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Introduction Advances in plant genome sequencing have improved our understanding of DNA sequence organisation, distribution, and expression on chromosomes (Heng et al. 2018). DNA probes specific for fluorescence in situ [...], Coffea spp. chromosomes are very small and accumulate a variety of repetitive DNA families around the centromeres. However, the proximal regions of Coffea chromosomes remain poorly understood, especially regarding the nature and organisation of the sequences. Taking advantage of the genome sequences of C. arabica (2n = 44), C. canephora, and C. eugenioides (C. arabica progenitors with 2n = 22) and good coverage genome sequencing of dozens of other wild Coffea spp., repetitive DNA sequences were identified, and the genomes were compared to decipher particularities of pericentromeric structures. The searches revealed a short tandem repeat (82 bp length) typical of Gypsy/TAT LTR retrotransposons, named Coffea_sat11. This repeat organises clusters with fragments of other transposable elements, comprising regions of non-coding RNA production. Cytogenomic analyses showed that Coffea_sat11 extends from the pericentromeres towards the middle of the chromosomal arms. This arrangement was observed in the allotetraploid C. arabica chromosomes, as well as in its progenitors. This study improves our understanding of the role of the Gypsy/TAT LTR retrotransposon lineage in the organisation of Coffea pericentromeres, as well as the conservation of Coffea_sat11 within the genus. The relationships between fragments of other transposable elements and the functional aspects of these sequences on the pericentromere chromatin were also evaluated. Highlights: A scattered short tandem repeat, typical of Gypsy/TAT LTR retrotransposons, associated with several fragments of other transposable elements, accumulates in the pericentromeres of Coffea chromosomes. This arrangement is preserved in all clades of the genus and appears to have a strong regulatory role in the organisation of chromatin around centromeres. Key words: centromeric retrotransposons, chromocentres, Coffea genomes, FISH, Gypsy LTR retrotransposons. Les chromosomes des espèces du genre Coffea sont très petits et présentent une accumulation de familles d'ADN répétitif autour des centromères. Les régions proximales des chromosomes du genre Coffea demeurent mal caractérisées, particulièrement en ce qui regarde la nature et l'organisation des séquences. En s'appuyant sur les séquences des génomes du C. arabica (2n = 44), du C. canephora et du C. eugenioides (les espèces ancestrales du C. arabica, chacune à 2n = 22), ainsi que du séquençage de profondeur adéquate pour des douzaines d'autres espèces sauvages au sein du genre Coffea, les séquences répétitives ont été identifiées et les génomes ont été comparés pour décoder les particularités des structures péricentromériques. Ces investigations ont révélé une courte séquence répétée en tandem (longue de 82 pb) qui est typique des rétrotransposons à LTR de type Gypsy/TAT et qui a été nommée Coffea_sat11. Cette séquence répétée est organisée en amas incluant des fragments d'autres éléments transposables, lesquels constituent des régions produisant des ARN non-codants. Des analyses cytogénétiques ont montré que Coffea_sat11 s'étend des régions péricentromériques jusqu'au milieu des bras chromosomiques. Cette disposition est observée tant au sein des chromosomes allotétraploïdes du C. arabica que chez ses ancêtres. La présente étude contribue à la compréhension du rôle de la lignée des rétrotransposons à LTR Gypsy/TAT dans l'organisation des régions péricentromériques chez le genre Coffea, ainsi que dans la conservation de Coffea_sat11 au sein du genre. Les relations avec des fragments d'autres éléments transposables et les aspects fonctionnels de ces séquences sur la chromatine péricentromérique sont également évalués. Mots-clés : rétrotransposons centromériques, chromocentres, génomes du genre Coffea, FISH, rétrotransoposons à LTR Gypsy.

Metazoan centromeres are specified epigenetically by the presence of CENP-A nucleosomes (3). Structural differences between CENP-A and histone H3 nucleosomes (2,4) and/or specific protein recognition elements in CENP-A seem to [...], During cell division, chromosomes are segregated to nascent daughter cells by attaching to the microtubules of the mitotic spindle through the kinetochore. Kinetochores are assembled on a specialized chromatin domain called the centromere, which is characterized by the replacement of nucleosomal histone H3 with the histone H3 variant centromere protein A (CENP-A). CENP-A is essential for centromere and kinetochore formation in all eukaryotes but it is unknown how CENP-A chromatin directs centromere and kinetochore assembly (1). Here we generate synthetic CENP-A chromatin that recapitulates essential steps of centromere and kinetochore assembly invitro. We show that reconstituted CENP-A chromatin when added to cell-free extracts is sufficient for the assembly of centromere and kinetochore proteins, microtubule binding and stabilization, and mitotic checkpoint function. Using chromatin assembled from histone H3/CENPA chimaeras, we demonstrate that the conserved carboxy terminus of CENP-A is necessary and sufficient for centromere and kinetochore protein recruitment and function but that the CENP-A targeting domain--required for new CENP-A histone assembly (2)--is not. These data show that two of the primary requirements for accurate chromosome segregation, the assembly of the kinetochore and the propagation of CENP-A chromatin, are specified by different elements in the CENP-A histone. Our unique cell-free system enables complete control and manipulation of the chromatin substrate and thus presents a powerful tool to study centromere and kinetochore assembly.

Mammalian centromere function depends upon a specialized chromatin organization where distinct domains of CENP-A and dimethyl K4 histone H3, forming centric chromatin, are uniquely positioned on or near the surface of the chromosome. These distinct domains are embedded in pericentric heterochromatin (characterized by H3 methylated at K9). The mechanisms that underpin this complex spatial organization are unknown. Here, we identify the essential histone variant H2A.Z as a new structural component of the centromere. Along linear chromatin fibers H2A.Z is distributed nonuniformly throughout heterochromatin, and centric chromatin where regions of nucleosomes containing H2A.Z and dimethylated K4 H3 are interspersed between subdomains of CENP-A. At metaphase, using the inactive X chromosome centromere as a model, complex folding of this fiber produces spatially positioned domains where H2A.Z/dimethylated K4 H3 chromatin juxtaposes one side of CENP-A chromatin, whereas a region of H2A/trimethyl K9 H3 borders the other side. A second region of H2A.Z is found, with trimethyl K9 H3 at the inner centromere. We therefore propose that H2A.Z plays an integral role in organizing centromere structure. centromere organization | chromosome structure | histone variants

The adociasulfate-2 (AS-2) binding regions of two human kinesins, centromere-associated protein E (CENP-E), a member of the kinesin-7 family, the Eg5, a member of the kinesin-5 family and the microtubules (MT)-binding region are investigated. The results have shown that AS-2 is a MT-competitive inhibitor of the human CENP-E motor domain.

In the pathogenic yeast Candida albicans, the 3-kb centromeric DNA regions (CEN) of each of the eight chromosomes have different and unique DNA sequences. The centromeric histone CaCse4p (CENP-A homolog) occurs only within these 3-kb CEN regions to form specialized centromeric chromatin. Centromere activity was maintained on small chromosome fragments derived in vivo by homologous recombination of a native chromosome with linear DNA fragments containing a telomere and a selectable marker. An in vivo derived 85-kb truncated chromosome containing the 3-kb CEN7 locus on 69 kb of chromosome 7 DNA was stably and autonomously maintained in mitosis, indicating that preexisting active CEN chromatin remains functional through many generations. This same 85-kb chromosome fragment, isolated as naked DNA (devoid of chromatin proteins) from C. albicans and reintroduced back into C. albicans cells by standard DNA transformation techniques, was unable to reform functional CEN chromatin and was mitotically unstable. Comparison of active and inactive CEN chromatin digested with micrococcal nuclease revealed that periodic nucleosome arrays are disrupted at active centromeres. Chromatin immunoprecipitation with antibodies against CaCse4p confirmed that CEN7 introduced into C. albicans cells as naked DNA did not recruit CaCse4p or induce its spread to a duplicate region only 7 kb away from active CEN7 chromatin. These results indicate that CaCse4p recruitment and centromere activation are epigenetically specified and maintained in C. albicans. CENP-A | centromere | Cse4p | kinetochore

The Shugoshin (Sgo) family of proteins plays a vital role in protection of centromeric cohesion in mitosis and meiosis from yeast to man, which is accomplished by recruiting the serine/threonine protein phosphatase 2A (PP2A) to centromeres. It is found that Sgo binds a fraction of PP2A, that the two proteins colocalize at the centromere from prophase to anaphase and that they both are required for preventing precocious separation of the sister chromatids.

Study is conducted in which a genetic loci is screened that modify the aberrant phenotypes caused by over expression of regulatory separase complex subunits Pimples/securin and Three rows in Drosophila. An interacting gene is found to encode a constitutive centromere protein.