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260–271 (2002).19. Kaksonen, M., Toret, C. P. & Drubin, D. G. Nature Rev.

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a reader for centromeric chromatinNikolina Sekulic and Ben E. Black

For nucleosome-encoded epigenetic information to be transmitted, an epigenetic mark requires a ‘reader’ for its physical recognition. CenP-n has now been identified as a reader of the centromere-specifying epigenetic mark that is generated by incorporation of the histone H3 variant CenP-a into centromeric nucleosomes.

Centromeres are defined epigenetically, and nucleosomes containing the histone H3 variant CENP-A are thought to provide the epigenetic information required to specify centromere location on the chromosome. As for other chromatin marks, it is important to understand how the epigenetic information is physically transmitted from the chromatin. On page 896 of this issue, Straight and colleagues take a step towards our understanding of the centromere by identifying CENP-N as a reader of the centromere mark comprising CENP-A-containing nucleosomes1.

How centromeres work has been a long-standing target of investigation due to their central role in directing the stable inheritance of the chromosome and all the genes present on it. Furthermore, the epigenetic centromere mark is perhaps the longest lived of all epige-netic marks, surviving through the germ line and (with rare exceptions) maintaining its location at the same chromosomal locus on the evolutionary timescales of speciation events. Yet, our understanding of the centromere mark lags behind its shorter-lived counterparts else-where on the chromosome, which are involved in other biological processes. In the case of its nearest neighbour, pericentromeric heterochro-matin, the identity of the critical nucleosomal mark (di- or tri-methylated K9 on the tail of histone H3) and reader (HP1) helped to develop early models for the spread and propagation of a chromosome domain2. Relative to our under-standing of the epigenetic pathways at the peri-centromere, our knowledge of the centromere lags behind; however, the centromeric field is

catching up with the identification of CENP-N as a reader1, along with other notable recent progress in describing the physical basis for marking the centromere3 and characterizing candidate molecules involved in cell-cycle-coupled replenishment of the mark4–7.

Before considering the reader itself, what is the nature of the CENP-A-generated mark? Many nucleosome-encoded marks elsewhere on the chromosome involve post-translational modifi-cation of the tails of conventional histones, with the tail of histone H3 serving as a particularly common target8. H3 is replaced by CENP-A in the centromeric nucleosome, which removes most of the common H3 tail modification sites and supplies a related but structurally distinct histone fold domain9. Many lines of evidence support the notion that the structural core of the CENP-A nucleosome is key to marking cen-tromeres. An engineered histone H3 chimaera (H3CATD), which contains the intact tail from H3 but harbours 22 amino-acid substitutions in the Loop 1 and α2 helix regions within the histone fold of CENP-A, is sufficient to target H3 to the centromere and replace the mitotic func-tion of CENP-A9,10. This domain, termed CATD (CENP-A targeting domain), confers nucleo-somal rigidity to CENP-A-containing nucleo-somes at the centromere3, providing a unique structure, which enables the reader to distinguish CENP-A-containing nucleosomes from bulk nucleosomes elsewhere on the chromosome that contain conventional H3 (Fig. 1a). The CATD may also provide a subset of exposed side-chains within its Loop 1, specific to the CENP-A nucleo-some, which could be directly recognized by the reader (Fig. 1b). In either case, the centromere reader is predicted to bypass the histone tails and hone in on the core of the CENP-A-containing nucleosome (Fig. 1c).

To investigate the reading of the centromere mark, Straight and colleagues rounded up the usual suspects, screening each of the 14 known constitutive non-histone human centromere proteins, one-by-one, for their ability to bind to CENP-A-containing mononucleosomes assem-bled from recombinant purified components1. While centromeres comprise a polynucleosome array of CENP-A-containing nucleosomes, the authors reasoned that one or more known cen-tromere components could directly recognize CENP-A-containing mononucleosomes, which represent the fundamental unit of centromere-specifying chromatin. The most likely candi-dates were the centromere proteins (CENP-B, CENP-C, CENP-H, CENP-M, CENP-N, CENP-T and CENP-U) that co-purify with CENP-A-containing nucleosomes, but not with their canonical counterparts containing conventional H3 (ref. 11).

There are several possible means by which each of these proteins could associate with the centromere-specifying nucleosome: by directly recognizing the α-satellite DNA sequence that wraps most CENP-A nucleosomes in the cell (as is well-established in the case of CENP-B association); by association with nucleosomes containing H3 that are directly adjacent to CENP-A-containing nucleosomes at the cen-tromere (as has been proposed for CENP-T and its close binding partner CENP-W12); by direct recognition of, and binding to, CENP-A nucleosomes independently of DNA sequence; or by indirect interaction (that is, in an arrange-ment bridged by another constitutive centro-mere component). Of the 14 candidates, only two, CENP-B and CENP-N, directly bound to the reconstituted CENP-A-containing mono-nucleosomes1. The association of CENP-B was, as expected, dependent on the α-satellite

Nikolina Sekulic and Ben E. Black are in the Department of Biochemistry and Biophysics, University of Pennsylvania, PA 19104–6059, USA.e-mail: blackbe@mail.med.upenn.edu

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sequences present in the nucleosomal DNA, but independent of the presence of CENP-A. On the other hand CENP-N, bound to CENP-A-containing nucleosomes independ-ently of DNA sequence. This critical finding makes CENP-N a prime candidate to represent a chromatin reader that specifically recognizes centromere-specifying nucleosomes from the presence of CENP-A in place of H3.

Straight and colleagues used SNAP pulse labelling, an approach used in earlier experi-ments to identify the telophase/G1 phase of new CENP-A assembly13, to demonstrate that CENP-N is required for the robust assembly of CENP-A nucleosomes1. The mechanism by which CENP-N and other constitutive centromere-associated network (CCAN) pro-teins participate in centromere reinforcement remains unclear, but may involve a high order of centromeric chromatin organization; pos-sibly required for efficient CENP-A nucleo-some assembly and telophase recruitment (directly or indirectly) of the Mis18 cen-tromere licensing complex4,5 or telophase/G1 recruitment of the HJURP chromatin assembly complex6,7. Disruption of CENP-N

by mutation or depletion yields mislocaliza-tion of some, but not all, CCAN constituents, culminating in defective kinetochores1. All of this supports the notion that the role of CENP-N in reading the centromere mark is a critical step in ensuring accurate chromo-some segregation at cell division.

In considering CENP-N as a candidate chromatin reader, it is important to distin-guish it from proteins that recognize a pre-nucleosomal histone complex destined for the centromere, as such proteins would be more likely to represent chromatin assembly proteins, chaperoning CENP-A–H4 complexes before incorporation into centromeric DNA. Recent candidates to represent the CENP-A-specific chromatin assembly proteins include HJURP in mammals6,7 and Sim3 and Scm3 in fission yeast14,15. Several lines of evidence argue against a role for CENP-N in chaper-oning pre-nucleosomal CENP-A complexes: CENP-N is present at the centromere through-out the cell cycle11 (as opposed to transient association following mitotic exit when new CENP-A nucleosomes are assembled), it is absent from the soluble HJURP-containing

complex that chaperones newly expressed CENP-A to the centromere6,7 and it does not seem to selectively bind to non-nucleosomal CENP-A1. Instead, CENP-N selectively binds to CENP-A-containing nucleosomes with an apparent Kd of 169 nM (ref. 1).

To determine how CENP-N reads the centromere-specifying nucleosome, Straight and colleagues compared its binding to three types of nucleosomes: conventional nucleo-somes containing the canonical histone octamer, nucleosomes in which the two cop-ies of H3 are replaced by CENP-A and those in which they are replaced by the chimaeric H3CATD. Although only background levels of binding to conventional nucleosomes were detected, CENP-N bound to CENP-A- and H3CATD-containing nucleosomes with nearly identical affinity1. Thus, CENP-N recognition of CENP-A-containing nucle-osomes involves not the tail of CENP-A, but instead the unique physical properties imparted by the CATD. It will be interest-ing to determine whether CENP-N reads the centromere-specifying nucleosome by recognizing CATD-directed nucleosomal

H4 2 helix

CENP-A 2 helixH4 3 helix

76

76

93

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Figure 1 Physical features that could be recognized by a reader of centromere-specifying nucleosomes. (a) Conformational rigidity of the CENP-A-containing nucleosome. The α2 helix within the CATD of CENP-A (red) and the α2 and α3 helices of H4 (blue) are protected from amide proton exchange3 indicative of structural rigidity that a reader could use to distinguish centromere-specifying nucleosomes from their conventional counterparts containing H3. (b) Surface-exposed side chains from a subset of residues of Loop 1 within the CATD of CENP-A in a molecular surface representation of a canonical nucleosome in the same orientation as in a with DNA in blue and histones in tan. Exposed regions corresponding to the predicted location of the CATD are coloured in red (CENP-A specific substitutions) and green (conserved between H3 and CENP-A). Note that only a small part of the C-terminal α2 helix is predicted to be solvent exposed (upper left green patch) where the surface residues are identical between H3 and CENP-A. A larger exposed area mostly consists of Loop 1 (circled). In the alignment (top), exposed positions in this region of the canonical nucleosome are highlighted in yellow, red letters indicate CENP-A-specific residues, black letters indicate H3-specific residues, and green letters indicate residues conserved between the two proteins. These side chains may be recognized by a centromere reader. Models in a and b are based on the crystal structure of a canonical nucleosome; PDB ID 1KX5. (c) Diagrams depicting distinct modes of reading tail modifications on conventional nucleosomes versus the predicted requirements for a candidate reader of CENP-A-containing nucleosomes. While epigenetic mark readers (orange) for conventional nucleosomes commonly recognize modified tail residues (black), a centromere mark reader (blue) is likely to recognize the rigid core of the CENP-A-containing nucleosome and/or specific residues protruding from its surface.

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rigidity (Fig. 1a), exposed side-chains spe-cific to the CATD (Fig. 1b) or a combina-tion of these unique physical signatures of CENP-A-containing nucleosomes.

Centromere-specific chromatin uses a dif-ferent strategy for its propagation than that used at the pericentromere (Fig. 2). In peri-centromeric heterochromatin propagation, the HP1 reader recruits the siRNA-contain-ing RITS complex and a methyltransferase (Clr4 in fission yeast, for instance) to meth-ylate neighbouring nucleosomes on H3 K9, as well as undergoing self–self interactions to recruit additional HP1 molecules2. In cen-tromere-specifying chromatin propagation, the CENP-N reader directly interacts with a network of constitutive centromere proteins

that together generate a chromatin compart-ment that is competent to recruit the Mis18 licensing complex and subsequently recruit the HJURP-containing centromeric chroma-tin assembly complex to load new CENP-A nucleosomes following mitotic exit.

In the context of centromeric chromatin — where there remain many biochemically uncharacterized components — this study provides the first glimpse at the reading of the mark generated by CENP-A nucleosomes, adding a key piece to our understanding of centromere identity and the assembly of a functional kinetochore. These findings will undoubtedly serve as a bridge to future stud-ies to elucidate the mechanisms used to mark, read and propagate centromeric chromatin.

1. Carroll, C. W., Silva, M. C. C., Godek, K. M., Jansen, L. E. T. & Straight, A. F. Nature Cell Biol. 11, 896–902 (2009).

2. Cam, H. P., Chen, E. S. & Grewal, S. I. Cell 136, 610–614 (2009).

3. Black, B. E., Brock, M. A., Bédard, S., Woods, V. L. & Cleveland, D. W. Proc. Natl Acad. Sci. USA 104, 5008–5013 (2007).

4. Fujita, Y. et al. Dev. Cell 12, 17–30 (2007).5. Maddox, P. S., Hyndman, F., Monen, J., Oegema, K. &

Desai, A. J. Cell Biol. 176, 757–763 (2007).6. Foltz, D. R. et al. Cell 137, 472–484 (2009).7. Dunleavy, E. M. et al. Cell 137, 485–497 (2009).8. Taverna, S. D., Li, H., Ruthenburg, A. J., Allis, C. D. &

Patel, D. J. Nature Struct. Mol. Biol. 14, 1025–1040 (2007).

9. Black, B. E. et al. Nature 430, 578–582 (2004).10. Black, B. E. et al. Mol. Cell 25, 309–322 (2007).11. Foltz, D. R. et al. Nature Cell Biol. 8, 458–469

(2006).12. Hori, T. et al. Cell 135, 1039–1052 (2008).13. Jansen, L. E., Black, B. E., Foltz, D. R. & Cleveland,

D. W. J. Cell Biol. 176, 795–805 (2007).14. Pidoux, A. L. et al. Mol. Cell 33, 299–311 (2009).15. Williams, J. S., Hayashi, T., Yanagida, M. & Russell, P.

Mol. Cell 33, 287–298 (2009).

Centromere Pericentromere

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Histone methyltransferase

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Recruitingpropagation

factors

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Figure 2 Distinct modes of epigenetic propagation at the pericentromere and centromere. At centromeric chromatin, CENP-A nucleosome are read by CENP-N. CCAN components are recruited by interactions with CENP-N, adjacent nucleosomes, and other CCAN components. To propagate the centromeric mark, the Mis18 complex is recruited to the centromere in telophase, followed by the recruitment of a prenucleosomal CENP-A/H4 histone complex chaperoned by HJURP to assemble new CENP-A-containing nucleosomes following mitotic exit every cell cycle. At pericentromeric heterochromatin, nucleosomes, marked by di- or tri-methylation of histone H3, are read by HP1-related proteins. Propagation factors, including histone methyltransferases and the siRNA-containing RITS complex are recruited by interactions with HP1 and each other, as well as by potential modes of recruitment not shown in this diagram including direct binding of the RITS complex by its Chp1 subunit and base pairing between the RITS-bound siRNA and nascent pericentromeric transcripts in fission yeast. To propagate the pericentromeric mark, neighbouring nucleosomes are methylated and local recruitment of additional HP1 molecules is facilitated by its self–self interactions.

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