Centromere-associated repeat arrays on Trypanosoma brucei chromosomes are much more extensive than predicted
© Echeverry et al; licensee BioMed Central Ltd. 2012
Received: 21 October 2011
Accepted: 18 January 2012
Published: 18 January 2012
African trypanosomes belong to a eukaryotic lineage which displays many unusual genetic features. The mechanisms of chromosome segregation in these diploid protozoan parasites are poorly understood. Centromeres in Trypanosoma brucei have been localised to chromosomal regions that contain an array of ~147 bp AT-rich tandem repeats. Initial estimates from the genome sequencing project suggested that these arrays ranged from 2 - 8 kb. In this paper, we show that the centromeric repeat regions are much more extensive.
We used a long-range restriction endonuclease mapping approach to more accurately define the sizes of the centromeric repeat arrays on the 8 T. brucei chromosomes where unambiguous assembly data were available. The results indicate that the sizes of the arrays on different chromosomes vary from 20 to 120 kb. In addition, we found instances of length heterogeneity between chromosome homologues. For example, values of 20 and 65 kb were obtained for the arrays on chromosome 1, and 50 and 75 kb for chromosome 5.
Our results show that centromeric repeat arrays on T. brucei chromosomes are more similar in size to those of higher eukaryotes than previously suspected. This information provides a firmer framework for investigating aspects of chromosome segregation and will allow epigenetic features associated with the process to be more accurately mapped.
Centromeres are the chromosomal loci that facilitate segregation in most eukaryotes. They are the site of assembly of the kinetochore, the nucleoprotein complex which anchors the microtubule spindles that separate sister chromatids and mediate their movement to the daughter nuclei. Most centromeres are "regional" and encompass large sections of DNA, spanning 0.06 - 5 Mb, in species as diverse as plants, insects and mammals [1–3]. Centromeric DNA is typically comprised of arrays of highly repeated sequences, interrupted by transposable elements [4, 5]. The repeats are generally restricted to centromeric regions and are often in the size range 150 - 180 bp. This length is similar to that of nucleosomes, a property that may be of functional significance . Although many features of centromeric DNA are widespread, there is little sequence conservation, even between closely related species , and most evidence suggests that centromeres are determined epigenetically [8, 9].
In human chromosomes, centromeres have a conserved core of α-satellite repeats (~170 bp) stretching over several megabases, which is flanked by extensive regions that contain multiple retrotransposon insertions . In eukaryotic microorganisms, centromeres can also encompass large regions of chromosomal DNA. Those of Schizosaccharomyces pombe for example, range from 35 - 110 kb  and are organised as chromosome-specific core elements, flanked by inverted arrays of 3 - 7 kb. These in turn are flanked by more extensive outer repeats. Unusually in Saccharomyces cerevisiae, the regions that specify kinetochore assembly are restricted to single 125 bp elements termed "point" centromeres . Some organisms, such as Caenorhabditis elegans, have holocentric chromosomes that lack specific centromeres . In these instances, microtubules bind along the entire length of the chromosome.
Protozoan parasites of the Trypanosoma brucei species complex are insect-transmitted pathogens that are of major medical and veterinary importance throughout sub-Saharan Africa. They belong to the Excavata, a eukaryotic lineage which includes the other trypanosomatid parasites Trypanosoma cruzi and Leishmania species. Several features of gene organisation and expression in these organisms are unusual. Protein coding genes lack conventional RNA polymerase II (pol II) promoters  and are organised in long co-directional clusters which can stretch for tens to hundreds of kilobases . Transcription is polycistronic, and processing involves a trans-splicing mechanism in which all mRNAs are modified post-transcriptionally by the addition of a 39-nucleotide spliced leader to their 5'-ends. T. brucei has a haploid genome content of 35 Mb, with 11 megabase pair chromosomes (0.9 - 5.7 Mb). Unusually, chromosome homologues can vary significantly in size . In addition, this parasite also contains two classes of atypical nuclear chromosomes; the intermediate-size chromosomes (300 - 900 kb) that contain some variant surface glycoprotein (VSG) genes, but no house-keeping genes, and the minichromosomes (50 - 100 kb), which appear to act as a reservoir of VSG sequences .
The T. brucei genome project was completed in 2005 . However, sequence elements characteristic of centromeric DNA in other eukaryotes were not described. Furthermore, candidates for the 'core' centromeric proteins and most of the other factors involved in kinetochore assembly could not be identified [14, 16]. This includes the variant histone CenH3, which specifies centromere location in eukaryotes and was thought to be ubiquitous . The first evidence on the nature and location of centromeric DNA in T. brucei came from a biochemical mapping approach based on etoposide-mediated topoisomerase-II cleavage [18, 19]. Topoisomerase-II has a major regulatory role in chromosome segregation and accumulates at centromeres during late metaphase, where it resolves the catenated DNA strands that provide the final structural link between sister kinetochores [20, 21]. This process requires double stranded DNA cleavage, passage of the uncut duplex through the gap and re-ligation to repair the break. Etoposide inhibits this re-ligation step leading to lesions in chromosomal DNA at sites of topoisomerase-II activity. In human chromosomes, etoposide-mediated cleavage sites occur within the α-satellite repeats that constitute centromeric DNA [22, 23]. In both T. cruzi and Plasmodium[25, 26], these sites have been delineated to chromosomal loci that confer mitotic stability. In Toxoplasma gondii, they co-locate with the binding sites of the centromeric histone CenH3 .
Using the etoposide mapping method, we identified the location of putative centromeric domains on the 8 T. brucei chromosomes that had been fully assembled . These loci, which occur once per chromosome, encompass regions between directional gene clusters that contain transposable elements and an array of AT-rich repeats predicted to extend between 2 and 8 kb. The tandem repeats are arranged in units of ~147 bp and share intra-chromosomal identities ranging from 50% to more than 90%. The units have a complex structure made up of degenerate sub-repeats of ~48 and ~30 bp (for a more detailed description of their make-up, see reference ). We also noted that the repeat arrays were located adjacent to ribosomal RNA genes on 5 of the chromosomes, although the significance of this is unknown. The intermediate and minichromosomes did not exhibit site-specific topoisomerase-II activity, suggesting that their segregation might involve a centromere-independent mechanism, a finding consistent with the "lateral-stacking" model .
In the initial analysis of the T. brucei centromeric domains, we identified discrepancies between the published sequence data of two chromosomes and our preliminary long range restriction mapping . We also found evidence of heterogeneity in the extent of these regions between chromosome homologues. However, it was unclear whether the differences arose from an under-estimation of the copy number of the tandem repeats, whether they were due to the gaps in the assembly of the adjacent regions, or whether this under-estimation of size was also the case with other T. brucei chromosomes. Here, we show that the centromeric repeats in T. brucei chromosomes are present at much higher copy number than predicted, with an organisation that is more typical of centromeric domains in higher eukaryotes than realised. These data provide a more complete model for T. brucei chromosome structure, an improved basis for investigating the mechanisms of segregation, and will enable more detailed functional mapping of this crucial chromosomal region to be undertaken.
Parasites and DNA preparation
T. brucei procyclic forms (genome project strain TREU 927/4) were grown in SDM-79 medium  with 10% heat-inactivated fetal bovine serum at 28°C. For preparation of intact chromosomal DNA, the agarose embedding technique was used . 108 procyclics were immobilized in 1% low melting-point agarose blocks and incubated at 48°C for 48 hours in proteinase K/sarcosyl buffer. Genomic DNA was extracted using the phenol-chloroform method .
In situ digestion and electrophoretic resolution
Prior to incubation with restriction endonucleases, agarose blocks were washed 3 times for 1 hour at 48°C in 50 volumes of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.4) containing 40 μg ml-1 phenylmethanesulphonylfluoride to inactivate proteinase K. After a minimum of 2 hours equilibration with the respective restriction enzyme buffer, blocks were incubated with restriction enzymes for 48 hours at 37°C. Fresh enzyme (5 - 10 units) was added at the 24 and 36 hour time points. The digested DNA was resolved by a CHEF (contour-clamped homogenous electric field) Mapper System (Bio-Rad) (typically quarter of a block was used per lane) using an auto-algorithm set to the designated molecular mass range. For resolution of DNA fragments less than 20 kb, genomic DNA (5 - 10 μg) was digested for 3 hours and fractionated on 0.5% agarose gels using standard electrophoresis techniques. As molecular size markers, a combination of Bio-Rad CHEF DNA standards 8 - 48 kb, lambda ladder 50 - 1000 kb and S. cerevisiae chromosomes from 225 - 2,200 kb were used. Southern blotting was performed using standard procedures as outlined previously .
Inferred sizes of the centromeric tandem arrays on chromosomes 1 - 8.
Size of centromeric
repeat array (GeneDB)
20 & 65 kb
30 & 55 kb
75 & 80 kb
50 & 75 kb
100 & 120 kb
Discussion and Conclusions
In eukaryotes, centromeric sequences are frequently organised as highly repetitive tandem arrays that stretch over extensive regions of chromosomal DNA. Their complete assembly has proven to be an intractable problem in most genome projects . Where detailed analysis has been undertaken, considerable intra-chromosomal size variation and sequence divergence has become apparent [3, 33]. This arises from the acquisition of point mutations and high rates of unequal homologous recombination. When the T. brucei genome was initially completed , regions subsequently identified as centromeres, were characterised by the presence of ~147 bp repeat arrays predicted to extend over 2 - 8 kb . In each of the 8 T. brucei chromosomes analysed here, our data suggest that these centromeric arrays are much larger (Table 1), varying from 20 kb (chromosome 1) to more than 100 kb (chromosome 7). We found a tendency for these regions to be more extensive in the larger chromosomes. This contrasts with S. pombe, where centromere length is inversely proportional to the chromosome length . In addition, we also observed several instances of heterogeneity between chromosome homologues (Table 1). Using data available on TriTrypDB, there is no evidence for single nucleotide polymorphisms contributing to the generation of larger than expected restriction fragments containing the arrays. Although we cannot demonstrate unambiguously that the missing segments of centromeric DNA are constituted by tandem repeats, it would be unusual if extensive segments of non-repetitive sequences had been missed from the corresponding regions of each chromosome during the genome project.
The nature of the centromere-kinetochore complex in trypanosomes and the role that the ~147 bp repeats play in recruitment are two of the most intriguing unsolved questions in parasite biology. Although segregation in trypanosomes appears to be mediated by a conventional microtubule - kinetochore attachment, the number of kinetochores seems to be less than the number of chromosomes [35, 36]. Trypanosomatids lack genes for the conserved "core" centromeric proteins, as well as the majority of other proteins involved in kinetochore assembly and function . They are distinct in lacking an obvious orthologue of the variant histone CenH3, which replaces the canonical histone H3 at centromeres in other eukaryotes. In T. brucei, the only histone H3 variant identified is non-essential, enriched at telomeres, and lacks the extended loop I region or characteristic carboxyl terminal domain that are diagnostic of CenH3 [37–39] .
In other eukaryotes, CenH3 is essential for kinetochore assembly and functions as an epigenetic marker for centromere location . By contrast, the centromeric repeat arrays with which they interact are not a pre-requisite. If normal centromere function is lost in some eukaryotes, neocentromeres can form in regions which lack these arrays, and once formed, the new location is stably inherited and specified epigentically by CenH3 binding [3, 40]. Centromeric repeats then accumulate in these regions over time, where they may have a role in providing an environment that favours or promotes the formation of centromeric chromatin. Traditionally, centromeric heterochromatin had been considered transcriptionally quiescent. However several recent studies, initially in fission yeast, have highlighted an essential role for short interfering RNAs (siRNAs) derived from centromeric sequences in the formation of heterochromatin and centromere function [41, 42]. Interestingly, a recent report has described a class of siRNAs derived from centromeric repeats in T. brucei, although their functional significance remains to be elucidated .
Our finding that the ~147 bp tandem repeats constitute a larger than expected component of T. brucei chromosomes provides an improved framework for investigating aspects of genome biology, including the determinants of centromere function. For example, the cell-cycle specific accumulation of topoisomerase-II at centromeres is required for regulated segregation of sister chromatids. Precise mapping of this decatenation activity onto T. brucei chromosomes was complicated by uncertainty over the size of the centromeric repeats arrays . Likewise, analysis of chromatin immunoprecipitation experiments to assess the extent of histone modifications associated with centromeric domains would be difficult to interpret in the absence of a more accurate chromosome map. To date most studies on chromosome segregation have focused on mammals, insects, plants and fungi. Analysis of the situation in trypanosomes demonstrates both similarities and differences from the standard model. We have now shown that the organisation of centromeric DNA repeats in T. brucei conforms to the "regional" class, typical of higher eukaryotes. In contrast, the protein factors which mediate segregation are unknown, and by inference, must be highly divergent. Further studies aimed at uncovering the mechanisms involved are crucial to ensure that our understanding the chromosome segregation takes full account of eukaryotic diversity.
List of Abbreviations
centromeric histone H3
contour-clamped homogenous electric field (electrophoresis)
short interfering RNA
variant surface glycoprotein.
Acknowledgements and funding
JMK was supported by the UK Biotechnology and Biological Sciences Research Council (grant number BB/C501292/1) and the Wellcome Trust (grant number 084175). MCE was the recipient of a Colombian Research Council (COLCIENCIAS) and Universidad Nacional de Colombia scholarship.
We acknowledge the work of our colleagues on the T. brucei Genome Project [ref. ] and thank Flora Logan (GeneDB; Wellcome Trust Sanger Institute) for assistance with accessing GeneDB sequence data and for useful discussions.
- Sun X, Wahlstrom J, Karpen G: Molecular structure of a functional Drosophila centromere. Cell. 1997, 91: 1007-1019. 10.1016/S0092-8674(00)80491-2.PubMed CentralView ArticlePubMed
- Sullivan BA, Blower MD, Karpen GH: Determining centromere identity: cyclical stories and forking paths. Nat Rev Genet. 2001, 2: 584-596. 10.1038/35084512.View ArticlePubMed
- Ma J, Wing RA, Bennetzen JL, Jackson SA: Plant centromere organization: a dynamic structure with conserved functions. Trends Genet. 2007, 23: 134-139. 10.1016/j.tig.2007.01.004.View ArticlePubMed
- Schueler MG, Higgins AW, Rudd MK, Gustashaw K, Willard HF: Genomic and genetic definition of a functional human centromere. Science. 2001, 294: 109-115. 10.1126/science.1065042.View ArticlePubMed
- Wong LH, Choo KH: Evolutionary dynamics of transposable elements at the centromere. Trends Genet. 2004, 20: 611-616. 10.1016/j.tig.2004.09.011.View ArticlePubMed
- Henikoff S, Ahmad K, Malik HS: The centromere paradox: Stable inheritance with rapidly evolving DNA. Science. 2001, 293: 1098-1102. 10.1126/science.1062939.View ArticlePubMed
- Malik HS, Henikoff S: Conflict begets complexity: the evolution of centromeres. Curr Opin Genet Dev. 2002, 12: 711-718. 10.1016/S0959-437X(02)00351-9.View ArticlePubMed
- Mehta GD, Agarwal MP, Ghosh SK: Centromere identity: a challenge to be faced. Mol Genet Genomics. 2010, 284: 75-94. 10.1007/s00438-010-0553-4.View ArticlePubMed
- Ekwall K: Epigenetic control of centromere behaviour. Annu Rev Genet. 2007, 41: 63-81. 10.1146/annurev.genet.41.110306.130127.View ArticlePubMed
- Steiner NC, Hahnenberger KM, Clarke L: Centromeres of the fission yeast Schizosaccharomyces pombe are highly variable genetic loci. Mol Cell Biol. 1993, 13: 4578-4587.PubMed CentralView ArticlePubMed
- Pidoux AL, Allshire RC: Kinetochore and heterochromatin domains of the fission yeast centromere. Chrom Res. 2004, 12: 521-534.View ArticlePubMed
- Maddox PS, Oegema K, Desai A, Cheeseman IM: "Holo"er than thou: Chromosome segregation and kinetochore function in C. elegens. Chrom Res. 2004, 12: 641-653.View ArticlePubMed
- Campbell DA, Thomas S, Sturm NR: Transcription in kinetoplastid protozoa: why be normal?. Microbes Infect. 2003, 5: 1231-1240. 10.1016/j.micinf.2003.09.005.View ArticlePubMed
- Berriman M, Ghedin E, Hertz-Fowler C, Blandin G, Renauld H, Bartholomeu DC, Lennard NJ, Caler E, Hamlin NE, Haas B: The genome of the African trypanosome Trypanosoma brucei. Science. 2005, 309: 416-422. 10.1126/science.1112642.View ArticlePubMed
- Gull K, Alsford S, Ersfield K: Segregation of minichromosomes in trypanosomes: implications for mitotic mechanisms. Trends Microbiol. 1998, 6: 319-323. 10.1016/S0966-842X(98)01314-6.View ArticlePubMed
- Foltz DR, Black BE, Bailey AO, Yates JR, Cleveland DW: The human CENP-A centromeric nucleosome-associated complex. Nature Cell Biol. 2006, 8: 458-469. 10.1038/ncb1397.View ArticlePubMed
- Malik HS, Henikoff S: Phylogenomics of the nucleosome. Nat Struct Biol. 2003, 10: 882-889. 10.1038/nsb996.View ArticlePubMed
- Obado SO, Bot C, Nilsson D, Andersson B, Kelly JM: Repetitive DNA is associated with centromeric domains in Trypanosoma brucei but not Trypanosoma cruzi. Genome Biol. 2007, 8: R37-10.1186/gb-2007-8-3-r37.PubMed CentralView ArticlePubMed
- Obado SO, Bot C, Echeverry MC, Bayona JC, Alvarez VE, Taylor MC, Kelly JM: Centromere-associated topoisomerase activity in bloodstream form Trypanosoma brucei. Nucl Acids Res. 2011, 39: 1023-1033. 10.1093/nar/gkq839.PubMed CentralView ArticlePubMed
- Baumann C, Körner R, Hofmann K, Nigg EA: PICH, a centromere-associated SNF2 family ATPase, is regulated by Plk1 and required for spindle checkpoint. Cell. 2007, 128: 101-114. 10.1016/j.cell.2006.11.041.View ArticlePubMed
- Chan K-L, North PS, Hickson ID: BLM is required for faithful chromosome segregation and its localization defines a class of ultrafine anaphase bridges. EMBO J. 2007, 26: 3397-3409. 10.1038/sj.emboj.7601777.PubMed CentralView ArticlePubMed
- Spence JM, Critcher R, Ebersole TA, Valdivia MM, Earnshaw WC, Fukagawa T, Farr CJ: Co-localization of centromere activity, proteins and topoisomerase-II within a subdomain of the major human × alpha-satellite array. EMBO J. 2002, 21: 5269-5280. 10.1093/emboj/cdf511.PubMed CentralView ArticlePubMed
- Jonstrup AT, Thomsen T, Wang Y, Knudsen BR, Koch J, Andersen AH: Hairpin structures formed by alpha satellite DNA of human centromeres are cleaved by human topoisomerase. Nucl Acids Res. 2008, 36: 6165-6174. 10.1093/nar/gkn640.PubMed CentralView ArticlePubMed
- Obado SO, Taylor MC, Wilkinson SR, Bromley EV, Kelly JM: Functional mapping of a trypanosome centromere by chromosome fragmentation identifies a 16 kb GC-rich transcriptional "strand-switch" domain as a major feature. Genome Res. 2005, 15: 36-43. 10.1101/gr.2895105.PubMed CentralView ArticlePubMed
- Kelly JM, McRobert L, Baker DA: Evidence on the chromosomal location of centromeric DNA in Plasmodium falciparum from etoposide-mediated topoisomerase-II cleavage. Proc Natl Acad Sci USA. 2006, 103: 6706-6711. 10.1073/pnas.0510363103.PubMed CentralView ArticlePubMed
- Iwanaga S, Khan SM, Kaneko I, Christodoulou Z, Newbold C, Yuda M, Janse CJ, Waters AP: Functional identification of the Plasmodium centromere and generation of a Plasmodium artificial chromosome. Cell Host Microbe. 2010, 7: 245-255. 10.1016/j.chom.2010.02.010.PubMed CentralView ArticlePubMed
- Brooks CF, Francia ME, Gissot M, Croken MM, Kim K, Striepen B: Toxoplasma gondii sequesters centromeres to a specific nuclear region throughout the cell cycle. Proc Natl Acad Sci USA. 2011, 108: 3767-3772. 10.1073/pnas.1006741108.PubMed CentralView ArticlePubMed
- Brun R, Jenni L: A new semi-defined medium for Trypansoma brucei sspp. Acta Tropica. 1977, 21-33. 34
- Gibson WC, Miles MA: The karyotype and ploidy of Trypanosoma cruzi. EMBO J. 1986, 5: 1299-1305.PubMed CentralPubMed
- Kelly JM: Isolation of DNA and RNA from Leishmania. Protocols in Molecular Parasitology. Edited by: Hyde JE. 1993, Humana Press, New Jersey, 21: 312-321. Methods in Molecular Biology (series editor Walker JM)View Article
- Benson G: Tandem repeats finder: a program to analyze DNA sequences. Nucl Acid Res. 1999, 27: 573-580. 10.1093/nar/27.2.573.View Article
- Alkan C, Cardone MF, Catacchio CR, Antonacci F, O'Brien SJ, Ryder OA, Purgato S, Zoli M, Della Valle G, Eichler EE, Ventura M: Genome-wide characterization of centromeric satellites from multiple mammalian genomes. Genome Res. 2011, 21: 137-145. 10.1101/gr.111278.110.PubMed CentralView ArticlePubMed
- Plohl M, Luchetti A, Mestrović N, Mantovani B: Satellite DNAs between selfishness and functionality: structure, genomics and evolution of tandem repeats in centromeric (hetero)chromatin. Gene. 2008, 409: 72-82. 10.1016/j.gene.2007.11.013.View ArticlePubMed
- Wood V, Gwilliam R, Rajandream M-A, Lyne M, Lyne R, Stewart A, Sgouros J, Peat N, Hayles J, Baker S: The genome sequence of Schizosaccharomyces pombe. Nature. 2002, 415: 871-880. 10.1038/nature724.View ArticlePubMed
- Ogbadoyi E, Ersfeld K, Robinson D, Sherwin T, Gull K: Architecture of the Trypanosoma brucei nucleus during interphase and mitosis. Chromosoma. 2000, 108: 501-513. 10.1007/s004120050402.View ArticlePubMed
- Ersfeld K, Gull K: Partitioning of large and minichromosomes in Trypanosoma brucei. Science. 1997, 276: 611-614. 10.1126/science.276.5312.611.View ArticlePubMed
- Alsford S, Horn D: Trypanosomatid histones. Mol Microbiol. 2004, 53: 365-372. 10.1111/j.1365-2958.2004.04151.x.View ArticlePubMed
- Lowell JE, Cross GA: A variant histone H3 is enriched at telomeres in Trypanosoma brucei. J Cell Sci. 2004, 117: 5937-5947. 10.1242/jcs.01515.View ArticlePubMed
- Guse A, Carroll CW, Moree B, Fuller CJ, Straight AF: In vitro centromere and kinetochore assembly on defined chromatin templates. Nature. 2011, 477: 354-358. 10.1038/nature10379.PubMed CentralView ArticlePubMed
- Stimpson KM, Sullivan BA: Epigenomics of centromere assembly and function. Curr Opin Cell Biol. 2010, 22: 772-780. 10.1016/j.ceb.2010.07.002.View ArticlePubMed
- Volpe TA, Kidner C, Hall IM, Teng G, Grewal SI, Martienssen RA: Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science. 2002, 297: 1833-1837. 10.1126/science.1074973.View ArticlePubMed
- Grewal SI: RNAi-dependent formation of heterochromatin and its diverse functions. Curr Opin Genet Dev. 2010, 20: 134-141. 10.1016/j.gde.2010.02.003.PubMed CentralView ArticlePubMed
- Patrick KL, Shi H, Kolev NG, Ersfeld K, Tschudi C, Ullu E: Distinct and overlapping roles for two Dicer-like proteins in the RNA interference pathways of the ancient eukaryote Trypanosoma brucei. Proc Natl Acad Sci USA. 2009, 106: 17933-17938. 10.1073/pnas.0907766106.PubMed CentralView ArticlePubMed
- Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, Rajandream MA, Barrell B: Artemis: sequence visualization and annotation. Bioinformatics. 2000, 16: 944-945. 10.1093/bioinformatics/16.10.944.View ArticlePubMed
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