Family matters: structural and functional conservation of centromere-associated proteins from yeast to humans

TICB-948; No. of Pages 10

Review

Family matters: structural and functional conservation of centromereassociated proteins from yeast to humans Stefan Westermann1 and Alexander Schleiffer2 1 2

Research Institute of Molecular Pathology (IMP), Dr. Bohr Gasse 7, 1030 Vienna, Austria IMP/IMBA Bioinformatics Core Facility, Research Institute of Molecular Pathology (IMP), Dr. Bohr Gasse 7, 1030 Vienna, Austria

Kinetochores form the fundamental link between chromosomal domains termed centromeres and spindle microtubules in all eukaryotes. This connection, provided by a large, multiprotein complex, is essential for precise chromosome segregation and thus ensures genetic stability. Here, we review recent insights into the composition and function of centromeric chromatin. Multiple approaches have converged to identify centromere-associated proteins from yeast to humans. Among them are newly characterized histone-fold family members that operate at the DNA–kinetochore interface and provide critical connections between chromosomes and microtubules. Together, these findings contribute to a unified view of how centromeric chromatin functions as a regulated scaffold for kinetochore assembly. Introduction All eukaryotes copy and distribute their genetic information in each cell division, ensuring that their progeny contains the correct blueprint for an entire organism. The cell accomplishes this process in different ways; following replication, sister chromatids are held together via the cohesin protein complex allowing their sorting as pairs on the mitotic spindle. After each pair is connected to opposite sides of the spindle – a process termed bi-orientation – cohesion is removed and sisters are segregated to the poles. Throughout this dynamic process, chromosomes undergo a complex array of different movements: initial capture on the side of microtubules, congression along fibers, sorting and oscillatory movements on the metaphase plate, and finally concerted pulling toward the spindle poles. It is an extraordinary achievement of the mitotic machinery that these highly complex movements almost always result in successful segregation of each chromosome pair. Kinetochores form the connection between two biological polymers: microtubules and DNA. Microtubules, formed by polymerization of ab tubulins, are highly conserved cytoskeletal elements. On the primary amino acid level, ab Corresponding author: Westermann, S. ([email protected]). Keywords: mitosis; CCAN; kinetochore; nucleosome; histones. 0962-8924/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tcb.2013.01.010

tubulins from yeast and humans are over 80% identical. Kinetochore–microtubule attachment sites among evolutionary distant organisms therefore differ only in the number of attached microtubules (one in budding yeast, two to four in fission yeast, 25–40 in human cells) but little in the structure of the polymer itself. Reflecting this conservation, essential parts of the kinetochore at this interface, in particular the KNL-1 MIS12 NDC80 (KMN) network, are highly conserved. The Ndc80 complex, for example, contacts the microtubule surface directly and point mutations in conserved amino acids of the microtubule-binding calponin homology domain of Ndc80 disrupt the attachment of chromosomes to the spindle in humans and yeast [1–4]. By contrast, all centromeres are marked by CENP-A (Box 1) but centromere DNA is highly divergent between species, bearing no resemblance in length or sequence among evolutionarily distant yeasts such as Saccharomyces cerevisiae and Schizosaccharomyces pombe or between yeast and human. A fundamental question is, therefore, how can highly divergent DNA sequences direct the assembly of conserved microtubule attachment machinery and how is this chromosomal domain maintained from one generation to the next? Discovery and conservation of centromere-associated proteins Centromere sequences were first discovered in yeast by exploiting their ability to direct the segregation of circular and linear DNA [5]. Proteins binding to these short, linear DNA sequences were isolated biochemically and constituted the first centromere-associated proteins [6]. The ‘point’ centromeres of budding yeast, however, deviate strongly from the regional centromeres of higher eukaryotes and the occurrence of protein complexes that bind to them in a sequence-specific manner is restricted to fungi (Box 2). To isolate proteins specifically enriched at vertebrate centromeres, recent approaches made use of the fact that eukaryotic centromeres are epigenetically marked by nucleosomes in which the canonical histone H3 is replaced by CENP-A (also called CenH3 or Cse4 in budding yeast). Comparative affinity purifications of CENP-A versus H3 nucleosomes from human cells [7,8] have established a group of at least 15 polypeptides that associates specifically Trends in Cell Biology xx (2012) 1–10

1

TICB-948; No. of Pages 10

Review

Trends in Cell Biology xxx xxxx, Vol. xxx, No. x

Box 1. CENP-A – the determinant of centromere identity and kinetochore assembly Centromeres are epigenetically marked by the presence of nucleosomes in which the canonical histone H3 is replaced by a variant termed CENP-A (yeast Cse4, Drosophila Cid). In humans, incorporation of novel CENP-A occurs in the G1 phase of the cell cycle and requires the CENP-A-specific chaperone HJURP [58], (Scm3 in budding yeast [64]) as well as additional factors such as Mis18 [65] and MgcRacGAP [66]. The structure of an octameric nucleosome assembled with CENP-A is similar to that of a H3 nucleosome, although with DNA entry and exit points appearing more accessible [67]. Structural differences within the histone fold, located in the a2 and a3 helices [termed the CENP-A targeting domain (CATD)] are required for the specific recognition of CENP-A nucleosomes by HJURP and thus for their deposition at centromeres. Binding of Scm3/HJURP to CENP-A is incompatible with CENP-A-H4 heterotetramer formation [68], demonstrating that HJURP cannot be part of the assembled CENP-A nucleosome. The CATD is also required for binding of the CCAN subunit CENP-N [69], which might be an important step through which CENP-A nucleosomes guide the assembly of the CCAN. The second CCAN subunit to specifically recognize CENP-A nucleosomes is CENP-C [70]. This interaction is independent of the CATD and instead requires the extreme Cterminal residues of CENP-A [71]. The specific recruitment of CENPC allows the in vitro assembly of kinetochores on synthetic chromatin templates in Xenopus extracts [71]. The structure of the centromeric nucleosome as it appears in vivo in the context of an assembled kinetochore is a matter of debate. The finding that only 80 bp of DNA are protected from MNase digest at yeast centromeres has been interpreted to be incompatible with a nucleosome that wraps DNA in a canonical manner [72]. Combined with biophysical data characterizing Drosophila and human CENP-A and biochemical assays indicating that centromeric nucleosomes might wrap DNA in a nonconventional left-handed manner in vitro, this observation has led to the proposal that these nucleosomes may contain only single copies of CENP-A, H4, H2A, and H2B, thus forming ‘hemisomes’. A possible way to reconcile the disparate models is that centromeric nucleosomes may change their structure between hemisomes and octamers in a cell cycle-dependent manner [73,74].

with centromeres (Table 1). Reciprocal and additional purifications have expanded the repertoire of centromere proteins in diverse organisms [9–13] and allowed a preliminary grouping into subcomplexes based on biochemical interactions and common localization dependencies [11,14]. The term constitutive centromere-associated network (CCAN) is now generally used to describe these proteins, although, as discussed below, it might be more accurate to replace ‘constitutive’ with ‘conserved’. Although the architecture of and connectivity among the CCAN proteins have not yet been fully established, several observations indicate that the CENP-A nucleosomes are an integral structural part of the inner kinetochore architecture. First, depletion of CCAN members leads in many cases to a reduction of CENP-A levels at centromeres [10]. An extreme example of this interdependency is found in the yeast Candida albicans, where depletion of the microtubule-binding outer kinetochore protein Dam1 leads to near-complete loss of CENP-A at the centromere [15]. Second, purification of the budding yeast KMN subunit Dsn1 from soluble extracts leads to coisolation of large assemblies of kinetochore components including Cse4CENP-A. This suggests that CENP-A nucleosomes can remain stably associated with other kinetochore components in the absence of DNA or chromatin [16]. Historically, the second major discovery tool for centromere proteins has been genetic screening in model organisms such as yeast [17,18]. This has been designed to detect proteins that enhance the transmission fidelity of circular or linear chromosomes (isolating ctf or mcm mutants in budding yeast, mis mutants in fission yeast). Although several phylogenetic relationships between CCAN members were established early [19], for other subunits, such as CENP-U/Ame1, their detection has required advanced bioinformatic tools, combining remote homology searches

Table 1. Overview of CCAN subunits in humans and budding yeast CCAN module

Human subunit CEN nucleosome CENP-A Histone H4 Histone H2A Histone H2B CENP-C CENP-C CCAN-HF

MNL

HIK

OPQUR

2

Budding yeast homolog Cse4 Hhf1/Hhf2 Hta1/Hta2 Htb1/Htb2 Mif2

Centromere localization dependencies Depends on HJURP/Scm3 – – – Depends on CENP-A/Cse4

CENP-T CENP-W CENP-S

Cnn1 Wip1 Mhf1

Depends on CENP-A Depends on CENP-T Depends on CENP-T

CENP-X CENP-N

Mhf2 Chl4

Depends on CENP-T Depends on CENP-A

CENP-L CENP-M CENP-H CENP-I CENP-K CENP-O CENP-P CENP-Q

Iml3 ? Mcm16 Ctf3 Mcm22 Mcm21 Ctf19 Okp1

CENP-U CENP-R

Ame1 ?

Common functions

Refs

Centromere specification and propagation

[58,75,76]

Recognizes CENP-A nucleosome, associates with Mis12 complex of KMN Associates with Ndc80-C of KMN Heterodimerizes with CENP-T Forms (CENP-S-CENP-X)2 tetramer, CENP-S-X-T-W heterotetramer Associates with FancM Associates with CENP-A nucleosome, Chl4 required for de novo assembly of centromere

[53,54,70,77,78] [20,46] [20,79] [41,80] [42,43] [69,81]

Depends on CENP-C, CENP-T Required for Ndc80 localization

[62,82]

Depends on CENP-C, CENP-T

[22] Okp1 essential for viability, CENP-Q binds microtubules? Ame1 essential for viability

[18,62]

TICB-948; No. of Pages 10

Review

Trends in Cell Biology xxx xxxx, Vol. xxx, No. x

with detailed secondary structure predictions, due to the lack of a high degree of sequence similarity among these proteins [20]. Structural features of centromere-associated proteins Figure 1 provides an overview of CCAN subunits based on secondary structure predictions, sequence conservation

scores between the respective human and yeast proteins, and structural domains. CCAN subunits lack catalytic domains, although CENP-M has a GTPase fold in which key residues are mutated, predicting that the protein is enzymatically inactive (A. Schleiffer and S. Westermann, unpublished). The main function of CCAN proteins is to provide the structural framework for the assembly of a

CENP-C

CENP-O

CCCC

CCCC

CENP-P

CCCC

CCCC

CENP-Q

CCCCCC

CENP-U

CCCCCCCCCC

CENP-R

CENP-H

CCCCC

CCCCCCCCCC CCCC

CCC

CCC

CCCCCCC

CENP-I

CENP-K

CCCCCCCCC

CCCC

CENP-A

CENP-T Key: CENP-W

Sequence conservaon CENP-T N-term mof CENP-T C-term mof

CENP-S

Histone core domain Cupin domain

CENP-X

α-helix β-strand Disordered coiled-coil CCC

CENP-M

CENP-N

100 AA

CENP-L TRENDS in Cell Biology

Figure 1. Conservation and structural features of constitutive centromere-associated network (CCAN) proteins. Schematic representations of the proteins indicate the distribution of secondary structural elements (JPRED), disordered regions (DISOPRED), and predicted coiled-coil domains (COILS). At the top, to evaluate the significance of the structural features, are plotted conservation scores between human and yeast proteins based on multiple sequence alignments. The cupin domain of CENP-C, the histone folds, and conserved motifs in CENP-T are highlighted.

3

TICB-948; No. of Pages 10

Review kinetochore. In this regard, it should be noted that many budding yeast CCAN subunits are not essential under standard laboratory conditions. This structural redundancy implies that the cell can assemble ‘reduced’ versions of kinetochores that retain the basic chromosome segregation function, whereas robustness and highfidelity performance require assembly of the entire machinery. This is consistent with the observation that some organisms, like Caenorhabditis elegans and Drosophila melanogaster, have lost most CCAN components and rely solely on CENP-A and -C for inner kinetochore assembly. Examining the secondary structures, the CCAN subunits CENP-C, -T, and, to a lesser extent, CENP-U, stand out because large segments of these proteins are predicted to be disordered. The most extreme example of this is CENP-C, where the disordered region has few interruptions and spans the entire length of the protein with the exception of a cupin fold at the C terminus. CENP-C is located close to the top of the assembly hierarchy of inner kinetochore proteins and might act as a scaffold that recruits multiple different downstream effectors to centromeres [21]. The disordered regions of CENP-U and -T are also localized at the N termini of the respective proteins, in CENP-T extending up to the histone-fold domain, whereas in CENP-U they are followed by a conserved coiled-coil domain that is likely to be important for association with other proteins of the CENP-Q, -P, -O, and -R group [22,23]. The most common structural feature of CCAN proteins is an alpha-helical coiled coil. Five proteins – CENP-Q, CENP-U, CENP-R, CENP-H, and CENP-K – are predicted to form mainly alpha helices and the coiled-coil region is a major, often central component of the conserved domain. Notably, the sequence of secondary-structure elements is identical between CENP-O and -P, and indeed, the crystal structure of the Kluyveromyces lactis Ctf19CENP-OMcm21CENP-P heterodimer illustrates the structural conservation between the two proteins [24]. An important implication from recent work is that six kinetochore proteins – Ctf19, Mcm21, Csm1 [25], Spc24, Spc25 [26], and Mad1 [27] – contain ‘RWD’ domains as fundamental building blocks. This versatile structural element can be combined into homodimers (in the case of Csm1 as part of the Monopolin complex, in the case of the checkpoint protein Mad1 via homodimerization of the C terminus) or heterodimers (Ctf19-Mcm21 or the Spc24-25 domain of the Ndc80 complex). In addition to the histone folds in CENP-T, -W, -S, and X, only the cupin domain in CENP-C is annotated as a defined globular structure. This well-conserved, ninestranded beta-jelly roll is used as a dimerization interface [28]. The DNA-binding activities of CCAN subunits are not fully characterized and apart from histone-fold domains in various subunits, which are discussed below, the only annotated domain is an ‘AT-hook’ DNA-binding motif. This is a small (nine amino acids) motif enriched in positively charged residues that binds to AT-rich DNA [29]. It was first described in high-mobility group (HMG) protein I [30] and is present in CENP-C [28] as well as in the CENP-U and -Q proteins of some species. 4

Trends in Cell Biology xxx xxxx, Vol. xxx, No. x

New histone-fold proteins at the centromere Eukaryotes compact and regulate their genomes with the help of histone proteins. The canonical octameric nucleosome, comprising histones H2A, H2B, H3, and H4, acts as the primary repeating subunit of chromatin. The core of the nucleosome is formed by the histone fold, an ancient structural element comprising three alpha-helices separated by two loops [31]. Proteins containing histone folds are already found in Archaea, placing them at the root of molecules that have evolved to interact with and compact DNA [32]. To acquire the ability to interact with the cytoskeleton, chromosomes may have functionalized this core-packaging machinery early in evolution. The universal importance of CENP-A proteins as the markers of centromere identity and the foundation for kinetochore assembly is now well established and was recently further substantiated by functional and structural analysis (reviewed in [33,34]) (Box 1). In addition to CENPA nucleosomes, centromere-proximal H3 nucleosomes also have important roles and act as recruiters of important effector molecules including the Chromosome-passenger Complex (CPC) [35,36], which regulates kinetochore–microtubule attachments [37]. An additional advance in our understanding of inner kinetochore assembly has been the discovery of new CCAN subunits that operate at the centromere–kinetochore interface. Similar to the constituents of canonical nucleosomes, the CCAN subunits CENP-T, -W, -S, and -X are histone fold-containing proteins (Figure 2). The presence of histone-fold proteins in non-nucleosomal protein complexes is not unprecedented: the TFIID complex, a major interaction partner of RNA polymerase II, contains many subunits (TAFs) that have histone folds. Further examples include transcription factors, like negative cofactor 2 (NC2a/NC2b), and the chromatin accessibility complex proteins (CHRAC14/CHARC16) [38,39]. Although in some Box 2. Sequence-specific centromere-binding proteins in budding yeast The simplest centromeres are found in Saccharomyces cerevisiae, comprising only 125 bp of DNA (point centromere) with three sequence elements that are conserved between the 16 chromosomes [19,83]. The centromeres of Schizosaccharomyces pombe, by contrast, are ‘regional’ because they are built on more complex DNA that contains repeated-sequence elements and has the hallmarks of heterochromatin. The sequence-defined point centromeres of budding yeast have been used to identify and isolate the first kinetochore complexes via DNA-affinity chromatography. The CBF3 complex that was isolated in this way has homologs in other point centromere-containing yeasts, but is not conserved outside fungi. Recently, structural studies have provided important insights into the function of this centromere-binding complex. The sequence specificity of CBF3 is derived from the Cep3 subunit, which contains a transcription factor-like Zn-finger domain and binds the CDEIII element [84,85]. Additional DNA-binding activity is present in the Ndc10 subunit, whose globular domain displays structural homology to Flp and Cre recombinases but lacks catalytic activity [86,87]. Its mode of interaction with DNA is based on positively charged amino acids that contact the sugar–phosphate backbone in a sequence-independent manner. Importantly, Ndc10 can bind directly to the CENP-A chaperone Scm3/HJURP and is required for its localization to kinetochores [64]. These findings establish a link between the defined sequence of yeast centromere DNA and the deposition of the CENP-A nucleosome.

TICB-948; No. of Pages 10

Review

Trends in Cell Biology xxx xxxx, Vol. xxx, No. x

(a)

(b)

H4–H3 / H3–H4

X–S / T–W

H4–H3 / H2B–H2A

CENP-T ext.

X–S / T–W

(c)

Negave

Posive Surface charge TRENDS in Cell Biology

Figure 2. Atomic details of canonical histones and constitutive centromere-associated network (CCAN) histone-fold proteins. (a) Structure of the histone H3-H4 heterotetramer (PDB ID: 1KX5, [88]) compared with the CENP-TWSX heterotetramer (PDB ID: 3VH5, [41]). Note the overall similarity of the structures. The CENP-T protein in the TWSX heterotetramer contains two additional alpha-helices (circled) that are important for centromere localization. (b) Half of the nucleosome core particle showing a H3-H4/H2A-H2B heterotetramer enclosed by a 73-bp DNA circle (PDB ID: 1KX5). To detect putative DNA interfaces in CCAN histone-fold proteins, the right panel shows a hypothetical model in which the CENP-TWSX heterotetramer is superimposed on the nucleosome core half and visualized with the same 73-bp DNA fragment. (c) Electrostatic surface potentials of the tetramers. The molecular visualization was performed in PyMOL. Note the high density of positively charged surface residues that form the putative DNA interface of the CENP-TWSX heterotetramer.

TAFs key residues responsible for DNA binding in canonical histones [40] are not preserved, CCAN histone-fold proteins have retained the ability to interact with DNA (Figure 2). Similar to canonical histones, these proteins follow a stepwise assembly process leading to the formation of different oligomers: CENP-T and -W associate into a heterodimer, which is required for their full association with kinetochores both in human cells and in yeast [20,41]. In the absence of CENP-T and -W, CENP-S and -X form a tetramer known to associate with components of the Fanconi anemia pathway [42,43]. When present in equimolar amounts, formation of a CENP-TWSX heterotetramer, which protects approximately 100 bp of DNA from MNase

digestion in vitro, is the preferred option. Comparing the structure of the CENP-TWSX particle to that of the subnucleosomal H3-H4 tetrasome, it is apparent that both particles use the central alpha-helix for heterodimerization (Figure 2a). A major difference is that the TWSX particle is structurally asymmetric, containing two additional short alpha helices that extend the histone fold in CENP-T and are positioned away from the potential DNAinteracting interface. The sequence of this region of CENPT is more highly conserved between yeast and human than the rest of the histone fold, suggesting that this is a functionally important part of the molecule. Indeed, elimination of these helices abolishes centromere localization of 5

TICB-948; No. of Pages 10

Review

Trends in Cell Biology xxx xxxx, Vol. xxx, No. x

CENP-T [41]; thus, they might be involved in interactions with other CCAN subunits.

than is CENP-A from H3 (Figure 3). In reconstitution experiments, the CCAN-HF proteins have not been observed to coassemble with canonical histones, but instead, as demonstrated for the chicken and human proteins, they associate with each other [41]. Whether this is also true for the yeast homologs remains to be established, because residues critical for the tetramerization are not obviously conserved in the yeast proteins. The formation of the TWSX heterotetramer is not strictly required for centromere localization of CENP-T, because

Do CCAN histone-fold proteins form a specialized nucleosome? Could CENP-T/W or CENP-S/X proteins, similar to the histone-fold protein CENP-A, be part of a specialized nucleosome that is present at centromeres? Phylogenetic sequence analysis has indicated that CENP-T, -W, -S, and -X are much more divergent from canonical histones

NC2β

TA F9

M Ho us m Xe Gall o n u op s Zy g S osa acco Da us n g c Sac char loss io om us ch Klu arom yces yve y rom ces yce s A Deb ary shbya Pyre omyce s nop Lept h osph ora a Botr eria yon ia Aspe rgillu s Ajello myces Gibbere lla Schizosacc haromyces Drosophila Uslago Homo

CENP-X

A H2

//

b. a l r. a id pa ces s y ce n d da Ca ndi ryom my s Ca ba erso yce klu. De heff rom yces Sc yve rom Klu cha c Sa bya g l a . s h As dida myce ces n y Ca charo arom Sac osacch Zyg ancea is h abd L ac norh Cae o Hom phila o Dros opsis id Arab ces aromy Sacch myces ccharo Schizosa Uslago myces Schizosaccharo Arabidospsi Caenorhabdis Saccharomyces

//

)

0.4

H4

Uslago Arabidopsis Drosophila Homo Caenorha bdis Yarrow ia Schizo sa Tuber ccharomyce s Usla go Lept o Pyre sphaeria n Pha ophora e Tal o s p h a a e Aje romyc ria Art llomy es c h e Ne rode s Ma urosp rma Gib gna ora Ne ber port Pic ctri ella he M hia a Cl eye pa av ro s. isp zy or ma a

H3

Phyto

CE NP -T ( fu ng i

Picea Vis phtho ra Ixodes Saccoglo ssus Mus Homo Xenopus Danio Trichomonas Micromonas Candida haromyces Schizosacc Ashbya yces Ajellom es myc ro la Ta illium Penic ces y rom ces a h c sac my Zygo ccharo uber T Sa gia Bru o a L is abd si s h r o op la n d e i b hi Ca Ara osop mo Dr Ho ces my o ro lag ha Us yces s cc  a om di os ar ab hiz ch orh Sc c Sa en Ca

r te s ac bu o b cus o l m og er coc ae oth ldo a ch n s Ar etha noc milu M etha o p u s a M tro hil is Ni osop abd s yce D r enorh p s i s om Ca bido char c A r a izos a Sch lago Us o s yce Hom harom c Sac idopsis Arab o Hom hila op Dros is rhabd Caeno myces ro a h cc Sa yces ccharom Schizosa Uslago s Saccharomyce Schizosaccharomyces

-A NP CE

B en las or toc Sc ha ys S hiz ac os cha Us bdi s ac ch rom lag s a o Ar rom yces ab ido yces ps Dro Hom is sop o hila

Ar c

ea ha

Aspergillus Saccharomyces Puccinia haromyces Schizosacc Oryza opsis Arabid lla omitre Physc Ixodes gia iopy T a e n Mus o Hom s opu Xen oma t o ios nch S a l m a l Bra tel tos lax ma hop ra c Ne Tri Hyd ora th a ph Cion a yto m Ph so m to liu his te Sc tyos c Di

Ca

CEN P-T (ve rt. )

Tuber Gibberella Chaetomium Neurospora Arabidop sis Uslag o Schizo sa Sacch ccharomyc es a Caen romyces orha Hom bdi s Dro o s Dan ophila Dic io e Cio ntrar chu Xen na s o Ga pus Ho l l u s M mo Na us Ha noa Ca lob rch Py ldiv act aeu ro irg eri m co um cc a us

H2B

P-S CEN

CEN P-W

NFYβ

TRENDS in Cell Biology

Figure 3. Phylogenetic distribution of constitutive centromere-associated network (CCAN) histone-fold proteins. Phylogenetic tree of CCAN-HF proteins (red), core histones (blue), and archeal histones (green). Other histone-fold proteins were added as outliers and colored yellow (TAF9, NC2b, NFYb). The scale bar in the center measures the evolutionary distance in amino acid substitutions per site. Due to the high degree of divergence and the low number of aligned residues, only a few branches are supported with high confidence and are marked with a red dot. For clarity, the branch length is not drawn to scale for some CENP-W variants. Unlike CENP-A, the CCAN-HF proteins group neither to one of the core histones nor to other histone-fold family proteins.

6

TICB-948; No. of Pages 10

Review

Trends in Cell Biology xxx xxxx, Vol. xxx, No. x

the CENP-S knockout in chicken cells retains CENP-T at kinetochores [44] and also deletion of the yeast CENP-S protein Mhf1 does not abolish kinetochore localization of Cnn1CENP-T (S. Westermann and A. Schleiffer, unpublished). These observations imply that CENP-T proteins can localize to kinetochores in a manner that does not involve heterotetramerization with CENP-S and -X. However, the phenotypes of CENP-S and -X knockouts, which have reduced Ndc80 levels at kinetochores and a perturbed outer plate structure, are consistent with the idea that CENP-T is not fully functional in these cells [44]. The emerging picture is that heterotetramerization might be required for the full function of these proteins at the centromere, but they are also integrated into the rest of the CCAN in a way that does not require the assembly of a particle with nucleosome-like characteristics. Structural characterization of the association of CENP-TWSX with DNA, as well as high-precision mapping of the interaction with centromeres in live cells, will be required to address this point further. Key connections between the centromere and the microtubule-binding interface The kinetochore complexes that constitute the interface with microtubules – Ndc80, KNL-1, and Mis12 (the KMN network) – do not possess direct DNA-binding activity; thus, they rely on specific centromere recruiters. Furthermore, biochemical isolation of kinetochores from yeast extracts using affinity chromatography of outer kinetochore components has demonstrated a stable association of centromere proteins, including CENP-A, even when purified away from DNA [16,45]. So far, two direct and functionally important connections between CCAN and KMN components have been established; among the (a)

CCAN-HF proteins, only CENP-T substantially extends the histone fold with a long N-terminal tail. This domain is important for kinetochore assembly in human and chicken cells [46] and contains a conserved peptide motif that is necessary and sufficient for yeast Cnn1CENP-T to interact with the Spc24-25 domain of the Ndc80 complex [20,47]. Ectopic targeting of the N-terminal tail of both CENP-T and Cnn1 to extrachromosomal lacO or tetO arrays is sufficient to nucleate a functional kinetochore that recruits the Ndc80 complex [20,46,48]. The distance between the Ndc80-binding motif and the histone fold is under evolutionary constraints, being never shorter than 123 residues (Pichia pastoris), which would translate to 43 nm of an extended polypeptide chain and could thus cover much of the length of a yeast or human kinetochore [49,50]. At least some of the inherent structural flexibility of the N tail of CENP-T is preserved in the context of an assembled kinetochore as it undergoes conformational changes in response to microtubule attachment [51]. Cdk1 phosphorylation of the N tail is required for initiation of kinetochore assembly in human cells, at both ectopic and endogenous loci [46,52]. Together, these findings establish the N tail of CENP-T as an important link in the structural framework of the kinetochore. A second connection between the inner and outer kinetochore is established via the CCAN subunit CENP-C, which binds to the Mis12 complex. The extreme N terminus of human CENP-C is sufficient to associate with the Mis12 complex directly and overexpression of an interaction-competent minimal CENP-C fragment has a dominant-negative phenotype by disrupting kinetochore organization and checkpoint signaling [53]. Corresponding results have been obtained for Drosophila CENP-C, whose N terminus is required for kinetochore assembly and, when

(b)

Microtubules Ndc80 complex

-

Kinetochore microtubule

Ndc80 complex Dam1 complex

Mis12 complex Ndc80 complex

CENP-C

CENP-A

+

Outer KT

CENP-TW CCAN Mis12 complex

Centromeric chroman Inner KT Biochemical interacon

CENP-C

Localizaon dependency TW SX

H3

H3

TW SX

Cse4 nucleosome TRENDS in Cell Biology

Figure 4. Principles of kinetochore architecture and the recruitment of the Ndc80 complex. (a) Schematic representation of parallel recruitment pathways for the microtubule-binding Ndc80 complex. (b) Illustration of kinetochore architecture highlighting the contributions of the constitutive centromere-associated network (CCAN) subunits CENP-A (Cse4), CENP-C, and CENP-T. Note that the illustration shows a single microtubule-attachment site and that the exact copy numbers of the kinetochore complexes present at such a site have not been conclusively established.

7

TICB-948; No. of Pages 10

Review targeted to non-centromeric locations, is also sufficient for initiating assembly of the KMN network [54]. The combined recruitment of the N termini of CENP-C and CENPT can direct kinetochore assembly on an ectopic locus and bypasses the essential role of the CENP-A nucleosome [46]. It is likely that additional important connections between the inner and outer kinetochore exist. In yeast, for example, the number of Mtw1 complexes per centromere exceeds that of Mif2CENP-C molecules [55], suggesting that additional CCAN subunits might make important contributions. Given the interactions described above, the CCAN subunits CENP-C and CENP-T are the origins of molecularly distinct connections to the microtubule-binding Ndc80 complex (Figure 4). Both the CENP-C-interacting Mis12 complex and the N terminus of CENP-T interact with the Spc24-25 domain of Ndc80-C and their binding is mutually exclusive [20,47,52]. The reason why kinetochores have evolved two distinct receptors for the same microtubulebinding molecule is currently unclear. In human cells, achieving normal levels of the Ndc80 complex at kinetochores requires the presence of both CENP-C and CENP-T. In budding yeast, however, Cnn1CENP-T is not essential and its deletion becomes problematic only when CENP-Cbased recruitment is compromised. The different functional contributions of these recruitment pathways remain to be established and may ultimately be understood only in the context of the larger kinetochore architecture. One hypothesis is that the kinetochore may position different populations of Ndc80 molecules to construct spatially and functionally distinct interfaces with the microtubule lattice. In this way, some Ndc80 molecules could be part of the force-generating machinery that controls movement, whereas other Ndc80 molecules might contribute to a frictional interface that allows persistent attachment [56]. Evidence for a dynamic centromere architecture The term ‘constitutive’ centromere associated network contrasts the behavior of the described centromere-associated proteins against the components of the microtubule-binding interface, which in human cells assemble only when cells enter mitotic prophase. There is accumulating evidence, however, that centromere proteins display dynamic behavior as well: newly synthesized CENPA, for example, is deposited during the G1 phase of the cell cycle and the pool of existing CENP-A at centromeres is distributed symmetrically to both daughter strands during replication [57,58]. In budding yeast, the CENPT homolog Cnn1 is present in low copy numbers at centromeres in G1, but its level increases strongly after replication and is maximal when cells enter anaphase [59]. Cell cycle-dependent loading of the CENP-T/W complex is also observed in human cells; here, CENP-T/W becomes stably associated with centromeres in late S phase and G2 and, unlike CENP-A, does not persist at centromeres over multiple generations [60]. The dynamic behavior underscores the critical role of CENP-T/W for the mitotic kinetochore and it is contrasted by the behavior of the CENP-A-interacting CCAN subunit CENP-N, which binds stably to centromeres only in S phase and dissociates again in G2 [61]. 8

Trends in Cell Biology xxx xxxx, Vol. xxx, No. x

Dynamic behavior of CCAN subunits can also be observed on even shorter timescales; the level of human CENP-I has been reported to fluctuate depending on whether it associates with a kinetochore that displays poleward or antipoleward motion [62]. This dependency on the polymerization state of kinetochore microtubules is reminiscent of the plus-end tracking protein EB1, which associates only with the plus ends of growing microtubules, and may indicate an involvement of CCAN subunits in the control of microtubule dynamics. This notion receives support from high-resolution fluorescence localization experiments demonstrating that the plus end of a microtubule is deeply embedded into the kinetochore and may be in a position to contact inner kinetochore proteins [49]. In addition, the CCAN subunit CENP-Q has been reported to display direct microtubule-binding activity in vitro [62]. Judging from the behavior of the autonomous plus-end tracker EB1, which targets polymerizing kinetochore microtubules in a manner similar to other microtubules [63], it appears, however, that the biochemical structure of the microtubule plus end in the context of a kinetochore is not dramatically altered. The mechanistic role of CCAN subunits in regulating microtubule dynamics thus requires further investigation. Concluding remarks and outlook Establishing a catalog of conserved proteins that operate at the centromere–kinetochore interface is only the beginning of their detailed analysis. The functional role of most of the CCAN subunits has not been established and to gain deep insights it will not be sufficient to study each subunit individually. What is the role of these proteins in the architecture of the kinetochore? How is their assembly regulated? What mechanisms underlie the dynamic behavior of CCAN subunits? To answer these questions, the ensemble properties of CCAN proteins will have to be investigated and studies in evolutionarily distant organisms must be compared to reveal architectural principles. Structural work has to define in atomic detail the interfaces between these proteins and their connections to the microtubule-binding components of the kinetochore. Biochemical assays that quantitatively study kinetochore assembly and allow probing the contributions of individual subunits will have to be developed. These approaches ultimately may permit the generation of mutations that separate structural from regulatory roles, to analyze them in vivo. We can expect important insights into the fundamental ability of chromosomes to direct their own segregation in the years to come. Acknowledgments The authors thank all members of the Westermann laboratory for discussions. They apologize to those authors whose work could not be cited due to space constraints. Research in the Westermann laboratory has received funding from the European Research Council under the European Community’s Seventh Framework Programme (FP7/20072013)/ERC grant agreement number 203499 and from the Austrian Science Fund FWF (SFB F34-B03).

References 1 Tooley, J.G. et al. (2011) The Ndc80 complex employs a tripartite attachment point to couple microtubule depolymerization to chromosome movement. Mol. Biol. Cell 22, 1217–1226

TICB-948; No. of Pages 10

Review 2 Sundin, L.J. et al. (2011) The NDC80 complex proteins Nuf2 and Hec1 make distinct contributions to kinetochore-microtubule attachment in mitosis. Mol. Biol. Cell 22, 759–768 3 Ciferri, C. et al. (2008) Implications for kinetochore-microtubule attachment from the structure of an engineered Ndc80 complex. Cell 133, 427–439 4 Lampert, F. et al. (2013) Molecular requirements for the formation of a kinetochore-microtubule interface by Dam1 and Ndc80 complexes. J. Cell Biol. 200, 21–30 5 Clarke, L. and Carbon, J. (1980) Isolation of a yeast centromere and construction of functional small circular chromosomes. Nature 287, 504–509 6 Lechner, J. and Carbon, J. (1991) A 240 kd multisubunit protein complex, CBF3, is a major component of the budding yeast centromere. Cell 64, 717–725 7 Obuse, C. et al. (2004) Proteomics analysis of the centromere complex from HeLa interphase cells: UV-damaged DNA binding protein 1 (DDB-1) is a component of the CEN-complex, while BMI-1 is transiently co-localized with the centromeric region in interphase. Genes Cells 9, 105–120 8 Foltz, D.R. et al. (2006) The human CENP-A centromeric nucleosomeassociated complex. Nat. Cell Biol. 8, 458–469 9 Izuta, H. et al. (2006) Comprehensive analysis of the ICEN (Interphase Centromere Complex) components enriched in the CENP-A chromatin of human cells. Genes Cells 11, 673–684 10 Okada, M. et al. (2006) The CENP-H-I complex is required for the efficient incorporation of newly synthesized CENP-A into centromeres. Nat. Cell Biol. 8, 446–457 11 Cheeseman, I.M. et al. (2002) Phospho-regulation of kinetochoremicrotubule attachments by the Aurora kinase Ipl1p. Cell 111, 163– 172 12 Liu, X. et al. (2005) Molecular analysis of kinetochore architecture in fission yeast. EMBO J. 24, 2919–2930 13 Shiroiwa, Y. et al. (2011) Mis17 is a regulatory module of the Mis6Mal2-Sim4 centromere complex that is required for the recruitment of CenH3/CENP-A in fission yeast. PLoS ONE 6, e17761 14 De Wulf, P. et al. (2003) Hierarchical assembly of the budding yeast kinetochore from multiple subcomplexes. Genes Dev. 17, 2902–2921 15 Thakur, J. and Sanyal, K. (2012) A coordinated interdependent protein circuitry stabilizes the kinetochore ensemble to protect CENP-A in the human pathogenic yeast Candida albicans. PLoS Genet. 8, e1002661 16 Akiyoshi, B. et al. (2010) Tension directly stabilizes reconstituted kinetochore-microtubule attachments. Nature 468, 576–579 17 Takahashi, K. et al. (1994) Fission yeast minichromosome loss mutants mis cause lethal aneuploidy and replication abnormality. Mol. Biol. Cell 5, 1145–1158 18 Ortiz, J. et al. (1999) A putative protein complex consisting of Ctf19, Mcm21, and Okp1 represents a missing link in the budding yeast kinetochore. Genes Dev. 13, 1140–1155 19 Meraldi, P. et al. (2006) Phylogenetic and structural analysis of centromeric DNA and kinetochore proteins. Genome Biol. 7, R23 20 Schleiffer, A. et al. (2012) CENP-T proteins are conserved centromere receptors of the Ndc80 complex. Nat. Cell Biol. 14, 604–613 21 Tanaka, K. et al. (2009) CENP-C functions as a scaffold for effectors with essential kinetochore functions in mitosis and meiosis. Dev. Cell 17, 334–343 22 Hori, T. et al. (2008) CENP-O class proteins form a stable complex and are required for proper kinetochore function. Mol. Biol. Cell 19, 843– 854 23 Hua, S. et al. (2010) CENP-U cooperates with Hec1 to orchestrate kinetochore-microtubule attachment. J. Biol. Chem. 286, 1627–1638 24 Schmitzberger, F. and Harrison, S.C. (2012) RWD domain: a recurring module in kinetochore architecture shown by a Ctf19-Mcm21 complex structure. EMBO Rep. 13, 216–222 25 Corbett, K.D. et al. (2010) The monopolin complex crosslinks kinetochore components to regulate chromosome-microtubule attachments. Cell 142, 556–567 26 Wei, R.R. et al. (2006) Structure of a central component of the yeast kinetochore: the Spc24p/Spc25p globular domain. Structure 14, 1003– 1009 27 Kim, S. et al. (2012) Structure of human Mad1 C-terminal domain reveals its involvement in kinetochore targeting. Proc. Natl. Acad. Sci. U.S.A. 109, 6549–6554

Trends in Cell Biology xxx xxxx, Vol. xxx, No. x

28 Cohen, R.L. et al. (2008) Structural and functional dissection of Mif2p, a conserved DNA-binding kinetochore protein. Mol. Biol. Cell 19, 4480– 4491 29 Aravind, L. and Landsman, D. (1998) AT-hook motifs identified in a wide variety of DNA-binding proteins. Nucleic Acids Res. 26, 4413– 4421 30 Reeves, R. and Nissen, M.S. (1990) The A.T-DNA-binding domain of mammalian high mobility group I chromosomal proteins. A novel peptide motif for recognizing DNA structure. J. Biol. Chem. 265, 8573–8582 31 Arents, G. and Moudrianakis, E.N. (1995) The histone fold: a ubiquitous architectural motif utilized in DNA compaction and protein dimerization. Proc. Natl. Acad. Sci. U.S.A. 92, 11170–11174 32 Talbert, P.B. and Henikoff, S. (2010) Histone variants–ancient wrap artists of the epigenome. Nat. Rev. Mol. Cell Biol. 11, 264–275 33 De Rop, V. et al. (2012) CENP-A: the key player behind centromere identity, propagation, and kinetochore assembly. Chromosoma 121, 527–538 34 Sekulic, N. and Black, B.E. (2012) Molecular underpinnings of centromere identity and maintenance. Trends Biochem. Sci. 37, 220–229 35 Wang, F. et al. (2010) Histone H3 Thr-3 phosphorylation by Haspin positions Aurora B at centromeres in mitosis. Science 330, 231–235 36 Kelly, A.E. et al. (2010) Survivin reads phosphorylated histone H3 threonine 3 to activate the mitotic kinase Aurora B. Science 330, 235–239 37 Liu, D. et al. (2009) Sensing chromosome bi-orientation by spatial separation of aurora B kinase from kinetochore substrates. Science 323, 1350–1353 38 Kamada, K. et al. (2001) Crystal structure of negative cofactor 2 recognizing the TBP-DNA transcription complex. Cell 106, 71–81 39 Hartlepp, K.F. et al. (2005) The histone fold subunits of Drosophila CHRAC facilitate nucleosome sliding through dynamic DNA interactions. Mol. Cell. Biol. 25, 9886–9896 40 Luger, K. and Richmond, T.J. (1998) DNA binding within the nucleosome core. Curr. Opin. Struct. Biol. 8, 33–40 41 Nishino, T. et al. (2012) CENP-T-W-S-X forms a unique centromeric chromatin structure with a histone-like fold. Cell 148, 487–501 42 Tao, Y. et al. (2012) The structure of the FANCM-MHF complex reveals physical features for functional assembly. Nat. Commun. 3, 782 43 Yan, Z. et al. (2010) A histone-fold complex and FANCM form a conserved DNA-remodeling complex to maintain genome stability. Mol. Cell 37, 865–878 44 Amano, M. et al. (2009) The CENP-S complex is essential for the stable assembly of outer kinetochore structure. J. Cell Biol. 186, 173–182 45 Gonen, S. (2012) The structure of purified kinetochores reveals multiple microtubule-attachment sites. Nat. Struct. Mol. Biol. 19, 925–929 46 Gascoigne, K.E. et al. (2011) Induced ectopic kinetochore assembly bypasses the requirement for CENP-A nucleosomes. Cell 145, 410–422 47 Malvezzi, F. et al. (2013) A structural basis for kinetochore recruitment of the Ndc80 complex via two distinct centromere receptors. EMBO J. 32, 409–423 48 Hori, T. et al. (2013) The CCAN recruits CENP-A to the centromere and forms the structural core for kinetochore assembly. J. Cell Biol. 200, 45–60 49 Wan, X. et al. (2009) Protein architecture of the human kinetochore microtubule attachment site. Cell 137, 672–684 50 Joglekar, A.P. et al. (2009) In vivo protein architecture of the eukaryotic kinetochore with nanometer scale accuracy. Curr. Biol. 19, 694–699 51 Suzuki, A. et al. (2011) Spindle microtubules generate tensiondependent changes in the distribution of inner kinetochore proteins. J. Cell Biol. 193, 125–140 52 Nishino, T. et al. (2013) CENP-T provides a structural platform for outer kinetochore assembly. EMBO J. 32, 424–436 53 Screpanti, E. et al. (2011) Direct binding of cenp-C to the mis12 complex joins the inner and outer kinetochore. Curr. Biol. 21, 391–398 54 Przewloka, M.R. et al. (2011) CENP-C is a structural platform for kinetochore assembly. Curr. Biol. 21, 399–405 55 Joglekar, A.P. et al. (2006) Molecular architecture of a kinetochoremicrotubule attachment site. Nat. Cell Biol. 8, 581–585 56 Dumont, S. et al. (2012) Deformations within moving kinetochores reveal different sites of active and passive force generation. Science 337, 355–358 9

TICB-948; No. of Pages 10

Review 57 Jansen, L.E. et al. (2007) Propagation of centromeric chromatin requires exit from mitosis. J. Cell Biol. 176, 795–805 58 Foltz, D.R. et al. (2009) Centromere-specific assembly of CENP-a nucleosomes is mediated by HJURP. Cell 137, 472–484 59 Bock, L.J. et al. (2012) Cnn1 inhibits the interactions between the KMN complexes of the yeast kinetochore. Nat. Cell Biol. 14, 614–624 60 Prendergast, L. et al. (2011) Premitotic assembly of human CENPs -T and -W switches centromeric chromatin to a mitotic state. PLoS Biol. 9, e1001082 61 Hellwig, D. et al. (2011) Dynamics of CENP-N kinetochore binding during the cell cycle. J. Cell Sci. 124, 3871–3883 62 Amaro, A.C. et al. (2010) Molecular control of kinetochore-microtubule dynamics and chromosome oscillations. Nat. Cell Biol. 12, 319–329 63 Tirnauer, J.S. et al. (2002) EB1 targets to kinetochores with attached, polymerizing microtubules. Mol. Biol. Cell 13, 4308–4316 64 Camahort, R. et al. (2007) Scm3 is essential to recruit the histone h3 variant cse4 to centromeres and to maintain a functional kinetochore. Mol. Cell 26, 853–865 65 Hayashi, T. et al. (2004) Mis16 and Mis18 are required for CENP-A loading and histone deacetylation at centromeres. Cell 118, 715–729 66 Lagana, A. et al. (2010) A small GTPase molecular switch regulates epigenetic centromere maintenance by stabilizing newly incorporated CENP-A. Nat. Cell Biol. 12, 1186–1193 67 Tachiwana, H. et al. (2011) Crystal structure of the human centromeric nucleosome containing CENP-A. Nature 476, 232–235 68 Cho, U.S. and Harrison, S.C. (2011) Recognition of the centromerespecific histone Cse4 by the chaperone Scm3. Proc. Natl. Acad. Sci. U.S.A. 108, 9367–9371 69 Carroll, C.W. et al. (2009) Centromere assembly requires the direct recognition of CENP-A nucleosomes by CENP-N. Nat. Cell Biol. 11, 896–902 70 Carroll, C.W. et al. (2010) Dual recognition of CENP-A nucleosomes is required for centromere assembly. J. Cell Biol. 189, 1143–1155 71 Guse, A. et al. (2011) In vitro centromere and kinetochore assembly on defined chromatin templates. Nature 477, 354–358 72 Krassovsky, K. et al. (2012) Tripartite organization of centromeric chromatin in budding yeast. Proc. Natl. Acad. Sci. U.S.A. 109, 243–248 73 Bui, M. et al. (2012) Cell-cycle-dependent structural transitions in the human CENP-A nucleosome in vivo. Cell 150, 317–326

10

Trends in Cell Biology xxx xxxx, Vol. xxx, No. x

74 Shivaraju, M. et al. (2012) Cell-cycle-coupled structural oscillation of centromeric nucleosomes in yeast. Cell 150, 304–316 75 Dunleavy, E.M. et al. (2009) HJURP is a cell-cycle-dependent maintenance and deposition factor of CENP-A at centromeres. Cell 137, 485–497 76 Mendiburo, M.J. et al. (2011) Drosophila CENH3 is sufficient for centromere formation. Science 334, 686–690 77 Westermann, S. et al. (2003) Architecture of the budding yeast kinetochore reveals a conserved molecular core. J. Cell Biol. 163, 215–222 78 Kwon, M.S. et al. (2007) CENP-C is involved in chromosome segregation, mitotic checkpoint function, and kinetochore assembly. Mol. Biol. Cell 18, 2155–2168 79 Hori, T. et al. (2008) CCAN makes multiple contacts with centromeric DNA to provide distinct pathways to the outer kinetochore. Cell 135, 1039–1052 80 Yang, H. et al. (2012) Saccharomyces cerevisiae MHF complex structurally resembles the histones (H3-H4)(2) heterotetramer and functions as a heterotetramer. Structure 20, 364–370 81 Mythreye, K. and Bloom, K.S. (2003) Differential kinetochore protein requirements for establishment versus propagation of centromere activity in Saccharomyces cerevisiae. J. Cell Biol. 160, 833–843 82 Cheeseman, I.M. et al. (2008) KNL1 and the CENP-H/I/K complex coordinately direct kinetochore assembly in vertebrates. Mol. Biol. Cell 19, 587–594 83 Westermann, S. et al. (2007) Structures and functions of yeast kinetochore complexes. Annu. Rev. Biochem. 76, 563–591 84 Purvis, A. and Singleton, M.R. (2008) Insights into kinetochore-DNA interactions from the structure of Cep3Delta. EMBO Rep. 9, 56–62 85 Bellizzi, J.J., III et al. (2007) Crystal structure of the yeast inner kinetochore subunit Cep3p. Structure 15, 1422–1430 86 Perriches, T. and Singleton, M.R. (2012) Structure of yeast kinetochore Ndc10 DNA-binding domain reveals unexpected evolutionary relationship to tyrosine recombinases. J. Biol. Chem. 287, 5173–5179 87 Cho, U.S. and Harrison, S.C. (2012) Ndc10 is a platform for inner kinetochore assembly in budding yeast. Nat. Struct. Mol. Biol. 19, 48–55 88 Davey, C.A. et al. (2002) Solvent mediated interactions in the structure of the nucleosome core particle at 1.9 a resolution. J. Mol. Biol. 319, 1097–1113