Skip to main content

New clues to understand how CENP-A maintains centromere identity

Abstract

The centromere is a specialized chromosomal region that directs the formation of the kinetochore, a huge protein assembly that acts as the attachment site for spindle microtubules and carries out chromosome movement during cell division. Centromere loss or the presence of extra centromeres adversely affect chromosome segregation and may result in aneuploidy, a condition found in many human tumors and a major cause of miscarriages and birth defects. Consequently, understanding the basis of centromere determination and propagation is of great relevance to both fundamental and clinical research. In recent years, it has become clear that centromeres are defined by the presence of a histone H3 variant known as Centromere Protein A, CENP-A, or CenH3. Much effort has been devoted to understanding the mechanisms that drive the assembly of CENP-A containing nucleosomes exclusively onto centromeric DNA, as well as the peculiar structure of these nucleosomes. We have recently developed an immunofluorescence-based assay that measures CENP-A incorporation in the centromeres of chromosomes assembled in Xenopus egg extracts. The spatial and temporal specificity of CENP-A deposition observed in human cells can be recapitulated in this in vitro system, making it suitable to dissect the precise role of the different factors that contribute to this pathway. Here, we discuss our results together with other recent advances in our understanding of the mechanisms that mediate centromere inheritance.

Introduction

Active centromeres are defined by the presence of nucleosomes containing a unique histone H3 variant known as CENP-A [1, 2]. Stretched centromeric chromatin from Drosophila, human and chicken DT40 cells shows the presence of interspersed blocks of CENP-A and canonical H3 nucleosomes [3, 4]. This chromatin fiber must then be folded to adopt the appropriate compact conformation over which the kinetochore is assembled [5, 6]. In proliferating cells, the amount of CENP-A present at centromeres has to be replenished at the time or after centromeric DNA is duplicated in order to propagate centromere identity. The mechanisms responsible for de novo CENP-A deposition specifically at centromeres and their regulation in the cell cycle have been an active field of research over the last years. Despite the lack of conservation of centromeric DNA sequences and of the timing of CENP-A nucleosome assembly among different organisms, there is a remarkable conservation of the major players involved in centromere propagation. Thus, research in different experimental systems greatly contributes to put the big picture in focus.

Discussion

Mechanisms of CENP-A incorporation mediated by HJURP

One central question of centromere biology is how is CENP-A deposited at centromeric chromatin. Members of a protein family named Scm3/HJURP (Holliday Junction Recognizing Protein), conserved from yeast to humans, have been proposed to act as CENP-A specific chaperones in yeast and human cells [715]. These proteins interact physically with CENP-A, can be found at centromeres and, most importantly, are required for CENP-A loading and maintenance. Many additional factors play a role in CENP-A incorporation, but how it actually happens remains unclear. To address this issue, we developed an assay to measure CENP-A incorporation using the Xenopus egg cell-free system [16]. In this immunofluorescence-based assay, nuclei assembled from sperm chromatin and taken at two different time points (e.g. mitosis and subsequent interphase) are combined and processed for immunofluorescence with a CENP-A specific antibody, imaged together, and the centromeric CENP-A signals measured to assess the average difference in intensity between the centromeres within each pair (see Figure 1). We first showed that, in the egg extracts, CENP-A incorporation occurs upon exit from mitosis but independently of DNA replication, same as in Drosophila embryos and human cells [1720]. Specific immunodepletion of proteins involved in the deposition of other histone H3 variants (CAF-1 and HIRA [21]), did not affect significantly the incorporation of CENP-A. In contrast, we found that CENP-A deposition depends on Xenopus HJURP (xHJURP) and that human HJURP can replace xHJURP. In fact, xHJURP is stored in the oocyte cytoplasm in association with CENP-A, which supports the idea that they form a pre-assembly complex. Analysis of the crystal structure of the CENP-A binding domain of Scm3 in complex with CENP-A and H4 reveals that the interaction of Scm3/HJURP with the CENP-A/H4 dimer is not compatible with binding to DNA further supporting that this is indeed a pre-assembly complex [22].

Figure 1
figure 1

An assay to measure CENP-A incorporation in Xenopus egg extracts. Step 1: Extract preparation and depletion. Extracts are prepared from laid eggs arrested in metaphase II (mitotic extracts) and subjected to immunodepletion with specific antibodies against factor X. Step 2: Nuclei assembly. Sperm chromatin is added to mitotic extracts in two different tubes. In one tube (top), incubation proceeds for 80-120 min to get mitotic chromosomes. In another tube (bottom), calcium is added to drive entry into interphase 40 min after sperm addition. Incubation proceeds for 80 min to get interphase nuclei that have undergone replication. Step 3: Nuclei isolation. Equal volumes of the reaction mixtures in both tubes are combined, fixed and centrifuged over a coverslip placed at the bottom of a glycerol cushion. Step 4: Immunofluorescence. Coverslips are processed for immunofluorescence with an antibody against CENP-A and DNA is stained with DAPI. Step 5: Image acquisition and analysis. Images of a mass of mitotic chromosomes next to an interphase nucleus are acquired and CENP-A signals are quantitated using ImageJ software (http://rsb.info.nih.gov/ij/). The average Integrated Density (ID = average pixel intensity × area) is first calculated from the IDs of individual centromeres within each interphase nucleus (IDI) and neighboring mass of mitotic chromosomes (IDM) and then a ratio between the IDs of each imaged pair is obtained (IDr). Finally, the average ID ratio (IDr) of at least 15 pairs is calculated. The relative CENP-A loading efficiency of a depleted extract with respect to a mock depleted extract (considered 100%) is calculated and represented. If depletion of a factor X prevents loading of CENP-A and the defect is rescued by adding back the factor to the depleted extract, we conclude that factor X is involved in CENP-A incorporation.

How HJURP works is still a mystery. It has been recently speculated that its action at centromeres could be coupled to DNA repair [23] or to transcription [24]. In vitro, Scm3 can incorporate CENP-A-containing octamers into plasmid DNA, but not octamers bearing canonical H3 [25]. This probably reflects the fact that the CENP-A targeting domain (CATD), comprised of loop1 and helix 2 of the histone fold domain, not present in H3, is required for the interaction with Scm3/HJURP [14, 26]. Importantly, the histone chaperone Nap1 can also do the job (this time for both CENP-A and H3-containing octamers), as it was previously shown for RbAp48 [27]. On the other hand, it is unclear what kind of nucleosomes Scm3/HJURP deposits onto DNA. In one study, nucleosome assembly was not observed upon incubation of Smc3/CENP-A/H4 with plasmid DNA unless H2A/H2B dimers were included in the mixture [25]. However, another group had previously achieved nucleosome reconstitution with this complex and linear lambda DNA [28] and evidence for CENP-A/H4 tetramer deposition on a plasmid template has also been reported for human HJURP [15]. Further studies will be required to explain these controversial results as well as the exact composition of CENP-A nucleosomes in vivo (tetrasome, hexasomes, octamers?) and whether they wrap DNA in the conventional left-handed manner or the opposite (for a recent review on these matters, see [29]).

The role of condensin II in CENP-A deposition and maintenance

Consistent with similar results in human cells, we observed that efficient CENP-A loading during interphase requires previous passage through mitosis [19]. We reasoned that maybe folding of centromeric chromatin during mitosis could be essential for subsequent CENP-A deposition. We thus decided to investigate whether condensin, a major mediator of chromosome condensation, had any role in CENP-A assembly, as suggested by a study in human cells [30]. There are two distinct condensin complexes in most organisms, condensin I and condensin II, which share the SMC2/SMC4 heterodimer but have distinct regulatory subunits. In Xenoupus egg extracts, condensin I is five fold more abundant than condensin II and its role in the assembly of mitotic chromosomes is clearly predominant [31]. However, condensin II accumulates at centromeres suggesting a specific function at this region. Indeed, we found that condensin II has two functions regarding CENP-A assembly. One, it stabilizes CENP-A nucleosomes preventing their eviction. Two, it allows efficient CENP-A incorporation. None of these functions are compensated by the presence of condensin I in extracts lacking condensin II.

The first function refers to the observation that a fraction of CENP-A nucleosomes are lost from centromeres in the absence of condensin II. Easier disassembly of CENP-A nucleosomes compared with canonical H3 nucleosomes could facilitate the clearance of those erroneously incorporated outside of centromeres [32, 33] and would be counterbalanced by some property of centromeric chromatin, presumably related to the presence of condensin II in this region. Importantly, this stabilization of CENP-A nucleosomes happens both in mitosis and in interphase.

Regarding the second function, we first suspected that the folding imposed by condensin II could contribute to the recognition of this region by HJURP. However, we did not observe changes in the localization of HJURP in the condensin II depleted extracts. Alternatively, targeting or function of some of the proteins acting downstream of HJURP to effectively incorporate CENP-A might require condensin II. Among those proteins could be remodeling complexes like FACT, RSF1 or CHD1 [34, 35] or some of the components of the constitutive centromere-associated network (CCAN) [36, 37]. Within this group of inner centromere proteins, CENP-C seems particularly important. On one hand, CENP-C has been proposed to connect CENP-A nucleosomes with CCAN components such as CENP-N [38]. On the other, it links CCAN and the Knl1-Mis12-Ndc80 complex (KMN) network of the outer kinetochore [3941]. Maybe as a consequence, CENP-C is required to stabilize the folded conformation of the mitotic centromere/kinetochore complex [4]. Since the KMN network, in addition to interacting with microtubules, recruits spindle assembly checkpoint proteins, loss of CENP-C results in multiple, adverse consequences for centromere architecture and function [39, 42]. Under this condition, decreased incorporation of CENP-A observed CENP-C knock down cells could be a secondary effect [38]. In this regard, one important advantage of our in vitro assay for CENP-A incorporation in Xenopus egg extracts is that one can analyze the effect of eliminating one specific factor after a single round of CENP-A loading without accumulation of errors from previous rounds or alteration of cell cycle progression. Thus, it will be important to investigate the functional relationship between CENP-C and condensin II in this experimental system.

How is CENP-A ectopic localization prevented?

Regulation of CENP-A transcription also contributes to specify CENP-A incorporation at centromeres. In human cells, mRNA levels of CENP-A are low during G1 and S phase and increase during G2, prior to its incorporation at the end of the next mitosis [17]. When CENP-A is overexpressed in yeast and Drosophila cells, it incorporates throughout the chromatin [33, 43]. However, proteasome-mediated degradation leads to elimination of this mislocalized CENP-A ensuring its exclusive presence at centromeres. Last year, two studies identified an E3 ubiquitin ligase, Psh1, that specifically targets CENP-A in budding yeast [44, 45]. Deletion of Psh1 increases CENP-A stability and causes its accumulation at euchromatic regions, but only if CENP-A is highly overexpressed. Like Scm3/HJURP, Psh1 recognizes the CATD of CENP-A. Thus, it is likely that binding of CENP-A to Scm3 at centromeres protects CENP-A from the action of Psh1. Identification of a similar proteolytic regulation in higher eukaryotes remains to be described. In Xenopus egg extracts, protein complexes are stored in the occyte cytoplasm so that the first twelve cycles following fertilization can happen with very little transcription. More than half of the soluble pool of CENP-A molecules in Xenopus egg extracts is bound to HJURP, which could be important to protect CENP-A from degradation. However, we also found that exogenous CENP-A added to these extracts is unable to compete with endogenous CENP-A for the binding of HJURP, but despite this, is stable [16]. Thus, the CENP-A degradation machinery is presumably not operative in the embryonic cycles.

In Drosophila, Cal1 was identified in a screening for regulators of the centromeric localization of CENP-A/Cid and CENP-C [46]. More recently, Schittenhelm et al. observed that the number of Cal1 molecules per centromere is much lower than CENP-A and CENP-C (2.5, 84 and 135 molecules, respectively) [47]. This, together with the observation that an increase in the centromeric signal of CENP-A is observed only if CENP-A is overexpressed along with Cal1 led the authors to propose that Cal1 restricts the deposition of CENP-A and CENP-C. Whether a similar mechanism exists in other organisms is unclear, since Cal1 orthologs have not been found outside Drosophilids.

Outlook

Despite recent advances in our understanding of how centromere identity is propagated, in particular the identification of Scm3/HJURP, we are only beginning to elucidate the molecular mechanisms involved and their careful regulation in the cell cycle. Fundamental questions need to be addressed. Does HJURP mediate CENP-A nucleosome assembly by itself or does it require the concerted action of additional chromatin remodelers? What restricts HJURP localization spatially (to centromeres) and temporally (early G1 in the case of human cells)? What is the role of epigenetic marks present on centromeric chromatin? The status of histone acetylation regulated by the Mis18 complex [48] and the presence of H3dimethylK4 [24] have both been proposed to affect CENP-A deposition, presumably by directing HJURP to centromeres, but the exact means of this regulation remains obscure. And what about centromeric RNA transcripts? HJURP and CENP-C accumulate at the nucleolus in interphase, an organelle in which nucleoprotein complexes are assembled [13, 49, 50]. In fact, CENP-C associates with RNA transcripts derived from centromeric repeats, and these transcripts are required for its centromeric targeting [50, 51]. Could HJURP have also an RNA component? Solving these and other exciting questions mentioned throughout this commentary will keep researchers busy in the years ahead.

Conclusions

Proper assembly of kinetochores at centromeres is essential for accurate chromosome segregation and thereby to ensure genome stability. Centromeres are determined epigenetically by the presence of the histone H3 variant, CENP-A. Several factors implicated in the deposition of this specialized histone have been identified, but the exact molecular mechanism of centromere inheritance remains unclear. We have recently developed an assay that measures CENP-A incorporation at centromeres of chromosomes assembled in Xenopus egg extracts and found that this in vitro system recapitulates the temporal restrictions observed in human cells, and that the major players involved, including HJURP, are highly conserved. We are therefore confident that this assay will be a useful tool to further dissect CENP-A deposition in molecular detail.

References

  1. Allshire RC, Karpen GH: Epigenetic regulation of centromeric chromatin: old dogs, new tricks? Nat Rev Genet 2008, 9: 923–937.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  2. Torras-Llort M, Moreno-Moreno O, Azorin F: Focus on the centre: the role of chromatin on the regulation of centromere identity and function. Embo J 2009, 28: 2337–2348. 10.1038/emboj.2009.174

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  3. Blower MD, Sullivan BA, Karpen GH: Conserved organization of centromeric chromatin in flies and humans. Dev Cell 2002, 2: 319–330. 10.1016/S1534-5807(02)00135-1

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Ribeiro SA, Vagnarelli P, Dong Y, Hori T, McEwen BF, Fukagawa T, Flors C, Earnshaw WC: A super-resolution map of the vertebrate kinetochore. Proc Natl Acad Sci USA 2010, 107: 10484–10489. 10.1073/pnas.1002325107

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Marshall OJ, Marshall AT, Choo KH: Three-dimensional localization of CENP-A suggests a complex higher order structure of centromeric chromatin. J Cell Biol 2008, 183: 1193–1202. 10.1083/jcb.200804078

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Santaguida S, Musacchio A: The life and miracles of kinetochores. Embo J 2009, 28: 2511–2531. 10.1038/emboj.2009.173

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  7. Camahort R, Li B, Florens L, Swanson SK, Washburn MP, Gerton JL: Scm3 is essential to recruit the histone h3 variant cse4 to centromeres and to maintain a functional kinetochore. Mol Cell 2007, 26: 853–865. 10.1016/j.molcel.2007.05.013

    Article  CAS  PubMed  Google Scholar 

  8. Mizuguchi G, Xiao H, Wisniewski J, Smith MM, Wu C: Nonhistone Scm3 and histones CenH3-H4 assemble the core of centromere-specific nucleosomes. Cell 2007, 129: 1153–1164. 10.1016/j.cell.2007.04.026

    Article  CAS  PubMed  Google Scholar 

  9. Pidoux AL, Choi ES, Abbott JK, Liu X, Kagansky A, Castillo AG, Hamilton GL, Richardson W, Rappsilber J, He X, Allshire RC: Fission yeast Scm3: A CENP-A receptor required for integrity of subkinetochore chromatin. Mol Cell 2009, 33: 299–311. 10.1016/j.molcel.2009.01.019

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. Sánchez-Pulido L, Pidoux AL, CP P, Allshire RC: Common ancestry of the CENP-A chaperones Scm3 and HJURP. Cell 2009, 137: 1173–1174. 10.1016/j.cell.2009.06.010

    Article  PubMed Central  PubMed  Google Scholar 

  11. Stoler S, Rogers K, Weitze S, Morey L, Fitzgerald-Hayes M, Baker RE: Scm3, an essential Saccharomyces cerevisiae centromere protein required for G2/M progression and Cse4 localization. Proc Natl Acad Sci USA 2007, 104: 10571–10576. 10.1073/pnas.0703178104

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  12. Williams JS, Hayashi T, Yanagida M, Russell P: Fission yeast Scm3 mediates stable assembly of Cnp1/CENP-A into centromeric chromatin. Mol Cell 2009, 33: 287–298. 10.1016/j.molcel.2009.01.017

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Dunleavy EM, Roche D, Tagami H, Lacoste N, Ray-Gallet D, Nakamura Y, Daigo Y, Nakatani Y, Almouzni-Pettinotti G: HJURP is a cell-cycle-dependent maintenance and deposition factor of CENP-A at centromeres. Cell 2009, 137: 485–497. 10.1016/j.cell.2009.02.040

    Article  CAS  PubMed  Google Scholar 

  14. Foltz DR, Jansen LE, Bailey AO, Yates JR, Bassett EA, Wood S, Black BE, Cleveland DW: Centromere-specific assembly of CENP-a nucleosomes is mediated by HJURP. Cell 2009, 137: 472–484. 10.1016/j.cell.2009.02.039

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Shuaib M, Ouararhni K, Dimitrov S, Hamiche A: HJURP binds CENP-A via a highly conserved N-terminal domain and mediates its deposition at centromeres. Proc Natl Acad Sci USA 2010, 107: 1349–1354. 10.1073/pnas.0913709107

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Bernad R, Sanchez P, Rivera T, Rodriguez-Corsino M, Boyarchuk E, Vassias I, Ray-Gallet D, Arnaoutov A, Dasso M, Almouzni G, Losada A: Xenopus HJURP and condensin II are required for CENP-A assembly. J Cell Biol 2011, 192: 569–582. 10.1083/jcb.201005136

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Shelby RD, Monier K, Sullivan KF: Chromatin assembly at kinetochores is uncoupled from DNA replication. J Cell Biol 2000, 151: 1113–1118. 10.1083/jcb.151.5.1113

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. 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. 10.1016/j.cub.2006.11.051

    Article  CAS  PubMed  Google Scholar 

  19. Jansen LE, Black BE, Foltz DR, Cleveland DW: Propagation of centromeric chromatin requires exit from mitosis. J Cell Biol 2007, 176: 795–805. 10.1083/jcb.200701066

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  20. Hemmerich P, Weidtkamp-Peters S, Hoischen C, Schmiedeberg L, Erliandri I, Diekmann S: Dynamics of inner kinetochore assembly and maintenance in living cells. J Cell Biol 2008, 180: 1101–1114. 10.1083/jcb.200710052

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  21. Tagami H, Ray-Gallet D, Almouzni G, Nakatani Y: Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis. Cell 2004, 116: 51–61. 10.1016/S0092-8674(03)01064-X

    Article  CAS  PubMed  Google Scholar 

  22. Zhou Z, Feng H, Zhou BR, Ghirlando R, Hu K, Zwolak A, Miller Jenkins LM, Xiao H, Tjandra N, Wu C, Bai Y: Structural basis for recognition of centromere histone variant CenH3 by the chaperone Scm3. Nature 2011, 472: 234–237. 10.1038/nature09854

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  23. Zeitlin SG, Chapados BR, Baker NM, Tai C, Slupphaug G, Wang JY: Uracil DNA N-glycosylase promotes assembly of human centromere protein a. PLoS ONE 2011, 6: e17151. 10.1371/journal.pone.0017151

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  24. Bergmann JH, Rodriguez MG, Martins NM, Kimura H, Kelly DA, Masumoto H, Larionov V, Jansen LE, Earnshaw WC: Epigenetic engineering shows H3K4me2 is required for HJURP targeting and CENP-A assembly on a synthetic human kinetochore. Embo J 2010, 30: 328–340.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  25. Shivaraju M, Camahort R, Mattingly M, Gerton JL: Scm3 is a centromeric nucleosome assembly factor. J Biol Chem 2011, 286: 12016–12023. 10.1074/jbc.M110.183640

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Black BE, Foltz DR, Chakravarthy S, Luger K, Woods VL Jr, Cleveland DW: Structural determinants for generating centromeric chromatin. Nature 2004, 430: 578–582. 10.1038/nature02766

    Article  CAS  PubMed  Google Scholar 

  27. Furuyama T, Dalal Y, Henikoff S: Chaperone-mediated assembly of centromeric chromatin in vitro. Proc Natl Acad Sci USA 2006, 103: 6172–6177. 10.1073/pnas.0601686103

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. Visnapuu ML, Greene EC: Single-molecule imaging of DNA curtains reveals intrinsic energy landscapes for nucleosome deposition. Nat Struct Mol Biol 2009, 16: 1056–1062. 10.1038/nsmb.1655

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  29. Black BE, Cleveland DW: Epigenetic centromere propagation and the nature of CENP-A nucleosomes. Cell 2011, 144: 471–479. 10.1016/j.cell.2011.02.002

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  30. Samoshkin A, Arnaoutov A, Jansen LE, Ouspenski I, Dye L, Karpova T, McNally J, Dasso M, Cleveland DW, Strunnikov A: Human condensin function is essential for centromeric chromatin assembly and proper sister kinetochore orientation. PLoS ONE 2009, 4: e6831. 10.1371/journal.pone.0006831

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  31. Ono T, Losada A, Hirano M, Myers MP, Neuwald AF, Hirano T: Differential contributions of condensin I and condensin II to mitotic chromosome architecture in vertebrate cells. Cell 2003, 115: 109–121. 10.1016/S0092-8674(03)00724-4

    Article  CAS  PubMed  Google Scholar 

  32. Conde e Silva N, Black BE, Sivolob A, Filipski J, Cleveland DW, Prunell A: CENP-A-containing nucleosomes: easier disassembly versus exclusive centromeric localization. J Mol Biol 2007, 370: 555–573. 10.1016/j.jmb.2007.04.064

    Article  CAS  PubMed  Google Scholar 

  33. Moreno-Moreno O, Torras-Llort M, Azorin F: Proteolysis restricts localization of CID, the centromere-specific histone H3 variant of Drosophila, to centromeres. Nucleic Acids Res 2006, 34: 6247–6255. 10.1093/nar/gkl902

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. Perpelescu M, Nozaki N, Obuse C, Yang H, Yoda K: Active establishment of centromeric CENP-A chromatin by RSF complex. J Cell Biol 2009, 185: 397–407. 10.1083/jcb.200903088

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  35. Okada M, Okawa K, Isobe T, Fukagawa T: CENP-H-containing Complex Facilitates Centromere Deposition of CENP-a in Cooperation with FACT and CHD1. Mol Biol Cell 2009, 20: 3986–3995. 10.1091/mbc.E09-01-0065

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Okada M, Cheeseman IM, Hori T, Okawa K, McLeod IX, Yates JR, Desai A, Fukagawa T: The CENP-H-I complex is required for the efficient incorporation of newly synthesized CENP-A into centromeres. Nat Cell Biol 2006, 8: 446–457. 10.1038/ncb1396

    Article  CAS  PubMed  Google Scholar 

  37. Hori T, Amano M, Suzuki A, Backer CB, Welburn JP, Dong Y, McEwen BF, Shang WH, Suzuki E, Okawa K, et al.: CCAN makes multiple contacts with centromeric DNA to provide distinct pathways to the outer kinetochore. Cell 2008, 135: 1039–1052. 10.1016/j.cell.2008.10.019

    Article  CAS  PubMed  Google Scholar 

  38. Carroll CW, Milks KJ, Straight AF: Dual recognition of CENP-A nucleosomes is required for centromere assembly. J Cell Biol 2010, 189: 1143–1155. 10.1083/jcb.201001013

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  39. Screpanti E, De Antoni A, Alushin GM, Petrovic A, Melis T, Nogales E, Musacchio A: Direct binding of cenp-C to the mis12 complex joins the inner and outer kinetochore. Curr Biol 2011, 21: 391–398. 10.1016/j.cub.2010.12.039

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  40. Milks KJ, Moree B, Straight AF: Dissection of CENP-C-directed centromere and kinetochore assembly. Mol Biol Cell 2009, 20: 4246–4255. 10.1091/mbc.E09-05-0378

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  41. Przewloka MR, Venkei Z, Bolanos-Garcia VM, Debski J, Dadlez M, Glover DM: CENP-C Is a Structural Platform for Kinetochore Assembly. Curr Biol 2011, 21: 399–405. 10.1016/j.cub.2011.02.005

    Article  CAS  PubMed  Google Scholar 

  42. Orr B, Sunkel CE: Drosophila CENP-C is essential for centromere identity. Chromosoma 2011, 120: 83–96. 10.1007/s00412-010-0293-6

    Article  CAS  PubMed  Google Scholar 

  43. Collins KA, Camahort R, Seidel C, Gerton JL, Biggins S: The overexpression of a Saccharomyces cerevisiae centromeric histone H3 variant mutant protein leads to a defect in kinetochore biorientation. Genetics 2007, 175: 513–525. 10.1534/genetics.106.064410

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  44. Hewawasam G, Shivaraju M, Mattingly M, Venkatesh S, Martin-Brown S, Florens L, Workman JL, Gerton JL: Psh1 is an E3 ubiquitin ligase that targets the centromeric histone variant Cse4. Mol Cell 2010, 40: 444–454. 10.1016/j.molcel.2010.10.014

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  45. Ranjitkar P, Press MO, Yi X, Baker R, MacCoss MJ, Biggins S: An E3 ubiquitin ligase prevents ectopic localization of the centromeric histone H3 variant via the centromere targeting domain. Mol Cell 2010, 40: 455–464. 10.1016/j.molcel.2010.09.025

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  46. Erhardt S, Mellone BG, Betts CM, Zhang W, Karpen GH, Straight AF: Genome-wide analysis reveals a cell cycle-dependent mechanism controlling centromere propagation. J Cell Biol 2008, 183: 805–818. 10.1083/jcb.200806038

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  47. Schittenhelm RB, Althoff F, Heidmann S, Lehner CF: Detrimental incorporation of excess Cenp-A/Cid and Cenp-C into Drosophila centromeres is prevented by limiting amounts of the bridging factor Cal1. J Cell Sci 2010, 123: 3768–3779. 10.1242/jcs.067934

    Article  CAS  PubMed  Google Scholar 

  48. 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. 10.1016/j.devcel.2006.11.002

    Article  CAS  PubMed  Google Scholar 

  49. Kato T, Sato N, Hayama S, Yamabuki T, Ito T, Miyamoto M, Kondo S, Nakamura Y, Daigo Y: Activation of Holliday junction recognizing protein involved in the chromosomal stability and immortality of cancer cells. Cancer Res 2007, 67: 8544–8553. 10.1158/0008-5472.CAN-07-1307

    Article  CAS  PubMed  Google Scholar 

  50. Wong LH, Brettingham-Moore KH, Chan L, Quach JM, Anderson MA, Northrop EL, Hannan R, Saffery R, Shaw ML, Williams E, Choo KH: Centromere RNA is a key component for the assembly of nucleoproteins at the nucleolus and centromere. Genome Res 2007, 17: 1146–1160. 10.1101/gr.6022807

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  51. Du Y, Topp CN, Dawe RK: DNA binding of centromere protein C (CENPC) is stabilized by single-stranded RNA. PLoS Genet 2011, 6: e1000835.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Research in our lab is supported by the Spanish Ministry of Science and Innovation (BFU2007-66627 and associated FPI fellowship, SAF2010-21517; Consolider CSD2007-00015 and FEDER) and the European Commission (FP7-PEOPLE-2008 ITN "Nucleosome 4D").

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Ana Losada.

Additional information

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

PS and AL wrote the manuscript together. Both authors read and approved the final manuscript.

Authors’ original submitted files for images

Below are the links to the authors’ original submitted files for images.

Authors’ original file for figure 1

Rights and permissions

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reprints and permissions

About this article

Cite this article

Sánchez, P., Losada, A. New clues to understand how CENP-A maintains centromere identity. Cell Div 6, 11 (2011). https://doi.org/10.1186/1747-1028-6-11

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/1747-1028-6-11

Keywords