CCAN Makes Multiple Contacts with Centromeric DNA to Provide Distinct Pathways to the Outer Kinetochore

Abstract

Kinetochore specification and assembly requires the targeted deposition of specialized nucleosomes containing the histone H3 variant CENP-A at centromeres. However, CENP-A is not sufficient to drive full-kinetochore assembly, and it is not clear how centromeric chromatin is established. Here, we identify CENP-W as a component of the DNA-proximal constitutive centromere-associated network (CCAN) of proteins. We demonstrate that CENP-W forms a DNA-binding complex together with the CCAN component CENP-T. This complex directly associates with nucleosomal DNA and with canonical histone H3, but not with CENP-A, in centromeric regions. CENP-T/CENP-W functions upstream of other CCAN components with the exception of CENP-C, an additional putative DNA-binding protein. Our analysis indicates that CENP-T/CENP-W and CENP-C provide distinct pathways to connect the centromere with outer kinetochore assembly. In total, our results suggest that the CENP-T/CENP-W complex is directly involved in establishment of centromere chromatin structure coordinately with CENP-A.

Human centromere genomics: now it's personal

“…The length and spatial organization of inner kinetochore proteins within these domains are considered critical for proper secondary chromosomal structure, kinetochore assembly, and interactions with the spindle apparatus (Blower et al 2002;Hori et al 2008). These observations emphasize an additional genomic pressure to organize centromere-competent sequences within a broader, yet regional, context, resulting in a measure of centromere-domain efficiency.…”

“…b These arrays, although capable of differentiating maternally and paternally derived chromosomes, are observed to have stable, Mendelian transmission within the context of three-generation pedigree studies DNA contact of a subset of constitutive, inner kinetochore proteins, including CENP-A, CENP-B, CENP-C, CENP-T/W, and CENP-S/X (Fig. 4) (Hori et al 2008). 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.…”

“…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).…”

“…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). Moreover, proper assembly of pericentromeric heterochromatin, typically correlated with functional kinetochores, may be influenced by sequence-based factors, such as transcriptional regulation and specialized chromatin compaction (Folco et al 2008).…”

The Centromere: Epigenetic Control of Chromosome Segregation during Mitosis

“…CENP-S/X plays noncentromeric roles in DNA repair independently of CENP-T/W (Singh et al 2010;Tao et al 2012;Zhao et al 2014), CENP-S/X depletion does not affect CENP-T centromere recruitment (Amano et al 2009), and CENP-S is dispensable in engineered neocentromeres that are positive for CENP-T in chicken DT-40 cells ). One possibility is that binding of CENP-S/X to CENP-T/W confers some centromere-specific function, but the ability of CENP-T to function independently from CENP-S suggests CENP-T/W/S/X does not form an obligate nucleosome-like particle (Hori et al 2008a;Amano et al 2009). Indeed, before identification of the CENP-T/W/S/X tetramer, CENP-T/W was suggested to associate primarily with histone H3 rather than CENP-A (Hori et al 2008a;Ribeiro et al 2010).…”