Functions of the centromere and kinetochore in chromosome segregation
Introduction
Eukaryotic organisms package their genomic DNA into a number of physically distinct chromosomes, which are replicated and equally segregated during each cell cycle. Accurate chromosome segregation requires each chromosome's centromere to build a kinetochore, a complex structure containing at least 100 different proteins that serves as the microtubule-binding site for the mitotic spindle. Correct centromere formation and specification is therefore essential to cell survival.
In contrast to kinetochores, which specifically assemble and function in mitosis, centromeric chromatin, and a group of 17 proteins that bind centromeric chromatin termed the constitutive centromere-associated network (CCAN), are present throughout the cell cycle [1]. A hallmark of centromeric chromatin in all eukaryotes is the presence of nucleosomes that contain the essential H3 variant CENP-A (CENtromere Protein-A) (Box 1). In metazoans, the underlying DNA appears to be mostly dispensable for centromere function. Instead, centromeric proteins epigenetically define each centromere. While the mechanisms of CENP-A assembly are yet to be fully defined, CENP-A is currently the most attractive candidate for the epigenetic mark.
Three broad criteria must be satisfied for centromere replication and function. First, as DNA replication dilutes CENP-A at centromeres, new CENP-A assembly during each cell cycle must maintain the correct amount of CENP-A chromatin. Second, CENP-A must facilitate CCAN protein recruitment to form the centromere. Third, CCAN proteins must provide the molecular platform for kinetochore formation to facilitate chromosome segregation during cell division.
The mechanisms of CENP-A assembly and CENP-A distribution during DNA replication have been extensively reviewed [2, 3, 4, 5, 6, 7, 8, 9]. Therefore, we focus on progress made in our understanding of the precise molecular links between the underlying DNA of the centromere, CENP-A, and the core CCAN. We then discuss how CCAN proteins promote kinetochore formation. Finally, we consider the implications of the recent advances in the understanding of CCAN dynamics.
Section snippets
The DNA–centromere interface: standing on two legs?
A major research focus in recent years has been to establish how core centromere proteins specifically assemble on centromeric DNA to provide a platform for mitotic kinetochore formation. Two constitutive centromere proteins, CENP-N and CENP-C, have been demonstrated to bind directly to CENP-A nucleosomes. CENP-N binds to reconstituted nucleosomes containing CENP-A/H3 chimeras that possess only the CENP-A Targeting Domain (CATD) [10] while CENP-C binds the unique C-terminal tail of CENP-A (
Kinetochore assembly on core CCAN components
In contrast to the CCAN, kinetochore proteins are recruited to centromeres as cells enter mitosis to build the kinetochore for chromosome segregation. Kinetochores attach to spindle microtubules [1], respond to tension generated by stable attachments [30], and when unattached, generate the spindle checkpoint signal that restrains anaphase onset [31]. Here, we focus on advances in our understanding of how the core CCAN components CENP-C and CENP-T recruit the KMN network, which mediates the
Recruitment and role of outer CCAN complexes
In addition to CENP-A, CENP-C, CENP-N and CENP-T/W/S/X, the other CCAN proteins CENP-H, CENP-I, CENP-K, CENP-L, CENP-M, CENP-O, CENP-P, CENP-Q, CENP-R, and CENP-U, form the extended centromere. These proteins can be divided into the CENP-L/M (and N, see Box 2) complex, CENP-H/I/K (the CENP-H complex) and CENP-O/P/Q/R/U (the CENP-O complex), based upon both biochemical interactions and phenotypic analyses (Figure 1c). CENP-N RNAi causes major defects in centromere function [10, 25, 40],
CCAN dynamics
Although we refer to the proteins of the CCAN as constitutive because they are detectable throughout the cell cycle at centromeres, recent data demonstrate that the CCAN is highly dynamic both in individual protein turnover and in overall composition. Both immunofluorescence microscopy and live-cell imaging of EGFP–CENP-N expressing cells suggest CENP-N levels at centromeres increases during S-phase and decreases before mitosis [40, 50]. Fluorescence Recovery After Photobleaching (FRAP) studies
Concluding remarks
Significant progress has been made in the understanding of how the constitutive centromere assembles and functions. CENP-A directly recruits CENP-C and CENP-N to centromeres, and CENP-C, together with CENP-T, recruits the KMN network. In parallel to this, the extended CCAN protein complexes regulate kinetochore microtubule attachments to promote bipolar spindle formation.
While this provides a simplified framework describing centromere function in humans, it is important to note that significant
Note added in proof
While in press, two independent studies were published describing the interaction between CENP-T and the Spc24/25 portion of the Ndc80 complex in Saccharomyces cerevisiae [60•] and in chicken DT40 cells [61•]. These reports confirmed that phosphorylation of CENP-T's N-terminal tail regulates the interaction with Spc24/25, and that CENP-T and the Mis12 complex compete for Spc24/25 binding, potentially revealing phosphorylation dependent changes in Ndc80 complex association with the centromere
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
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