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The histone variant CENP-A and centromere specification
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The histone variant CENP-A and centromere specification Ben E Black and Emily A Bassett The centromere is the chromosomal locus that guides faithful inheritance. Centromeres are specified epigenetically, and the histone H3 variant CENP-A has emerged as the best candidate to carry the epigenetic centromere mark. Recent advances demonstrate the physical basis for this epigenetic mark whereby CENP-A confers conformational rigidity to the nucleosome it forms with other core histones. This nucleosome is recognized by a multisubunit complex of constitutive centromere proteins, termed the CENP-ANAC. Evidence from two CENP-A relatives in diverse eukaryotes suggests that the histone complexes they form adopt highly unconventional arrangements on DNA. Centromere identity, itself, is propagated during mitotic exit and early G1, and it relies upon a cis-acting targeting domain within CENP-A and a proposed centromere ‘priming’ reaction. Addresses Department of Biochemistry and Biophysics and Program in Biochemistry and Molecular Biophysics, University of Pennsylvania, Philadelphia, PA 19104, United States Corresponding author: Black, Ben E ([email protected])
Current Opinion in Cell Biology 2008, 20:91–100 This review comes from a themed issue on Cell structure and dynamics Edited by Yixian Zheng and Karen Oegema Available online 15th January 2008 0955-0674/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2007.11.007
Introduction The faithful transmission of the genome relies upon a cell division mechanism whereby each replicated chromosome pair, termed sister chromatids, are separated and equally partitioned into daughter cells. This process involves bi-orientation of mitotic chromosomes on the microtubule-based spindle. The centromere is the locus on the chromosome that serves as the spindle connection point, and lessons from chromosomal rearrangements that result in either the duplication or elimination of the centromere strongly suggest the rule that one-and-onlyone centromere is tolerated per chromosome (Figure 1a). More than one centromere will lead to chromosome breakage by the spindle. Conversely, the lack of a centromere will lead an entire chromosome to missegregate. Either of these events is a genetic disaster, and as such, epigenetic mechanisms are in place to maintain centromere identity [1,2]. www.sciencedirect.com
Nucleosomes in which CENP-A replaces canonical H3 are the fundamental units of the chromatin at the foundation of the kinetochore, the mitotic protein assembly that mediates attachment to the mitotic spindle and generates a mitotic checkpoint signal that arrests cells from entering anaphase until all chromosomes are properly aligned on the metaphase plate. CENP-A nucleosomes are found at active but not at inactive centromeres and are strong candidates to carry the epigenetic centromere mark. Many other chromatin-based post-translational modifications and pericentromeric heterochromatin domains are required for full centromere function, which includes controlling sister chromatin cohesion [3]. In this review, however, we will focus on recent advances in understanding the physical basis of how CENP-A-containing nucleosomes distinguish themselves from bulk chromatin as the site for centromere and kinetochore formation, and we will highlight emerging work from diverse eukaryotic systems that sheds light on the cellular pathway leading to cell-cycle-coupled replenishment of the CENP-A mark.
How does CENP-A mark centromeres? CENP-A physically marks centromeres by assembling into a nucleosomal structure that is distinguishable from its canonical counterparts that contain histone H3. In the human version of the CENP-A nucleosome, the a2-helix (Figure 1b, red) and the preceding loop (L1) form the CENP-A targeting domain (CATD) that confers conformational rigidity — a 10-fold slowing of hydrogen exchange along the peptide backbone — to the interface it forms with its binding partner histone H4 (Figure 1b, the a2 and a3 helices of H4 are shown in blue) [4]. The CATD also confers centromere targeting and an essential mitotic function to CENP-A, and together this supports a model where a structurally divergent nucleosome containing CENP-A is the fundamental unit of the chromatin that specifies centromere location [5]. The centromere must accommodate extraordinary physical constraints: firstly, specialized higher-order chromatin folding; secondly, assembly of the mitotic kinetochore complex; and thirdly, mitotic spindle forces [6,7,8]. One particular site in the CENP-A nucleosome that may be important for accommodating these constraints is the entry/exit DNA of the nucleosome. Indeed, Lys49 of CENP-A causes a weakening of its interaction with the entry/exit DNA of the nucleosome compared to within the canonical nucleosome where H3 has an arginine at the corresponding position in its aN helix (Figure 1b; Arg49 in green) [9]. The steady-state unwrapping of 7 bp of DNA at the entry/exit may provide centromeric chromatin arrays with the ability to adopt atypical higherCurrent Opinion in Cell Biology 2008, 20:91–100
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Figure 1
CENP-A nucleosomes are the fundamental unit of centromeric chromatin. (a) CENP-A is found at all active centromeres including those on neocentromeric marker chromosomes (right) that lack the a-satellite repeats typically found in megabase stretches at normal human centromeres (left) [62]. In addition, CENP-A vacates inactive centromeres, such as those found epigenetically silenced in the case of pseudodicentric chromosomes (center). (b) Model of the canonical H3-containing nucleosome (Protein Data Bank number 1kx5; [63]) with the corresponding region highlighted that displays conformational rigidity when assembled with CENP-A in place of histone H3 (the a2 helix of CENP-A in red; the a2 and a3 helices of H4 in blue). Only one pair of CENP-A and H4 is highlighted for reasons of clarity. The side-chain of Arg49 from histone H3 (green) that intercalates into the entry/exit DNA is changed to a lysine in the corresponding position in CENP-A and leads to steady state unwrapping of 7 bp on nucleosomes assembled onto DNA minicircles [9]. Recent studies have indicated diverse arrangements of nucleosomes and histone complexes containing CENP-A (or one of its relatives) in humans (c), flies (d), and budding yeast (e). Although human CENP-A has a tail of identical length to canonical H3, in its counterparts CID (d) and Cse4p (e), large domains that do not display evolutionary conservation extend >100 amino acids outside the predicted nucleosome core. The N-terminal extension includes the END domain in Cse4p that provides an essential function to budding yeast centromeres [64]. Scm3p is an essential component of budding yeast centromeres [13,15,16] that has the ability to displace H2A/H2B dimers from (Cse4p/H4)2 heterotetramers [13], raising the possibility that (Cse4p/H4/Scm3p)2 heterohexamers (panel e, left) wrap centromeric DNA into a nucleosome-like structure (panel e, right).
Current Opinion in Cell Biology 2008, 20:91–100
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order folding to enhance centromere function. The majority of human CENP-A nucleosomes are homotypic (i.e. two copies of CENP-A, see Figure 1c, left) [10], and purification of CENP-A nucleosomes also isolated a smaller pool that are heterotypic (i.e. one copy of CENP-A and one copy of H3, Figure 1c, right) and represents 10% of the total pool of CENP-A nucleosomes [7]. It remains unclear if there is any functional difference between homotypic or heterotypic CENP-A nucleosomes, or whether or not the heterotypic nucleosomes represent a transient form during the genesis of homotypic nucleosomes. Nucleosomes containing the CENP-A fruit fly relative CID were able to form a minor cross-linkable species consistent with an octameric histone arrangement in mitotic chromosome preparations, but they were underrepresented in interphase nuclei preparations [11]. This finding, along with evidence from atomic force microscopy for a structure half the height of typical nucleosomes, as well as micrococcal nuclease protection on immunopurified CID-containing arrays that suggested a compact nucleosomal conformation relative to H3-containing counterparts, led to the proposal that CID is present in a highly unstable ‘half-nucleosome’ arrangement (Figure 1d, left) [11]. The ‘half-nucleosome’ conclusion is heavily reliant upon a single approach: the failure to detect a band at the position expected for an octamer after treatment with the alkali-dependent crosslinker dimethyl suberimidate. Failure to crosslink an octamer is expected, however, because dimethyl suberimidate is specific to primary amines, which are absent from the region that is predicted to hold the two ‘halves’ of the octameric CID nucleosome together (Figure 1d, center and right; the corresponding region in H3 has two primary amines: a lysine in loop 2 (L2) and another in the a3 helix). An alternative interpretation of these data also consistent with the evidence is that CID nucleosomes in interphase nuclei preparations are somewhat more difficult to crosslink into octamers than their mitotic counterparts or H3-containing nucleosomes, and that they generate a compact nucleosomal architecture with larger linker DNA lengths (Figure 1d, possibility #2). Either way, CID probably marks chromatin by conferring structural rigidity to the complexes into which it assembles — in a manner similar to how human CENP-A rigidifies (CENP-A/H4)2 heterotetramers and the octameric nucleosomes it forms [4] — to specify the location of the fruit fly centromere. Additionally, the possibility that there may be interphase tetramer forms (i.e. not nucleosomes) of histone complexes containing CID, may also reflect an ‘immature’ form of centromeric chromatin occurring after DNA replication. At the ‘point’ centromere of budding yeast, an individual nucleosome containing the CENP-A relative Cse4p is found [12], but H2A and H2B are not detectable by www.sciencedirect.com
ChIP [13], despite earlier biochemical evidence to indicate the presence of H2A and H2B in a yeast nucleosome containing Cse4p [14]. Scm3p, an essential budding yeast centromere component [13,15,16], has the ability to displace H2A/H2B from an (H2A/H2B/ Cse4p/H4)2 octamer [13]. Taken together, these findings suggest that an atypical ‘nucleosome’ containing Scm3p in place of H2A/H2B dimers forms at yeast centromeres (Figure 1e) [13]. An equally interesting possibility is that Cse4p is maintained at centromeres outside a normal nucleosome for most of the cell cycle by Scm3p, transiently assembling into an octameric (H2A/ H2B/Cse4p/H4)2 nucleosome configuration required for mitosis.
What does the CENP-A nucleosomal array recruit to centromeres? The CENP-A-containing nucleosome in human cells is physically associated with a six subunit protein complex, termed the CENP-A Nucleosome Associated Complex (NAC), that contains CENP-C, CENP-H, CENP-M, CENP-N, CENP-T, and CENP-U(50) [7]. At least a subset of CENP-ANAC components are tethered to a complex that is more distal to the CENP-A nucleosome and contains CENP-I, CENP-K, CENP-L, CENP-O, CENP-P, CENP-Q, CENP-R, and CENP-S [7,17,18]. The position of the CENP-A nucleosome — wrapping centromeric DNA — suggests it sits atop the centromere/kinetochore-building hierarchy. Depletion experiments of various centromere components, including CENP-A itself, from diverse eukaryotes support this view [19–21]. Furthermore, the initial step of CENP-ANAC recruitment provides a molecular linkage from the CENP-A nucleosome outward to the kinetochore.
Coalescing CENP-A nucleosomes to form a three-dimensional array A chromosome stretching experiment from Bill Brinkley and colleagues initially suggested a multi-subunit model for centromere organization [22]. This arrangement was more recently shown to extend down to the CENP-A nucleosome array (Figure 2a), as physically unfolded centromeric subunits are interspersed with chromatin spacers of nucleosomes containing canonical H3 [6]. Furthermore, CENP-A shows a strong preference for assembling at specific locations within a neocentromere [23], and constitutive centromere components CENP-C and CENP-H are specifically found in close proximity to the CENP-A-rich regions [24]. Although it is not clear what drives positioning of CENP-A nucleosomes at preferred sites in nonrepetitive DNA, these findings suggest a nonrandom arrangement along linear DNA. CENP-A nucleosome coalescence then organizes the arrays into the higher-order chromatin structure that functions as the foundation of the mitotic kinetochore. It is currently unclear whether physical interactions between neighboring CENP-A nucleosomes self-direct the coalescence of Current Opinion in Cell Biology 2008, 20:91–100
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Figure 2
Achieving the higher-order chromatin structure found at the centromere. (a) Centromere stretching experiment indicating that the array of CENP-A nucleosomes, coalesced in three-dimensional space, are not contiguous along the DNA but are interrupted by spacers containing blocks of H3containing nucleosomes. The image was kindly provided by Gary Karpen and reproduced from [6]. CENP-A nucleosome coalescence could be entirely self-directed (b) or may necessitate the action of bridging factors — perhaps components of the CENP-ANAC—to organize into the array that forms the foundation of the mitotic kinetochore (c). For simplicity, centromeric chromatin is shown in a coiled conformation, but no data have emerged to indicate the actual higher-order fold that culminates in the coalesced array of CENP-A nucleosomes at the foundation of the mitotic kinetochore.
the CENP-A array (Figure 2b), or if one or more CENPANAC proteins (or perhaps other centromere proteins) act as bridging factors to guide this process (Figure 2c). Reinforcing the epigenetic mark by preserving centromere and kinetochore structure is likely to involve several Current Opinion in Cell Biology 2008, 20:91–100
proteins, and indeed CENP-A levels are negatively affected at human centromeres after depletion of Mis12 [25], reminiscent of the phenotypes observed after mutation of several fission yeast genes including Mis6, Mis12, and Sim4 (reviewed elsewhere [3]). www.sciencedirect.com
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Seeding new centromeres: the chicken and the egg Once formed, centromeres are epigenetically maintained at the same locus, independent of DNA sequence, cell division after cell division and generation after generation [26,27,28,29], so why are megabase stretches of these repeats found at most eukaryotic centromeres? Clues come from human artificial chromosome (HAC) constructs whose establishment in cells require intact a-satellites (the human version of the centromere repeat containing the 17-mer CENP-B binding box) as well as the expression of CENP-B — a protein that is otherwise dispensable for centromere function and epigenetic maintenance [30,31]. Future experiments with HACs will further elucidate the initial events in centromere formation, defined by the generation of an array of CENP-A nucleosomes to specify the centromere as well as a crucial adjacent chromatin domain resembling bona fide pericentromeric heterochromatin domains (i.e. enriched with nucleosomes where histone H3 is modified with a trimethylation at Lys9) [32]. In principle, another method for seeding new centromeres is to deliver the epigenetic mark to a noncentromeric region of the chromosome. Indeed, transient overexpression of CID, so that it replaces histone H3 in euchromatic regions of fruit fly chromosomes, leads to the occasional recruitment of one or more centromere/kinetochore proteins [33], so it may be possible to form a fully functional centromere by targeting CENP-A to an ectopic site on the chromosome arm.
Maintaining the mark in the germline The original purification of CENP-A protein was performed out of bovine sperm [34], providing an initial indication that the CENP-A persists at centromeres in the germline. More recent localization studies from diverse species indicate that CENP-A typically is positioned on the chromosome to mark the location of the centromere throughout gametogenesis [35,36]. On chromosome arms, epigenetic regulation of specific genes by altering local DNA methylation patterns results in modulated transcription in germ cells and in developing embryos. Centromeric DNA in diverse species is also often methylated, but the role of DNA methylation in centromere identity and function in somatic and germ cells remains unclear. In most eukaryotes, including mammals, ‘regional’ centromeres (i.e. discrete megabase regions of the chromosome, see Figure 1a) are recombination-free regions and when attached to the meiotic spindle can be used to successfully segregate recombined homologous chromosomes. Holocentric organisms with the centromere running from end-to-end of the chromosome would seemingly have a hopeless problem with organizing recombined homologous chromosomes on the spindle during meiosis I. The nematode Caenorhabditis elegans and its relatives have evolved an elaborate chromosome pairing center system to accommodate the loss of a www.sciencedirect.com
discrete, regional centromere [37], as well as a system to transiently uncouple centromeric chromatin containing a subset of centromere components including CENP-A and CENP-C from the spindle attachment site in meiosis I [38]. Furthermore, CENP-A is removed from C. elegans chromosomes in meiosis II [38], before centromeres are ‘reset’ for mitotic divisions. The mechanism whereby centromere identity is reacquired before the first mitotic division after worm fertilization remains unclear, but its elucidation may shed light on the rare occurrence of de novo establishment of regional centromeres in other eukaryotes.
Crunch time: chromosome segregation and epigenetic centromere marking intertwined Conventional wisdom would suggest that because CENP-A nucleosomes are the foundational ‘building blocks’ of the kinetochore, then the full complement would be required at the centromere during mitosis. Examples from yeast, red algae, and plant species support this notion, because the CENP-A counterpart in each species appears to assemble at centromeres before the entry into mitosis [39–42]. In animal cells, however, centromeres proceed through chromosome segregation having filled only half of the available sites for CENP-A nucleosome assembly [43,44]. Although centromeric DNA is replicated in S-phase, human CENP-A is not expressed until G2 [10] and then sequestered until an assembly reaction at centromeres that begins in telophase and lasts several hours into the subsequent G1 phase (Figure 3) [44]. The temporal coupling of CENP-A assembly at centromeres to mitotic exit suggests a system of epigenetic feedback to faithfully maintain centromere identity. Furthermore, since newly expressed CENP-A is excluded from the chromatin at the foundation of the kinetochore, perhaps centromeric chromatin undergoes maturation steps that require passage through an entire cell cycle for proper mitotic function. The control of temporal loading of CENP-A into centromeres is mediated, at least in part, by the action of the Mis18a/b proteins and KNL2 (also termed M18BP1), because their depletion causes CENP-A to mislocalize [45,46]. KNL2 contains a Myb DNA binding motif, suggesting a role at the level of centromeric DNA, itself. Strikingly, Mis18a/b and KNL2 are recruited to centromeres in the same time window as CENP-A loading, assembling at centromeres in telophase and remaining there for several hours into the subsequent G1 phase (Figure 3) [45,46]. One clue to how these proteins may function comes from the finding that CENP-A mislocalization, upon Mis18a depletion, is rescued by the histone deacetylase inhibitor, Trichostatin A [45]. This suggests a ‘priming’ event whereby the CENP-Acontaining nucleosome, an adjacent nucleosome containing canonical histone H3, or another centromere protein, requires acetylation to license the centromere for Current Opinion in Cell Biology 2008, 20:91–100
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Figure 3
Cell cycle timing of new CENP-A synthesis in G2 and deposition into centromeres during mitotic exit and early G1.
cell-cycle-dependent targeting of newly synthesized CENP-A. It is also noteworthy that both Mis18a/b proteins can interact with the general chromatin assembly factor 1 (CAF-1)-complex members CAF1p46/p48 (also referred to as RbAp46/48) [45] that bind directly to histone H4 [47]. CAF1p46/p48 are capable of loading both canonical H3 and the CENP-A counterpart CID onto plasmid DNA in vitro [48]. In human cells, the entire CAF-1 complex, including CAF1p46/p48, co-purifies with H3.1-containing nucleosomes but not those containing CENP-A [7], raising the intriguing possibility that the centromere priming event in telophase/early G1 involves the distinct pool of centromeric H3-containing nucleosomes that are immediately adjacent to CENP-A nucleosomes and specifically modified with a dimethylation on lysine 4 of the H3 tail [49].
The path to the centromere The centromere targeting determinants for CENP-A were originally mapped to its histone fold domain, as opposed to its N-terminal tail that extends beyond nucleosomal DNA [50]. Indeed, mutations in the Loop 1 region or the adjacent a2 helix of the histone fold domain disrupt centromere localization of CENP-A, but neither region alone is sufficient to serve as a centromere targeted domain capable of converting canonical histone H3 to a centromere targeting histone (Figure 4a) [10,51]. Rather, both regions are required for centromere targeting (Figure 4b) [52]. Furthermore, the CATD confers an essential mitotic function to CENP-A nucleosomal arrays as well as the conformational rigidity observed in individual nucleosomes in which CENP-A replaces H3 [5]. Together, these findings point to a self-perpetuating mechanism to maintain the epigenetic centromere mark. Current Opinion in Cell Biology 2008, 20:91–100
How, then, might newly made CENP-A be distinguished from H3.1 and H3.3 destined for bulk chromatin assembly throughout the rest of the nucleosome? Canonical prenucleosomal histone complexes are found associated with the chromatin assembly protein Asf1 as H3/H4 dimers [53,54,55], and distinct deposition pathways exist where the CAF-1 complex mediates replication-dependent assembly of H3.1 and the HIRA complex mediates replication-independent chromatin assembly of H3.3 (Figure 4c and d) [53]. Besides the aforementioned CAF1p46/p48, other candidates to assemble CENP-A or one of its relatives among known chromatin assembly proteins based on biochemical and genetic evidence include the FACT complex and Hrp1, respectively [7,56]. A simple prediction based on the pathways used by other H3 variants is that the CENP-A chromatin assembly pathway includes a module for recognition of the CENP-A/H4 complex via the CATD (Figure 4e).
Too much of a good thing: limiting CENP-A misincorporation into chromosome arms If the number of sites for CENP-A assembly at centromeres or the CATD-mediated targeting pathway is overfilled, then mistargeting of CENP-A to chromosome arms will occur. Experimentally, this is observed upon overexpression, and overproduction in cells represents a potential mechanism to generate chromosomal instability. Indeed, CENP-A overexpression is observed in a high frequency of aneuploid colon cancer tumors [57]. One mechanism that has been proposed to limit CENP-A spreading beyond centromeres is boundaries made up of classical pericentromeric heterochromatin [3], and examination of distinct chromatin domains forming on HACs supports this notion [58]. A complimentary mechwww.sciencedirect.com
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Figure 4
cis-Acting targeting determinants for CENP-A are provided by the CATD. (a) Targeting of CENP-A requires an intact CATD, because neither the L1 nor the a2 helix is sufficient to confer centromere targeting [10,51,52]. (b) Centromere targeting of H3CATD (green, lower panel). Nuclei are shown in blue and centromeres in red (image reproduced from [52]). Known chromatin assembly complexes for H3.1 (c) and H3.3 (d) variants. Both CAF-1 and HIRA assembly complexes contain dimeric forms of H3.1/H4 or H3.3/H4, respectively, as well as the Asf1 chromatin assembly protein [53] whose binding blocks the H3/H3 interface required for (H3/H4)2 tetramerization. The trimeric H3/H4/Asf1 model is from Protein Data Bank number 2hue [54], and the highlighted residues within the a2 helix of H3 proteins are the replication timing determinants for chromatin assembly that differentiate the pathways used for H3.1/H4 (c) or H3.3/H4 (d), respectively [53,65]. (e) The centromere chromatin assembly pathway uses the CATD (red; shown here in the CENP-A/H4 homology model) to assemble CENP-A nucleosomes at centromeres during mitotic exit and early G1.
anism to constrain CENP-A to the centromere is proteolysis to selectively degrade excess CENP-A. Evidence for such a mechanism comes from diverse experimental systems where a CENP-A-related protein is overexpressed and subsequently degraded in a proteosome-dependent manner [39,59,60]. It is unclear, however, what role, if any, proteolysis plays in animal cells under normal expression conditions where the G2 synthesized pool of CENP-A protein is targeted to centromeres in telophase and G1 and represents the major route for centromere maintenance [43,44].
Outlook Centromere specification is at the nexus of several arenas of biological research: speciation/evolutionary www.sciencedirect.com
biology, artificial chromosome technology, chromatin structure/biochemistry, epigenetics, and cell biology. Although the ‘rules’ governing the establishment and maintenance of centromere identity remain to be entirely elucidated, it is clear that once marked, epigenetic determinants are sufficient to drive inheritance in perpetuity. Centromere re-positioning along the chromosome arm correlates with speciation events in mammals [61], linking centromere identity to genomic events spanning evolutionary timescales. Experiments to expand our understanding of the epigenetic centromere mark are focused on the specialized nucleosome containing CENP-A that wraps centromeric DNA. Lessons learned from the centromere mark — perhaps the longest-lived of all epigenetic marks — will probably Current Opinion in Cell Biology 2008, 20:91–100
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provide insight into other epigenetic pathways that modulate diverse chromosome functions, mostly related to gene expression.
Acknowledgements We apologize to colleagues whose work could not be cited here due to space limitations. BEB is supported in part by a Career Award in the Biomedical Sciences from the Burroughs Wellcome Fund.
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42. Maruyama S, Kuroiwa H, Miyagishima SY, Tanaka K, Kuroiwa T: Centromere dynamics in the primitive red alga Cyanidioschyzon merolae. Plant J 2007, 49:1122-1129.
27. Baum M, Sanyal K, Mishra PK, Thaler N, Carbon J: Formation of functional centromeric chromatin is specified epigenetically in Candida albicans. Proc Natl Acad Sci U S A 2006, 103:14877-14882. Truncated minichromosomes that are mitotically stable in C. albicans are insufficient to establish centromeres de novo when purified and reintroduced to yeast cells as naked DNA, strongly arguing that epigenetic determinants within centromeric chromatin containing a CENP-A relative (CaCse4p) are driving inheritance in this pathogenic yeast.
43. Schuh M, Lehner CF, Heidmann S: Incorporation of Drosophila CID/CENP-A and CENP-C into centromeres during early embryonic anaphase. Curr Biol 2007, 17:237-243. Measurements in syncytial fruit fly embryos undergoing rapid synchronous mitotic divisions demonstrate an increase in CID levels at centromeres in anaphase, suggesting a chromatin assembly event for this CENP-A relative late in mitosis.
28. Han F, Lamb JC, Birchler JA: High frequency of centromere inactivation resulting in stable dicentric chromosomes of maize. Proc Natl Acad Sci U S A 2006, 103:3238-3243. Centromere inactivation in the absence of strong selective pressure was observed at high frequency in maize lines containing dispensable ‘translocation’ chromosomes. 29. Nasuda S, Hudakova S, Schubert I, Houben A, Endo TR: Stable barley chromosomes without centromeric repeats. Proc Natl Acad Sci U S A 2005, 102:9842-9847. 30. Ohzeki J, Nakano M, Okada T, Masumoto H: CENP-B box is required for de novo centromere chromatin assembly on human alphoid DNA. J Cell Biol 2002, 159:765-775. 31. Schueler MG, Higgins AW, Rudd MK, Gustashaw K, Willard HF: Genomic and genetic definition of a functional human centromere. Science 2001, 294:109-115. 32. Okamoto Y, Nakano M, Ohzeki J, Larionov V, Masumoto H: A minimal CENP-A core is required for nucleation and maintenance of a functional human centromere. EMBO J 2007, 26:1279-1291. 33. Heun P, Erhardt S, Blower MD, Weiss S, Skora AD, Karpen GH: Mislocalization of the Drosophila centromere-specific histone CID promotes formation of functional ectopic kinetochores. Dev Cell 2006, 10:303-315. Forcing CID into euchromatin by overexpression is sufficient to seed chromosome arm sites that show centromere activity by occasionally recruiting one or more centromere proteins. 34. Palmer DK, O’Day K, Trong HL, Charbonneau H, Margolis RL: Purification of the centromere-specific protein CENP-A and demonstration that it is a distinctive histone. Proc Natl Acad Sci U S A 1991, 88:3734-3738.
44. Jansen LE, Black BE, Foltz DR, Cleveland DW: Propagation of centromeric chromatin requires exit from mitosis. J Cell Biol 2007, 176:795-805. Temporal labeling of a CENP-A fusion protein containing the SNAP tag is used to demonstrate that the G2 synthesized pool of CENP-A in human tissue culture cells is not immediately loaded onto DNA, but waits until telophase to start loading. Centromere loading of newly expressed CENP-A is restricted to telophase and the first portion of the subsequent G1, pointing to a cell-cycle-coupled centromere targeting mechanism. 45. Fujita Y, Hayashi T, Kiyomitsu T, Toyoda Y, Kokubu A, Obuse C, Yanagida M: Priming of centromere for CENP-A recruitment by human hMis18alpha, hMis18beta, and M18BP1. Dev Cell 2007, 12:17-30. This study explores centromere proteins hMis18a/b and KNL2 (M18BP1; see also [46]) that are recruited to centromeres late in mitosis (and subsequently evicted in G1) and are required for CENP-A accumulation at centomeres, probably participating in a centromere ‘priming’ event that is necessary to maintain centromere identity. 46. Maddox PS, Hyndman F, Monen J, Oegema K, Desai A: Functional genomics identifies a Myb domain-containing protein family required for assembly of CENP-A chromatin. J Cell Biol 2007, 176:757-763. See annotation to [45]. 47. Henikoff S: Versatile assembler. Nature 2003, 423:814-815 817. 48. Furuyama T, Dalal Y, Henikoff S: Chaperone-mediated assembly of centromeric chromatin in vitro. Proc Natl Acad Sci U S A 2006, 103:6172-6177. 49. Sullivan BA, Karpen GH: Centromeric chromatin exhibits a histone modification pattern that is distinct from both euchromatin and heterochromatin. Nat Struct Mol Biol 2004, 11:1076-1083. 50. Sullivan KF, Hechenberger M, Masri K: Human CENP-A contains a histone H3 related histone fold domain that is required for targeting to the centromere. J Cell Biol 1994, 127:581-592.
35. Zhang X, Li X, Marshall JB, Zhong CX, Dawe RK: Phosphoserines on maize CENTROMERIC HISTONE H3 and histone H3 demarcate the centromere and pericentromere during chromosome segregation. Plant Cell 2005, 17:572-583.
51. Vermaak D, Hayden HS, Henikoff S: Centromere targeting element within the histone fold domain of Cid. Mol Cell Biol 2002, 22:7553-7561.
36. Zeitlin SG, Patel S, Kavli B, Slupphaug G: Xenopus CENP-A assembly into chromatin requires base excision repair proteins. DNA Repair (Amst) 2005, 4:760-772.
52. Black BE, Foltz DR, Chakravarthy S, Luger K, Woods VL, Cleveland DW: Structural determinants for generating centromeric chromatin. Nature 2004, 430:578-582.
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54. English CM, Adkins MW, Carson JJ, Churchill ME, Tyler JK: Structural basis for the histone chaperone activity of Asf1. Cell 2006, 127:495-508. This study and [55] provide atomic detail to the mechanism by which Asf1 separates (H3/H4)2 tetramers into H3/H4 dimers as part of the assembly pathways for bulk chromatin. This strongly implies that the H3/H4 dimer, as opposed to the tetramer, is the unit of H3-carried and H4-carried epigenetic marks that persists chromatin reassembly after DNA metabolic events, such as replication.
39. Collins KA, Furuyama S, Biggins S: Proteolysis contributes to the exclusive centromere localization of the yeast Cse4/CENP-A histone H3 variant. Curr Biol 2004, 14: 1968-1972. 40. Pearson CG, Yeh E, Gardner M, Odde D, Salmon ED, Bloom K: Stable kinetochore-microtubule attachment constrains centromere positioning in metaphase. Curr Biol 2004, 14:1962-1967. 41. Lermontova I, Schubert V, Fuchs J, Klatte S, Macas J, Schubert I: Loading of Arabidopsis centromeric histone CENH3 occurs mainly during G2 and requires the presence of the histone fold domain. Plant Cell 2006, 18:2443-2451. www.sciencedirect.com
55. Antczak AJ, Tsubota T, Kaufman PD, Berger JM: Structure of the yeast histone H3-ASF1 interaction: implications for chaperone mechanism, species-specific interactions, and epigenetics. BMC Struct Biol 2006, 6:26. See annotation to [54]. 56. Walfridsson J, Bjerling P, Thalen M, Yoo EJ, Park SD, Ekwall K: The CHD remodeling factor Hrp1 stimulates CENP-A loading to centromeres. Nucleic Acids Res 2005, 33:2868-2879. Current Opinion in Cell Biology 2008, 20:91–100
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58. Lam AL, Boivin CD, Bonney CF, Rudd MK, Sullivan BA: Human centromeric chromatin is a dynamic chromosomal domain that can spread over noncentromeric DNA. Proc Natl Acad Sci U S A 2006, 103:4186-4191.
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The histone variant CENP-A and centromere specification Ben E Black and Emily A Bassett The centromere is the chromosomal locus that guides faithful inheritance. Centromeres are specified epigenetically, and the histone H3 variant CENP-A has emerged as the best candidate to carry the epigenetic centromere mark. Recent advances demonstrate the physical basis for this epigenetic mark whereby CENP-A confers conformational rigidity to the nucleosome it forms with other core histones. This nucleosome is recognized by a multisubunit complex of constitutive centromere proteins, termed the CENP-ANAC. Evidence from two CENP-A relatives in diverse eukaryotes suggests that the histone complexes they form adopt highly unconventional arrangements on DNA. Centromere identity, itself, is propagated during mitotic exit and early G1, and it relies upon a cis-acting targeting domain within CENP-A and a proposed centromere ‘priming’ reaction. Addresses Department of Biochemistry and Biophysics and Program in Biochemistry and Molecular Biophysics, University of Pennsylvania, Philadelphia, PA 19104, United States Corresponding author: Black, Ben E ([email protected])
Current Opinion in Cell Biology 2008, 20:91–100 This review comes from a themed issue on Cell structure and dynamics Edited by Yixian Zheng and Karen Oegema Available online 15th January 2008 0955-0674/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2007.11.007
Introduction The faithful transmission of the genome relies upon a cell division mechanism whereby each replicated chromosome pair, termed sister chromatids, are separated and equally partitioned into daughter cells. This process involves bi-orientation of mitotic chromosomes on the microtubule-based spindle. The centromere is the locus on the chromosome that serves as the spindle connection point, and lessons from chromosomal rearrangements that result in either the duplication or elimination of the centromere strongly suggest the rule that one-and-onlyone centromere is tolerated per chromosome (Figure 1a). More than one centromere will lead to chromosome breakage by the spindle. Conversely, the lack of a centromere will lead an entire chromosome to missegregate. Either of these events is a genetic disaster, and as such, epigenetic mechanisms are in place to maintain centromere identity [1,2]. www.sciencedirect.com
Nucleosomes in which CENP-A replaces canonical H3 are the fundamental units of the chromatin at the foundation of the kinetochore, the mitotic protein assembly that mediates attachment to the mitotic spindle and generates a mitotic checkpoint signal that arrests cells from entering anaphase until all chromosomes are properly aligned on the metaphase plate. CENP-A nucleosomes are found at active but not at inactive centromeres and are strong candidates to carry the epigenetic centromere mark. Many other chromatin-based post-translational modifications and pericentromeric heterochromatin domains are required for full centromere function, which includes controlling sister chromatin cohesion [3]. In this review, however, we will focus on recent advances in understanding the physical basis of how CENP-A-containing nucleosomes distinguish themselves from bulk chromatin as the site for centromere and kinetochore formation, and we will highlight emerging work from diverse eukaryotic systems that sheds light on the cellular pathway leading to cell-cycle-coupled replenishment of the CENP-A mark.
How does CENP-A mark centromeres? CENP-A physically marks centromeres by assembling into a nucleosomal structure that is distinguishable from its canonical counterparts that contain histone H3. In the human version of the CENP-A nucleosome, the a2-helix (Figure 1b, red) and the preceding loop (L1) form the CENP-A targeting domain (CATD) that confers conformational rigidity — a 10-fold slowing of hydrogen exchange along the peptide backbone — to the interface it forms with its binding partner histone H4 (Figure 1b, the a2 and a3 helices of H4 are shown in blue) [4]. The CATD also confers centromere targeting and an essential mitotic function to CENP-A, and together this supports a model where a structurally divergent nucleosome containing CENP-A is the fundamental unit of the chromatin that specifies centromere location [5]. The centromere must accommodate extraordinary physical constraints: firstly, specialized higher-order chromatin folding; secondly, assembly of the mitotic kinetochore complex; and thirdly, mitotic spindle forces [6,7,8]. One particular site in the CENP-A nucleosome that may be important for accommodating these constraints is the entry/exit DNA of the nucleosome. Indeed, Lys49 of CENP-A causes a weakening of its interaction with the entry/exit DNA of the nucleosome compared to within the canonical nucleosome where H3 has an arginine at the corresponding position in its aN helix (Figure 1b; Arg49 in green) [9]. The steady-state unwrapping of 7 bp of DNA at the entry/exit may provide centromeric chromatin arrays with the ability to adopt atypical higherCurrent Opinion in Cell Biology 2008, 20:91–100
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Figure 1
CENP-A nucleosomes are the fundamental unit of centromeric chromatin. (a) CENP-A is found at all active centromeres including those on neocentromeric marker chromosomes (right) that lack the a-satellite repeats typically found in megabase stretches at normal human centromeres (left) [62]. In addition, CENP-A vacates inactive centromeres, such as those found epigenetically silenced in the case of pseudodicentric chromosomes (center). (b) Model of the canonical H3-containing nucleosome (Protein Data Bank number 1kx5; [63]) with the corresponding region highlighted that displays conformational rigidity when assembled with CENP-A in place of histone H3 (the a2 helix of CENP-A in red; the a2 and a3 helices of H4 in blue). Only one pair of CENP-A and H4 is highlighted for reasons of clarity. The side-chain of Arg49 from histone H3 (green) that intercalates into the entry/exit DNA is changed to a lysine in the corresponding position in CENP-A and leads to steady state unwrapping of 7 bp on nucleosomes assembled onto DNA minicircles [9]. Recent studies have indicated diverse arrangements of nucleosomes and histone complexes containing CENP-A (or one of its relatives) in humans (c), flies (d), and budding yeast (e). Although human CENP-A has a tail of identical length to canonical H3, in its counterparts CID (d) and Cse4p (e), large domains that do not display evolutionary conservation extend >100 amino acids outside the predicted nucleosome core. The N-terminal extension includes the END domain in Cse4p that provides an essential function to budding yeast centromeres [64]. Scm3p is an essential component of budding yeast centromeres [13,15,16] that has the ability to displace H2A/H2B dimers from (Cse4p/H4)2 heterotetramers [13], raising the possibility that (Cse4p/H4/Scm3p)2 heterohexamers (panel e, left) wrap centromeric DNA into a nucleosome-like structure (panel e, right).
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CENP-A and centromere specification Black and Bassett 93
order folding to enhance centromere function. The majority of human CENP-A nucleosomes are homotypic (i.e. two copies of CENP-A, see Figure 1c, left) [10], and purification of CENP-A nucleosomes also isolated a smaller pool that are heterotypic (i.e. one copy of CENP-A and one copy of H3, Figure 1c, right) and represents 10% of the total pool of CENP-A nucleosomes [7]. It remains unclear if there is any functional difference between homotypic or heterotypic CENP-A nucleosomes, or whether or not the heterotypic nucleosomes represent a transient form during the genesis of homotypic nucleosomes. Nucleosomes containing the CENP-A fruit fly relative CID were able to form a minor cross-linkable species consistent with an octameric histone arrangement in mitotic chromosome preparations, but they were underrepresented in interphase nuclei preparations [11]. This finding, along with evidence from atomic force microscopy for a structure half the height of typical nucleosomes, as well as micrococcal nuclease protection on immunopurified CID-containing arrays that suggested a compact nucleosomal conformation relative to H3-containing counterparts, led to the proposal that CID is present in a highly unstable ‘half-nucleosome’ arrangement (Figure 1d, left) [11]. The ‘half-nucleosome’ conclusion is heavily reliant upon a single approach: the failure to detect a band at the position expected for an octamer after treatment with the alkali-dependent crosslinker dimethyl suberimidate. Failure to crosslink an octamer is expected, however, because dimethyl suberimidate is specific to primary amines, which are absent from the region that is predicted to hold the two ‘halves’ of the octameric CID nucleosome together (Figure 1d, center and right; the corresponding region in H3 has two primary amines: a lysine in loop 2 (L2) and another in the a3 helix). An alternative interpretation of these data also consistent with the evidence is that CID nucleosomes in interphase nuclei preparations are somewhat more difficult to crosslink into octamers than their mitotic counterparts or H3-containing nucleosomes, and that they generate a compact nucleosomal architecture with larger linker DNA lengths (Figure 1d, possibility #2). Either way, CID probably marks chromatin by conferring structural rigidity to the complexes into which it assembles — in a manner similar to how human CENP-A rigidifies (CENP-A/H4)2 heterotetramers and the octameric nucleosomes it forms [4] — to specify the location of the fruit fly centromere. Additionally, the possibility that there may be interphase tetramer forms (i.e. not nucleosomes) of histone complexes containing CID, may also reflect an ‘immature’ form of centromeric chromatin occurring after DNA replication. At the ‘point’ centromere of budding yeast, an individual nucleosome containing the CENP-A relative Cse4p is found [12], but H2A and H2B are not detectable by www.sciencedirect.com
ChIP [13], despite earlier biochemical evidence to indicate the presence of H2A and H2B in a yeast nucleosome containing Cse4p [14]. Scm3p, an essential budding yeast centromere component [13,15,16], has the ability to displace H2A/H2B from an (H2A/H2B/ Cse4p/H4)2 octamer [13]. Taken together, these findings suggest that an atypical ‘nucleosome’ containing Scm3p in place of H2A/H2B dimers forms at yeast centromeres (Figure 1e) [13]. An equally interesting possibility is that Cse4p is maintained at centromeres outside a normal nucleosome for most of the cell cycle by Scm3p, transiently assembling into an octameric (H2A/ H2B/Cse4p/H4)2 nucleosome configuration required for mitosis.
What does the CENP-A nucleosomal array recruit to centromeres? The CENP-A-containing nucleosome in human cells is physically associated with a six subunit protein complex, termed the CENP-A Nucleosome Associated Complex (NAC), that contains CENP-C, CENP-H, CENP-M, CENP-N, CENP-T, and CENP-U(50) [7]. At least a subset of CENP-ANAC components are tethered to a complex that is more distal to the CENP-A nucleosome and contains CENP-I, CENP-K, CENP-L, CENP-O, CENP-P, CENP-Q, CENP-R, and CENP-S [7,17,18]. The position of the CENP-A nucleosome — wrapping centromeric DNA — suggests it sits atop the centromere/kinetochore-building hierarchy. Depletion experiments of various centromere components, including CENP-A itself, from diverse eukaryotes support this view [19–21]. Furthermore, the initial step of CENP-ANAC recruitment provides a molecular linkage from the CENP-A nucleosome outward to the kinetochore.
Coalescing CENP-A nucleosomes to form a three-dimensional array A chromosome stretching experiment from Bill Brinkley and colleagues initially suggested a multi-subunit model for centromere organization [22]. This arrangement was more recently shown to extend down to the CENP-A nucleosome array (Figure 2a), as physically unfolded centromeric subunits are interspersed with chromatin spacers of nucleosomes containing canonical H3 [6]. Furthermore, CENP-A shows a strong preference for assembling at specific locations within a neocentromere [23], and constitutive centromere components CENP-C and CENP-H are specifically found in close proximity to the CENP-A-rich regions [24]. Although it is not clear what drives positioning of CENP-A nucleosomes at preferred sites in nonrepetitive DNA, these findings suggest a nonrandom arrangement along linear DNA. CENP-A nucleosome coalescence then organizes the arrays into the higher-order chromatin structure that functions as the foundation of the mitotic kinetochore. It is currently unclear whether physical interactions between neighboring CENP-A nucleosomes self-direct the coalescence of Current Opinion in Cell Biology 2008, 20:91–100
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Figure 2
Achieving the higher-order chromatin structure found at the centromere. (a) Centromere stretching experiment indicating that the array of CENP-A nucleosomes, coalesced in three-dimensional space, are not contiguous along the DNA but are interrupted by spacers containing blocks of H3containing nucleosomes. The image was kindly provided by Gary Karpen and reproduced from [6]. CENP-A nucleosome coalescence could be entirely self-directed (b) or may necessitate the action of bridging factors — perhaps components of the CENP-ANAC—to organize into the array that forms the foundation of the mitotic kinetochore (c). For simplicity, centromeric chromatin is shown in a coiled conformation, but no data have emerged to indicate the actual higher-order fold that culminates in the coalesced array of CENP-A nucleosomes at the foundation of the mitotic kinetochore.
the CENP-A array (Figure 2b), or if one or more CENPANAC proteins (or perhaps other centromere proteins) act as bridging factors to guide this process (Figure 2c). Reinforcing the epigenetic mark by preserving centromere and kinetochore structure is likely to involve several Current Opinion in Cell Biology 2008, 20:91–100
proteins, and indeed CENP-A levels are negatively affected at human centromeres after depletion of Mis12 [25], reminiscent of the phenotypes observed after mutation of several fission yeast genes including Mis6, Mis12, and Sim4 (reviewed elsewhere [3]). www.sciencedirect.com
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Seeding new centromeres: the chicken and the egg Once formed, centromeres are epigenetically maintained at the same locus, independent of DNA sequence, cell division after cell division and generation after generation [26,27,28,29], so why are megabase stretches of these repeats found at most eukaryotic centromeres? Clues come from human artificial chromosome (HAC) constructs whose establishment in cells require intact a-satellites (the human version of the centromere repeat containing the 17-mer CENP-B binding box) as well as the expression of CENP-B — a protein that is otherwise dispensable for centromere function and epigenetic maintenance [30,31]. Future experiments with HACs will further elucidate the initial events in centromere formation, defined by the generation of an array of CENP-A nucleosomes to specify the centromere as well as a crucial adjacent chromatin domain resembling bona fide pericentromeric heterochromatin domains (i.e. enriched with nucleosomes where histone H3 is modified with a trimethylation at Lys9) [32]. In principle, another method for seeding new centromeres is to deliver the epigenetic mark to a noncentromeric region of the chromosome. Indeed, transient overexpression of CID, so that it replaces histone H3 in euchromatic regions of fruit fly chromosomes, leads to the occasional recruitment of one or more centromere/kinetochore proteins [33], so it may be possible to form a fully functional centromere by targeting CENP-A to an ectopic site on the chromosome arm.
Maintaining the mark in the germline The original purification of CENP-A protein was performed out of bovine sperm [34], providing an initial indication that the CENP-A persists at centromeres in the germline. More recent localization studies from diverse species indicate that CENP-A typically is positioned on the chromosome to mark the location of the centromere throughout gametogenesis [35,36]. On chromosome arms, epigenetic regulation of specific genes by altering local DNA methylation patterns results in modulated transcription in germ cells and in developing embryos. Centromeric DNA in diverse species is also often methylated, but the role of DNA methylation in centromere identity and function in somatic and germ cells remains unclear. In most eukaryotes, including mammals, ‘regional’ centromeres (i.e. discrete megabase regions of the chromosome, see Figure 1a) are recombination-free regions and when attached to the meiotic spindle can be used to successfully segregate recombined homologous chromosomes. Holocentric organisms with the centromere running from end-to-end of the chromosome would seemingly have a hopeless problem with organizing recombined homologous chromosomes on the spindle during meiosis I. The nematode Caenorhabditis elegans and its relatives have evolved an elaborate chromosome pairing center system to accommodate the loss of a www.sciencedirect.com
discrete, regional centromere [37], as well as a system to transiently uncouple centromeric chromatin containing a subset of centromere components including CENP-A and CENP-C from the spindle attachment site in meiosis I [38]. Furthermore, CENP-A is removed from C. elegans chromosomes in meiosis II [38], before centromeres are ‘reset’ for mitotic divisions. The mechanism whereby centromere identity is reacquired before the first mitotic division after worm fertilization remains unclear, but its elucidation may shed light on the rare occurrence of de novo establishment of regional centromeres in other eukaryotes.
Crunch time: chromosome segregation and epigenetic centromere marking intertwined Conventional wisdom would suggest that because CENP-A nucleosomes are the foundational ‘building blocks’ of the kinetochore, then the full complement would be required at the centromere during mitosis. Examples from yeast, red algae, and plant species support this notion, because the CENP-A counterpart in each species appears to assemble at centromeres before the entry into mitosis [39–42]. In animal cells, however, centromeres proceed through chromosome segregation having filled only half of the available sites for CENP-A nucleosome assembly [43,44]. Although centromeric DNA is replicated in S-phase, human CENP-A is not expressed until G2 [10] and then sequestered until an assembly reaction at centromeres that begins in telophase and lasts several hours into the subsequent G1 phase (Figure 3) [44]. The temporal coupling of CENP-A assembly at centromeres to mitotic exit suggests a system of epigenetic feedback to faithfully maintain centromere identity. Furthermore, since newly expressed CENP-A is excluded from the chromatin at the foundation of the kinetochore, perhaps centromeric chromatin undergoes maturation steps that require passage through an entire cell cycle for proper mitotic function. The control of temporal loading of CENP-A into centromeres is mediated, at least in part, by the action of the Mis18a/b proteins and KNL2 (also termed M18BP1), because their depletion causes CENP-A to mislocalize [45,46]. KNL2 contains a Myb DNA binding motif, suggesting a role at the level of centromeric DNA, itself. Strikingly, Mis18a/b and KNL2 are recruited to centromeres in the same time window as CENP-A loading, assembling at centromeres in telophase and remaining there for several hours into the subsequent G1 phase (Figure 3) [45,46]. One clue to how these proteins may function comes from the finding that CENP-A mislocalization, upon Mis18a depletion, is rescued by the histone deacetylase inhibitor, Trichostatin A [45]. This suggests a ‘priming’ event whereby the CENP-Acontaining nucleosome, an adjacent nucleosome containing canonical histone H3, or another centromere protein, requires acetylation to license the centromere for Current Opinion in Cell Biology 2008, 20:91–100
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Figure 3
Cell cycle timing of new CENP-A synthesis in G2 and deposition into centromeres during mitotic exit and early G1.
cell-cycle-dependent targeting of newly synthesized CENP-A. It is also noteworthy that both Mis18a/b proteins can interact with the general chromatin assembly factor 1 (CAF-1)-complex members CAF1p46/p48 (also referred to as RbAp46/48) [45] that bind directly to histone H4 [47]. CAF1p46/p48 are capable of loading both canonical H3 and the CENP-A counterpart CID onto plasmid DNA in vitro [48]. In human cells, the entire CAF-1 complex, including CAF1p46/p48, co-purifies with H3.1-containing nucleosomes but not those containing CENP-A [7], raising the intriguing possibility that the centromere priming event in telophase/early G1 involves the distinct pool of centromeric H3-containing nucleosomes that are immediately adjacent to CENP-A nucleosomes and specifically modified with a dimethylation on lysine 4 of the H3 tail [49].
The path to the centromere The centromere targeting determinants for CENP-A were originally mapped to its histone fold domain, as opposed to its N-terminal tail that extends beyond nucleosomal DNA [50]. Indeed, mutations in the Loop 1 region or the adjacent a2 helix of the histone fold domain disrupt centromere localization of CENP-A, but neither region alone is sufficient to serve as a centromere targeted domain capable of converting canonical histone H3 to a centromere targeting histone (Figure 4a) [10,51]. Rather, both regions are required for centromere targeting (Figure 4b) [52]. Furthermore, the CATD confers an essential mitotic function to CENP-A nucleosomal arrays as well as the conformational rigidity observed in individual nucleosomes in which CENP-A replaces H3 [5]. Together, these findings point to a self-perpetuating mechanism to maintain the epigenetic centromere mark. Current Opinion in Cell Biology 2008, 20:91–100
How, then, might newly made CENP-A be distinguished from H3.1 and H3.3 destined for bulk chromatin assembly throughout the rest of the nucleosome? Canonical prenucleosomal histone complexes are found associated with the chromatin assembly protein Asf1 as H3/H4 dimers [53,54,55], and distinct deposition pathways exist where the CAF-1 complex mediates replication-dependent assembly of H3.1 and the HIRA complex mediates replication-independent chromatin assembly of H3.3 (Figure 4c and d) [53]. Besides the aforementioned CAF1p46/p48, other candidates to assemble CENP-A or one of its relatives among known chromatin assembly proteins based on biochemical and genetic evidence include the FACT complex and Hrp1, respectively [7,56]. A simple prediction based on the pathways used by other H3 variants is that the CENP-A chromatin assembly pathway includes a module for recognition of the CENP-A/H4 complex via the CATD (Figure 4e).
Too much of a good thing: limiting CENP-A misincorporation into chromosome arms If the number of sites for CENP-A assembly at centromeres or the CATD-mediated targeting pathway is overfilled, then mistargeting of CENP-A to chromosome arms will occur. Experimentally, this is observed upon overexpression, and overproduction in cells represents a potential mechanism to generate chromosomal instability. Indeed, CENP-A overexpression is observed in a high frequency of aneuploid colon cancer tumors [57]. One mechanism that has been proposed to limit CENP-A spreading beyond centromeres is boundaries made up of classical pericentromeric heterochromatin [3], and examination of distinct chromatin domains forming on HACs supports this notion [58]. A complimentary mechwww.sciencedirect.com
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Figure 4
cis-Acting targeting determinants for CENP-A are provided by the CATD. (a) Targeting of CENP-A requires an intact CATD, because neither the L1 nor the a2 helix is sufficient to confer centromere targeting [10,51,52]. (b) Centromere targeting of H3CATD (green, lower panel). Nuclei are shown in blue and centromeres in red (image reproduced from [52]). Known chromatin assembly complexes for H3.1 (c) and H3.3 (d) variants. Both CAF-1 and HIRA assembly complexes contain dimeric forms of H3.1/H4 or H3.3/H4, respectively, as well as the Asf1 chromatin assembly protein [53] whose binding blocks the H3/H3 interface required for (H3/H4)2 tetramerization. The trimeric H3/H4/Asf1 model is from Protein Data Bank number 2hue [54], and the highlighted residues within the a2 helix of H3 proteins are the replication timing determinants for chromatin assembly that differentiate the pathways used for H3.1/H4 (c) or H3.3/H4 (d), respectively [53,65]. (e) The centromere chromatin assembly pathway uses the CATD (red; shown here in the CENP-A/H4 homology model) to assemble CENP-A nucleosomes at centromeres during mitotic exit and early G1.
anism to constrain CENP-A to the centromere is proteolysis to selectively degrade excess CENP-A. Evidence for such a mechanism comes from diverse experimental systems where a CENP-A-related protein is overexpressed and subsequently degraded in a proteosome-dependent manner [39,59,60]. It is unclear, however, what role, if any, proteolysis plays in animal cells under normal expression conditions where the G2 synthesized pool of CENP-A protein is targeted to centromeres in telophase and G1 and represents the major route for centromere maintenance [43,44].
Outlook Centromere specification is at the nexus of several arenas of biological research: speciation/evolutionary www.sciencedirect.com
biology, artificial chromosome technology, chromatin structure/biochemistry, epigenetics, and cell biology. Although the ‘rules’ governing the establishment and maintenance of centromere identity remain to be entirely elucidated, it is clear that once marked, epigenetic determinants are sufficient to drive inheritance in perpetuity. Centromere re-positioning along the chromosome arm correlates with speciation events in mammals [61], linking centromere identity to genomic events spanning evolutionary timescales. Experiments to expand our understanding of the epigenetic centromere mark are focused on the specialized nucleosome containing CENP-A that wraps centromeric DNA. Lessons learned from the centromere mark — perhaps the longest-lived of all epigenetic marks — will probably Current Opinion in Cell Biology 2008, 20:91–100
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provide insight into other epigenetic pathways that modulate diverse chromosome functions, mostly related to gene expression.
Acknowledgements We apologize to colleagues whose work could not be cited here due to space limitations. BEB is supported in part by a Career Award in the Biomedical Sciences from the Burroughs Wellcome Fund.
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