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ChIP-seq in oat-maize additional lines using CenH3 antibody

ABSTRACT: Many existing centromeres may have originated as neocentromeres that activated de novo from non-centromeric regions. However, the evolutionary path from a neocentromere to a mature centromere has been elusive. Here we analyzed the centromeres of six chromosomes that were transferred from maize into oat as the result of an inter-species cross. Centromere size and location were assayed by chromatin immunoprecipitation for the histone variant CENH3, which is a defining feature of functional centromeres. Maize and oat are highly divergent and differ in genome size by four fold. Two isolates of maize chromosome proved to contain neocentromeres in the sense that they had moved from the original site, whereas the remaining seven centromeres (1, 2, 5, 6, 8, 9 and 10) were retained in the same area in both species. In all cases the CENH3-binding domains were dramatically expanded to encompass a larger area in the oat background (~4 Mb) than the average centromere size in maize (~2 Mb). The expansion of maize centromeres appeared to be restricted by the transcription of genes located in regions flanking the original centromeres. The results from the current study provide evidence that (1) centromere size is regulated; (2) centromere sizes tend to be uniform within a species regardless of chromosome size or origin of the centromere; and (3) neocentromeres emerge and expand preferentially in gene poor regions. Our results, together with data from several animal species, suggest that centromere size expansion may be a key factor in the survival of neocentric chromosomes in natural populations.

ORGANISM(S): Avena sativa/Zea mays addition line

PROVIDER: GSE47342 | GEO | 2013/09/01

SECONDARY ACCESSION(S): PRJNA205183

REPOSITORIES: GEO

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Functional centromere lost centromeric specific sequences but gain new sequences from nearby chromosomal arm.

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2013-06-25 | GSE43140 | GEO

Functional centromere lost centromeric specific sequences but gain new sequences from nearby chromosomal arm.

2013-06-25 | E-GEOD-43140 | biostudies-arrayexpress

The CentO satellite confers translational and rotational phasing on cenH3 nucleosomes in rice centromeres

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2013-11-06 | E-GEOD-50755 | biostudies-arrayexpress

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2020-04-17 | GSE136854 | GEO

Holocentromeres are dispersed point centromeres localized at transcription factor hotspots

2014-04-08 | GSE44412 | GEO

Epigenetic characteristics and gene-expression analysis of a de novo centromere in maize

Project description:De novo centromeres originate occasionally from non-centromeric regions of chromosomes, providing an excellent model system to study centromeric chromatin. The maize mini-chromosome Dp3a contains a de novo centromere, which was derived from a euchromatic site on the long arm of chromosome 3 that lacks traditional centromeric repeat sequences. Our previous study found that the CENH3 binding domain of this de novo centromere is only 350 kb with a high-density gene distribution with low-density of transposons. Here we analyzed the kinetochore complex assembled on the de novo centromere, which revealed that it resembles the native centromeres. Meiotic analyses showed that the Dp3a mini-chromosome formed bi-orientation during meiosis I and could stably transmit through meiosis. We applied next generation sequencing technology to analyze gene transcription, DNA methylation and histone modifications for this region. Our RNA-seq data revealed that active chromatin is not a barrier for de novo centromere formation. The gene expression level within the Dp3a breakpoints of native chromosome 3 indicated a gene dosage effect due to the additional copy of Dp3a mini-chromosome region in chromosome 3. Bisulfite-ChIP-seq results indicate a slightly increased DNA methylation level after de novo centromere formation, reaching the level of a native centromere. No strand-specific bias of DNA methylation was found at maize native and Dp3a de novo centromeres. H2A-T133 phosphorylation occurs in the CENH3 nucleosome. These results provide insight into the mechanism of de novo centromere formation and subsequent consequences.

2016-12-01 | GSE64641 | GEO

Fine structure analysis and epigenetic properties of a de novo centromere on a chromosome fragment in maize

Project description:A small fragment from maize chromosome 3 was created by irradiation by Stadler and Roman and named Duplication 3a (or Dp3a). This small chromosome does not contain any detectable CentC and CRM sequences, but when molecular features of functional centromeres such as CENH3 and CENP-C were examined, they were present. Immunolocalization analysis of phosphorylation of Ser-10 of histone H3 levels on Dp3a shows a pattern typical of a functional centromere. Meiotic analysis revealed that sister chromatids divided equationally at meiosis I as do all small chromosomes examined to date in maize. To examine the sequences associated with CENH3, chromatin immunoprecipitation (ChIP) was carried out with anti-CENH3 antibodies using material from young seedlings with and without Dp3 chromosome as the tissue source. The ChIPed DNA sample was then labeled for FISH detection and prepared for Illumina sequencing.The ChIP-Seq reads were mapped to the B73 reference genome and a significant peak was detected in the Dp3a sample that span 350 kb of the long arm of chromosome 3, which is the candidate region for association with CENH3. ChIP-bisulfite-seq results indicated that there is a slightly increased DNA methylation level after the centromere formation, approaching the level similar to normal centromere regions. Collectively, the results suggest the formation of a de novo centromere on this fragment that initially must have started at the time of X-irradiation release from the progenitor chromosome. These observations add further evidence for the epigenetic nature of centromere function in maize. ChIP-seq was carried out with anti-CENH3 antibodies using material from young seedlings with and without Dp3a chromosome. For Dp3a, some ChIPed DNA was treated with sodium bisulfite and prepared for Illumina sequencing to test its methylation level.

2013-06-25 | E-GEOD-43138 | biostudies-arrayexpress

The deposition of CENH3 is stringently regulated in Maize

Project description:The centromere, as an essential element to control chromosome segregation, is epigenetically determined by CENH3-containing nucleosomes as a functional marker, therefore the accurate deposition of CENH3 is crucial to chromosome transmission. We characterized the deposition of CENH3 in maize by over-expression and mutational analysis. Our results revealed that over-expressing CENH3 in callus is lethal while over-expressing GFP-CENH3 and CENH3-YFP in callus and plants is not and can be partly deposited normally. Different mutations of GFP-CENH3 demonstrated that CENH3-Thr4 in the N terminus was needed for the deposition as a positive phosphorylation site and the last five amino acids in the C terminus are necessary for deposition. The C terminal tail of CENH3 is confirmed to be responsible for the interaction of CENH3 and histone H4, which indicates that CENH3 maintains deposition in centromeres via interacting with H4 to form stable nucleosomes. For GFP-CENH3 and CENH3-YFP, the fused tags at the termini probably affect the structure of CENH3 and reduce its interaction with other proteins, which in turn could decrease proper deposition. Taken together, multiple amino acids or motifs were shown to play essential roles in CENH3 deposition, which is suggested to be affected by numerous factors in maize.

2020-06-01 | GSE133578 | GEO

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2013-06-25 | GSE43138 | GEO

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