T-W-S-X heterotetramer. High-resolution structural analysis of the individual complexes and the heterotetramer reveals similarity to other histone fold-containing complexes including canonical histones within a nucleosome. The CENP-T-W-S-X heterotetramer binds to and supercoils DNA. Mutants designed to compromise heterotetramerization or the DNA-protein contacts around the heterotetramer strongly reduce the DNA binding and supercoiling activities in vitro and compromise kinetochore assembly in vivo. These data suggest that the CENP-T-W-S-X complex forms a unique nucleosome-like structure to generate contacts with DNA, extending the “histone code” beyond canonical nucleosome proteins.

Human centromere genomics: now it's personal

“…Apart from CENP-B, which binds specifically to a 17-bp motif (CENP-B box) (Masumoto et al 1989), the remaining inner kinetochore proteins appear to bind centromeric DNA in a complex manner independent of a clear DNA binding site. From the perspective of the underlying DNA, these interactions introduce a combination of multi-protein interactions assuming sequence flexibility to tolerate the induced supercoiling, spacing, and bending necessary for nucleosome wrapping to accommodate centromere-specific chromatin (Furuyama and Henikoff 2009;Hori et al 2008;Nishino et al 2012). In addition to direct sequence-based interactions with inner kinetochore proteins, centromeric sequences also accommodate a hierarchy of constitutive and dynamic protein-based interactions that bridge the inner kinetochore and the outer kinetochore in a protein-mediated transfer of tension from spindle forces to those proteins that directly bind DNA (Hori et al 2008;Screpanti et al 2011).…”

The Centromere: Epigenetic Control of Chromosome Segregation during Mitosis

“…3). In vitro, a heterotetramer of CENP-T, -W, -S, and -X was proposed to protect 100 bp of DNA from nuclease digestion, in a similar manner to H3 and CENP-A nucleosomes (Nishino et al 2012). More recently, a CENP-T/W/S/X octamer has been shown to bind 100 bp of DNA and potentially induce positive supercoils (as opposed to negative supercoils induced by nucleosomes) (Takeuchi et al 2014).…”

“…5). Several models have been proposed for a "placeholder," a marking mechanism that identifies where to assemble new CENP-A nucleosomes, including naked DNA, histone H3.3 nucleosomes (Dunleavy et al 2011), hybrid CENP-A/H3.3 nucleosomes, a CENP-T/W/ S/X complex (Nishino et al 2012), hemisomes (Bui et al 2012), or an unidentified component. The chromatin-associated factors that dictate the positions of new CENP-A assembly will most likely be targets for some of the assembly factors discussed above.…”

Principles and practice of nucleosome positioningin vitro

“…A number of other protein complexes have since been shown to share the same histone fold complex structure, although not all of these necessarily bind DNA (Kamada et al 2001;Hartlepp et al 2005;Nishino et al 2012).…”

“…A large majority of nucleosome crystal structures are based on a palindromic 73 bp region from the human alpha satellite repeat (Harp et al 1996). However, the functionally relevant nucleosome positioning is unclear and complicated by active debate over the repeating chromatin unit size, the wrapping topology, and whether relevant centromeric chromatin is wrapped as either H3 and CENP-A containing nucleosomes or both (Tachiwana et al 2011), or possibly even other histone fold structures of centromeric proteins (Nishino et al 2012).…”