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

ABSTRACT: We identified a newly formed dicentric chromosome (sDic-15) in maize from intrachromosomal recombination and BFB cycles, in which only one centromere is active. The centromeres lost CentC sequences and dramatic reduced the CRM sequences, but when the molecular features of functional centromeres such as CENH3 was examined, they were present. Immunolocalization analysis of phosphorylation of H3T3, H3ser-10 and H2A levels on this new centromere shows a pattern typical of a functional centromere. Meiotic analysis revealed that this dicentric chromosome is table and transmit very well. To examine the new sequences associated with CENH3 in this centromere, chromatin immunoprecipitation (ChIP) was carried out with anti-CENH3 antibodies and material from young seedlings with or without dicentric chromosome. We mapped the ChIP-Seq reads to the reference genome and found a 723kb region from the short arm of maize chromosome 9 involve the new centromere formation. This region is gene-poor and full of TEs, but genes in this region are transcribed. The original 723kb region shows a high DNA methylation level as native centromeres but had no significant change when it involved into new centromere formation. The reactivation of this newly formed centromere indicated that centromere reactivation may not dependent on the relatively intact DNA sequences or topology of original inactive centromere. ChIP-seq was carried out with anti-CENH3 antibodies using material from young seedlings with and without sDic-15 chromosome. For sDic-15, some ChIPed DNA was treated with sodium bisulfite and prepared for Illumina sequencing to test its methylation level.

ORGANISM(S): Zea mays

SUBMITTER: Pang Junling

PROVIDER: E-GEOD-43140 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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

Sequential centromere shift via de novo formation in maize

Project description:The ability of centromeres to alternate between active and inactive states indicates significant epigenetic elements controlling centromere assembly and centromere function. In maize (Zea mays), misdivision of the B chromosome centromere on a translocation with the short arm of chromosome 9 (TB-9Sb) can produce many variants with varying centromere sizes and centromeric DNA sequences. In derivatives of TB-9Sb, we found a de novo centromere on chromosome telo-3-3, which has no canonical centromeric repeat sequences. This centromere is derived from a 288-kb region on the short arm of chromosome 9, and is 19 megabases (Mb) removed from the translocation breakpoint of chromosome 9 in TB-9Sb. This centromere is much smaller than normal ones but can be maintained through meiosis. The functional B centromere in progenitor telo2-2 is deleted from telo3-3 but some B-repeat sequences remain. The de novo centromere of telo3-3 becomes inactive in three further derivatives with new centromeres being formed elsewhere on the chromosomes. One such de novo centromere contains only 200-kb CENH3 binding domain. This 200-kb centromere is located 3 Mb removed from the translocation breakpoint in a new location. The deleted B centromere in telo3-3 is activated in two derivatives. Our results suggest that de novo centromere formation is more common than previously thought and can persist on chromosomal fragments without a canonical centromere providing implications for karyotype evolution. ChIP-seq was carried out with anti-CENH3 antibodies using material from young leaves with control, telo3-3 and its derivate.

2015-03-03 | E-GEOD-59124 | biostudies-arrayexpress

Epigenetic characteristics and gene-expression analysis of Derivative 3-3 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 Derivative 3-3 contains a de novo centromere, which was derived from a euchromatic site on the short arm of chromosome 9 that lacks traditional centromeric repeat sequences. Our previous study found that the CENH3 binding domain of this de novo centromere is only 288 kb with a high-density gene distribution with low-density of transposons. Here we applied next generation sequencing technology to analyze gene transcription, DNA methylation for this region. Our RNA-seq data revealed that active chromatin is not a barrier for de novo centromere formation. Bisulfite-ChIP-seq results indicate a slightly increased DNA methylation level after de novo centromere formation, reaching the level of a native centromere. These results provide insight into the mechanism of de novo centromere formation and subsequent consequences. RNA-seq was carried out using material from seedling and young leaves between control and Derivative 3-3. Bisulfite-ChIP-seq was carried out with anti-CENH3 antibodies using material from young leaves in Derivative 3-3.

2016-08-05 | E-GEOD-76533 | biostudies-arrayexpress

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

Project description:We identified a newly formed dicentric chromosome (sDic-15) in maize from intrachromosomal recombination and BFB cycles, in which only one centromere is active. The centromeres lost CentC sequences and dramatic reduced the CRM sequences, but when the molecular features of functional centromeres such as CENH3 was examined, they were present. Immunolocalization analysis of phosphorylation of H3T3, H3ser-10 and H2A levels on this new centromere shows a pattern typical of a functional centromere. Meiotic analysis revealed that this dicentric chromosome is table and transmit very well. To examine the new sequences associated with CENH3 in this centromere, chromatin immunoprecipitation (ChIP) was carried out with anti-CENH3 antibodies and material from young seedlings with or without dicentric chromosome. We mapped the ChIP-Seq reads to the reference genome and found a 723kb region from the short arm of maize chromosome 9 involve the new centromere formation. This region is gene-poor and full of TEs, but genes in this region are transcribed. The original 723kb region shows a high DNA methylation level as native centromeres but had no significant change when it involved into new centromere formation. The reactivation of this newly formed centromere indicated that centromere reactivation may not dependent on the relatively intact DNA sequences or topology of original inactive centromere.

2013-06-25 | GSE43140 | GEO

Holocentromeres are dispersed point centromeres localized at transcription factor hotspots

Project description:The centromere is a defining feature of eukaryotic chromosomes and is essential for the segregation of chromosomes during cell division. Centromeres are universally marked by the histone variant cenH3 and are restricted to specialized chromatin that most commonly localized to a single position along the chromosome. However, the DNA on which centromeric nucleosomes assemble is not conserved and varies greatly in size and composition. It ranges from genetically defined point centromeres that assemble a single cenH3-containing nucleosome to epigenetically defined regional centromeres embedded in megabases of tandemly repeated DNA to holocentromeres that extend along the length of the entire chromosomes. The organization of regional and holocentric centromeres has so far been elusive, as the precise locations of cenH3-containing sequences could not be determined. Our results show that the point centromere is the basic unit of holocentromeres and provide a basis for understanding how centromeric chromatin is maintained. We use high-resolution mapping of cenH3-associated DNA to show that Caenorhabditids elegans holocentromeres are organized as dispersed but discretely localized point centromeres.

2014-04-08 | E-GEOD-44412 | biostudies-arrayexpress

Centromere misdivision promotes centromeric sequence changes, de novo centromere formation, and centromere expansion

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).

2016-04-02 | E-GEOD-63752 | biostudies-arrayexpress

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

Project description:Plant and animal centromeres comprise megabases of highly repeated satellite sequences, yet centromere function can be specified epigenetically on single-copy DNA by the presence of nucleosomes containing a centromere-specific variant of histone H3 (cenH3). We determined the positions of cenH3 nucleosomes in rice (Oryza sativa), which has centromeres composed of both the 155-bp CentO repeat and single-copy non-CentO sequences. We find that cenH3 nucleosomes protect 90-100 bp of DNA from micrococcal nuclease digestion, sufficient for only a single wrap of DNA around the cenH3 nucleosome core. cenH3 nucleosomes are translationally phased with 155-bp periodicity on CentO repeats, but not on non-CentO sequences. CentO repeats have a ~10-bp periodicity in WW dinucleotides and in micrococcal nuclease cleavage, providing evidence for rotational phasing of cenH3 nucleosomes on CentO, and suggesting that satellites evolve for translational and rotational stabilization of centromeric nucleosomes. Examination of measured size of rice centromere nucleosome

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

Genomic and genetic characterization of rice Cen3 reveals evolutionary remnants of a complex centromere (small RNA data)

Project description:The centromere is the chromosomal site for assembly of the kinetochore where spindle fibers attach during cell division. In most muticellular eukaryotes, centromeres are composed of long tracks of satellite repeats that are recalcitrant to sequencing and fine scale genetic mapping. Here we report the genomic and genetic characterization of the complete centromere of rice chromosome 3. Using a DNA fiber-fluorescence in situ hybridization approach, we demonstrated that the centromere of chromosome 3 (Cen3) contains ~414 kb of the centromeric satellite repeat CentO. Cen3 includes a ~1,851-kb domain associated with CENH3, the centromere-specific histone H3 variant. This CENH3-associated chromatin domain is embedded within a 3,083-kb region that lacks genetic recombination. We detected several active genes within the CENH3-binding domain based on a comprehensive annotation and a survey for matches with mRNA signatures. However, the gene density in the CENH3-binding domain is significantly lower than in the pericentromeric domains. In contrast, the CENH3-binding domain contains a higher percentage of repetitive DNA sequences than the pericentromeric regions. These results suggest that Cen3 is in the process of evolving from a genic region toward an accumulation of satellite repeats and transposable elements that is more characteristic of centromeres in most complex eukaryotic species. Keywords: MPSS, small RNA, rice, centromere, Cen3, chromosome 3

2006-05-26 | GSE4912 | GEO

Genomic and genetic characterization of rice Cen3 reveals evolutionary remnants of a complex centromere (mRNA data)

2006-05-26 | GSE4920 | GEO

Centromere-Like Regions in Budding Yeast

Project description:Accurate chromosome segregation requires centromeres (CENs), the chromosomal sites where kinetochores form, to bridge DNA and attach to microtubules. In contrast to most eukaryotes, Saccharomyces cerevisiae possesses sequence-defined point centromeres. Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) of four kinetochore components reveals regions of overlapping, extra-centromeric protein localization upon overproduction of the centromeric histone, Cse4 (CENP-A or CenH3). These identified sequences enhance proper plasmid and chromosome segregation, and are termed Centromere-like Regions (CLRs). CLRs form in close proximity to S. cerevisiae CENs and share characteristics typical of point and regional centromeres. CLR sequences are conserved among related budding yeasts, suggesting a role in vivo. These studies provide new insights into the origin and evolution of centromeres. ChIP-Seq analysis of the kinetochore components Cse4, Mif2, Ndc10 and Ndc80 in budding yeast strains (Saccharomyces cerevisiae) with normal and elevated levels of Cse4

2013-01-18 | E-GEOD-31466 | biostudies-arrayexpress

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