Project description:Centromeres typically contain repeat sequences, but centromere function does not necessarily depend on these sequences. In aneuploid wheat (Triticum aestivum) and wheat distant hybridization offspring, we found functional centromeres with dramatic changes to centromeric retrotransposon of wheat (CRW) sequences. CRW sequences were greatly reduced in the ditelosomic lines 1BS, 5DS, 5DL, and a wheat-Thinopyrum elongatum addition line. CRWs were completely lost in the ditelosomic line 4DS, but a 994 kb ectopic genomic DNA sequence was involved in de novo centromere formation on the 4DS chromosome. In addition, two ectopic sequences were incorporated in a de novo centromere in a wheat-Th. intermedium addition line. Centromeric sequences were also expanded to the chromosome arm in wide hybridizations. Stable alien chromosomes with two and three regions containing centromeric sequences were found in wheat-Th. elongatum hybrid derivatives, but only one is functional. In wheat-rye (Secale cereale) hybrids, rye centromere specific sequences spread to the chromosome arm and may cause centromere expansion. Thus, distant wheat hybridizations cause frequent and significant changes to the centromere via centromere misdivision, which may affect retention or loss of alien chromosomes in hybrids. ChIP-seq was carried out with anti-CENH3 antibody using material 4DS and control (Chinese Spring, CS as short).
Project description:Centromeres typically contain repeat sequences, but centromere function does not necessarily depend on these sequences. In aneuploid wheat (Triticum aestivum) and wheat distant hybridization offspring, we found functional centromeres with dramatic changes to centromeric retrotransposon of wheat (CRW) sequences. CRW sequences were greatly reduced in the ditelosomic lines 1BS, 5DS, 5DL, and a wheat-Thinopyrum elongatum addition line. CRWs were completely lost in the ditelosomic line 4DS, but a 994 kb ectopic genomic DNA sequence was involved in de novo centromere formation on the 4DS chromosome. In addition, two ectopic sequences were incorporated in a de novo centromere in a wheat-Th. intermedium addition line. Centromeric sequences were also expanded to the chromosome arm in wide hybridizations. Stable alien chromosomes with two and three regions containing centromeric sequences were found in wheat-Th. elongatum hybrid derivatives, but only one is functional. In wheat-rye (Secale cereale) hybrids, rye centromere specific sequences spread to the chromosome arm and may cause centromere expansion. Thus, distant wheat hybridizations cause frequent and significant changes to the centromere via centromere misdivision, which may affect retention or loss of alien chromosomes in hybrids.
Project description:Centromere identity is defined and maintained epigenetically by the presence of the histone variant CENP-A. How centromeric CENP-A position is specified and precisely maintained through DNA replication is not fully understood. The recently released Telomere-to-Telomere (T2T-CHM13) genome assembly containing the first complete human centromere sequences provides a new resource for examining CENP-A position. Mapping CENP-A position in clones of the same cell line to T2T-CHM13 identified highly similar CENP-A position following multiple cell divisions. In contrast, centromeric CENP-A epialleles were evident at several centromeres of different human cell lines, demonstrating the location of CENP-A enrichment and site of kinetochore recruitment varies among human cells. Across the cell cycle, CENP-A molecules deposited in G1 phase are maintained at their precise position through DNA replication. Thus, despite CENP-A dilution during DNA replication, CENP-A is precisely reloaded onto the same sequences within the daughter centromeres, maintaining unique centromere identity among human cells.
Project description:The histone H3 variant, CENP-ACnp1, is normally assembled upon canonical centromeric sequences, but there is no apparent obligate coupling of sequence and assembly, suggesting that centromere location can be epigenetically determined. To explore the tolerances and constraints on CENP-ACnp1 deposition we investigated whether certain locations are favoured when additional CENP-ACnp1 is present in fission yeast cells. Our analyses show that additional CENP-ACnp1 accumulates within and close to heterochromatic centromeric outer repeats, and over regions adjacent to rDNA and telomeres. The use of minichromosome derivatives with unique DNA sequences internal to chromosome ends shows that telomeres are sufficient to direct CENP-ACnp1 deposition. However, chromosome ends are not required as CENP-ACnp1 deposition also occurs at telomere repeats inserted at an internal locus and correlates with the presence of H3K9 methylation near these repeats. The Ccq1 protein, which is known to bind telomere repeats and recruit telomerase, was found to be required to induce H3K9 methylation and thus promote the incorporation of CENP-A near telomere repeats. These analyses demonstrate that at non-centromeric chromosomal locations the presence of heterochromatin influences the sites at which CENP-A is incorporated into chromatin and thus, potentially the location of centromeres. For CENP-A/Cnp1 chromatin immunoprecipitation: DNA immunoprecipitated with anti-Cnp1 serum using chromatin extracts from mutants and wild type control cells in biological duplicates normalized to input DNA from each strain.
Project description:Centromeres are the regions of eukaryotic chromosomes where kinetochores are assembled and direct the correct segregation of chromosomes. Active centromeres are defined by presence of nucleosomes containing CENP-A, a histone H3 variant, which alone is sufficient to direct kinetochore assembly. Once assembled at a location CENP-A chromatin and the kinetochore is maintained at that location though a positive feedback loop where kinetochore proteins recruited by CENP-A promote deposition of new CENP-A following replication. Although CENP-A chromatin itself is a heritable entity, it is normally associated with specific sequences such as human alpha satellite arrays. Such analyses suggest that properties of centromeric DNA itself may favour assembly of CENP-A rather than H3 nucleosomes. To investigate the innate properties of centromeric DNA we have examined histone dynamics on this DNA assembled in CENP-A chromatin at endogenous centromeres and when assembled only in H3 chromatin at an ectopic location. We demonstrate that H3 occupancy on centromeric DNA is innately low while H3 turnover is high. Moreover, even at an ectopic location centromeric DNA programs H3 deposition in S phase and its eviction during G2 when CENP-A is otherwise deposited. G2 accumulation of RNAPII on centromeric DNA during G2 is consistent with transcription-coupled destabilisation of H3 nucleosomes to favour CENP-A deposition.
Project description:Centromeres are the regions of eukaryotic chromosomes where kinetochores are assembled and direct the correct segregation of chromosomes. Active centromeres are defined by presence of nucleosomes containing CENP-A, a histone H3 variant, which alone is sufficient to direct kinetochore assembly. Once assembled at a location CENP-A chromatin and the kinetochore is maintained at that location though a positive feedback loop where kinetochore proteins recruited by CENP-A promote deposition of new CENP-A following replication. Although CENP-A chromatin itself is a heritable entity, it is normally associated with specific sequences such as human alpha satellite arrays. Such analyses suggest that properties of centromeric DNA itself may favour assembly of CENP-A rather than H3 nucleosomes. To investigate the innate properties of centromeric DNA we have examined histone dynamics on this DNA assembled in CENP-A chromatin at endogenous centromeres and when assembled only in H3 chromatin at an ectopic location. We demonstrate that H3 occupancy on centromeric DNA is innately low while H3 turnover is high. Moreover, even at an ectopic location centromeric DNA programs H3 deposition in S phase and its eviction during G2 when CENP-A is otherwise deposited. G2 accumulation of RNAPII on centromeric DNA during G2 is consistent with transcription-coupled destabilisation of H3 nucleosomes to favour CENP-A deposition.
Project description:Centromeres are the regions of eukaryotic chromosomes where kinetochores are assembled and direct the correct segregation of chromosomes. Active centromeres are defined by presence of nucleosomes containing CENP-A, a histone H3 variant, which alone is sufficient to direct kinetochore assembly. Once assembled at a location CENP-A chromatin and the kinetochore is maintained at that location though a positive feedback loop where kinetochore proteins recruited by CENP-A promote deposition of new CENP-A following replication. Although CENP-A chromatin itself is a heritable entity, it is normally associated with specific sequences such as human alpha satellite arrays. Such analyses suggest that properties of centromeric DNA itself may favour assembly of CENP-A rather than H3 nucleosomes. To investigate the innate properties of centromeric DNA we have examined histone dynamics on this DNA assembled in CENP-A chromatin at endogenous centromeres and when assembled only in H3 chromatin at an ectopic location. We demonstrate that H3 occupancy on centromeric DNA is innately low while H3 turnover is high. Moreover, even at an ectopic location centromeric DNA programs H3 deposition in S phase and its eviction during G2 when CENP-A is otherwise deposited. G2 accumulation of RNAPII on centromeric DNA during G2 is consistent with transcription-coupled destabilisation of H3 nucleosomes to favour CENP-A deposition.
Project description:Centromeres are the regions of eukaryotic chromosomes where kinetochores are assembled and direct the correct segregation of chromosomes. Active centromeres are defined by presence of nucleosomes containing CENP-A, a histone H3 variant, which alone is sufficient to direct kinetochore assembly. Once assembled at a location CENP-A chromatin and the kinetochore is maintained at that location though a positive feedback loop where kinetochore proteins recruited by CENP-A promote deposition of new CENP-A following replication. Although CENP-A chromatin itself is a heritable entity, it is normally associated with specific sequences such as human alpha satellite arrays. Such analyses suggest that properties of centromeric DNA itself may favour assembly of CENP-A rather than H3 nucleosomes. To investigate the innate properties of centromeric DNA we have examined histone dynamics on this DNA assembled in CENP-A chromatin at endogenous centromeres and when assembled only in H3 chromatin at an ectopic location. We demonstrate that H3 occupancy on centromeric DNA is innately low while H3 turnover is high. Moreover, even at an ectopic location centromeric DNA programs H3 deposition in S phase and its eviction during G2 when CENP-A is otherwise deposited. G2 accumulation of RNAPII on centromeric DNA during G2 is consistent with transcription-coupled destabilisation of H3 nucleosomes to favour CENP-A deposition.