Chickens possess centromeres with both extended tandem repeats and short non-tandem-repetitive sequences

Abstract

The centromere is essential for faithful chromosome segregation by providing the site for kinetochore assembly. Although the role of the centromere is conserved throughout evolution, the DNA sequences associated with centromere regions are highly divergent among species and it remains to be determined how centromere DNA directs kinetochore formation. Despite the active use of chicken DT40 cells in studies of chromosome segregation, the sequence of the chicken centromere was unclear. Here, we performed a comprehensive analysis of chicken centromere DNA which revealed unique features of chicken centromeres compared with previously studied vertebrates. Centromere DNA sequences from the chicken macrochromosomes, with the exception of chromosome 5, contain chromosome-specific homogenous tandem repetitive arrays that span several hundred kilobases. In contrast, the centromeres of chromosomes 5, 27, and Z do not contain tandem repetitive sequences and span non-tandem-repetitive sequences of only approximately 30 kb. To test the function of these centromere sequences, we conditionally removed the centromere from the Z chromosome using genetic engineering and have shown that that the non-tandem-repeat sequence of chromosome Z is a functional centromere.

Figure 1.

Identification of centromere DNA in chicken. Using DNAs precipitated with CENPA, FISH analysis were performed. Probes are indicated in each panel. FISH signals are shown in red. Centromeres are stained with anti-CENPT antibodies (green). CenDNAs of chromosomes 1, 2, 3, 4, 7, 8, and 11 are identified. These sequences are chromosome-specific. The clone containing CNM repeats hybridized with microchromosomes and chromosomes 6 and 9.

Figure 2.

DNA sequence of centromere of each macro-chromosome is specific. (A) Using DNA clones, which hybridized with centromere region by FISH, DNA sequence of each repeat-unit was characterized. Each DNA sequence was deposited in DDBJ/EMBL/GenBank DNA database. Accession numbers of Cen1, Cen2, Cen3, Cen4, Cen7, Cen8, and Cen11 are AB556722, AB556723, AB556724, AB556725, AB556726, AB556727, and AB556728, respectively. Length of each repeat-unit was determined by Southern hybridization. Probes are indicated in each panel. (B) Homology region in each repeat-unit sequence with known interspersed repeats is shown. Percentage of identity and position of repeats are also shown. Sequences of interspersed repeats are from RepBase. RepBase IDs of CR1-C, Satellite CR1, and GGXHOI sequence are CR1-C, Sat-CR1-GG, and GGXHOI, respectively. Li and Leung (2006) have previous shown that Cen4 contains a CR-1 repeat.

Figure 3.

Genome organization of centromere region of chromosome 1 and chromosome 2. (A) DNAs from BAC clones hybridized with Cen1 were digested with HindIII and characterized by Southern analysis. Various BAC clones containing unique sequences and the 1.8-kb repeat-unit sequence were obtained. (B) A pulsed-field gel electrophoresis (PFGE) analysis was performed to determine the length consisting of the repeat-unit. Information of restriction enzyme sites is shown in C. (C) Genome organization around Cen1 region. Sequences of contigs from BAC002H09 and BAC003G11 are deposited in the DDBJ/EMBL/GenBank database with accession numbers AB556732–AB556734. Sequencing of BAC clones identified the exact boundary between unique region and repeat region. The length consisting of the repeat-unit was determined by a PFGE analysis. (D) DNAs from BAC clones hybridized with Cen2 were digested with HindIII and characterized by Southern analysis. Various BAC clones containing unique sequences and the 3.0-kb repeat-unit sequence were obtained. (E) A PFGE analysis was performed to determine the length consisting of the repeat-unit. Information of restriction enzyme sites is shown in F. (F) Genome organization around Cen2 region. Sequences of contigs from BAC074F14 and BAC285H02 are deposited in the DDBJ/EMBL/GenBank database with accession numbers AB556735 and AB556736, respectively. Sequencing of BAC clones identified the exact boundary between unique region and repeat region. The length consisting of the repeat-unit was determined by a PFGE analysis.

Figure 4.

Centromere region is devoid of tandem-repeated sequences in chromosomes 5 and Z. (A) A distribution of DNA precipitated with CENPA in entire chromosome Z. Major peak is around position of the 42 Mb. High-resolution profile in 1000-bp windows indicates that the binding region of CENPA is restricted in the 30-kb region. Detail sequence information is available in several BAC contigs. As part of sequence information around the 42-Mb position was absent in the NCBI database, we remapped the DNA against BAC contig (387 kb) covering these genomic region and the CENPA associated-DNA was mapped in the 30-kb region, which does not contain tandem-repetitive sequence. Gene ID around CENPA-associated DNA is shown. (B) FISH analysis using a BAC clone (134D03) containing CENPA binding region of chromosome Z (red in top panel). Centromeres are immunostained with anti-CENPT antibodies (green). FISH signals of the 134D03BAC colocalized with centromeres, while FISH signals of the MHM repeat sequence (BamHI 2.2-kb unit) (red in bottom panel) did not localize with CENPT. (C) A distribution of DNA precipitated with CENPA in entire chromosome 5. Major peak is around position of the 3 Mb. High-resolution profile in 1000-bp windows indicates that the binding region of CENPA is restricted in the 30-kb region. Gene IDs around CENPA associated DNA are shown. Clone numbers of BACs are indicated. (D) FISH analysis using a BAC clone (231H07) containing CENPA binding region of chromosome 5 (red in top panel). Centromeres are immunostained with anti-CENPT antibodies (green). FISH signals of the 231H07 BAC colocalized with CENPT, while FISH signals of a BAC clone (429G11) around chromosome 5 satellite repeat sequence (red of bottom panel) did not localize with CENPT.

Figure 5.

Deletion of centromere region of chromosome Z results in chromosome instability. (A) To delete entire centromere region of chromosome Z, loxP sequences were inserted into both flanking sites of the centromere region using homologous recombination. CENPA binding region is also shown. (B) Expected genotype after recombination between both loxP sites upon activation of Cre recombinase. The 127-kb region should be removed after addition of OHT. (C) Confirmation of loxP insertions at both flanking sites of the centromere region. Information of restriction map and probes (#1 and #4) for Southern hybridization are shown in A. (D) Confirmation of recombination between both loxP sites. PFGE analysis was performed. Information for the restriction map and the probe (Bleo) for Southern hybridization is shown in B. After recombination a novel 97-kb band is observed. (E) Growth curve of DT40 cells after removal of centromere DNA. (F) Representative images of abnormal metaphase cells in which Z chromosome is not aligned at metaphase plate. Z chromosome was painted with a Z-specific macrosatellite DNA probe (red). (G) Representative images of abnormal anaphase cells in which sister Z chromosomes are left at midzone. Z chromosome was painted with a Z-specific macrosatellite DNA probe (red). (H) Representative images of FISH analysis with a Z-specific macrosatellite DNA probe (red). Cells with two copies of chromosome Z or lacking chromosome Z are observed. (I) Numbers of cells containing chromosome Z after removal of centromere sequence. As a control, numbers of cells containing chromosome 1 were scored. Total cell numbers (n) for the measurements are also shown.