Genomic and genetic characterization of rice Cen3 reveals evolutionary remnants of a complex centromere (mRNA data)
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ABSTRACT: 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, mRNA, transcriptome, rice, centromere, Cen3, chromosome 3
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
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. MPSS was performed to sequence small RNAs that derived from 16 untreated and 6 abiotic-treated diverse tissue libraries. The method for the MPSS sequencing of mRNAs is described in Brenner et al. (Nat Biotechnol. 2000 18:630).
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. MPSS was performed to sequence small RNAs that derived from inflorescence and seedling. The method for the MPSS sequencing of small RNAs are described in the paper associated with this dataset (Lu et al., 2005).
Project description:Specification and propagation of the centromeres of eukaryotic chromosomes is determined by epigenetic mechanisms. Unfortunately, the epigenetic characteristics of centromeric DNA and chromatin are difficult to define because the centromeres are composed of highly repetitive DNA sequences in most eukaryotic species. Several rice centromeres have been fully sequenced, making rice an excellent model for centromere research. We conducted genome-wide mapping of cytosine methylation using methylcytosine immunoprecipitation combined with Illumina sequencing. The DNA sequences in the core domains of rice Cen4, Cen5, and Cen8 showed elevated methylation levels compared to the sequences in the pericentromeric regions. In addition, elevated methylation levels were associated with the DNA sequences in the CENH3-binding subdomains compared to the sequences in the flanking H3 subdomains. In contrast, the centromeric domain of Cen11, which is composed exclusively of centromeric satellite DNA, is hypomethylated compared to the pericentromeric domains. Thus, the DNA sequences associated with functional centromeres can be either hypomethylated or hypermethylated. The methylation patterns of centromeric DNA appear to be correlated with the composition of the associated DNA sequences. We propose that both hypomethylation and hypermethylation of CENH3-associated DNA sequences can serve as epigenetic marks to distinguish where CENH3 deposition will occur within the surrounding H3 chromatin. mCIP-seq of one sample of rice seedling
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
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.
Project description:Specification and propagation of the centromeres of eukaryotic chromosomes is determined by epigenetic mechanisms. Unfortunately, the epigenetic characteristics of centromeric DNA and chromatin are difficult to define because the centromeres are composed of highly repetitive DNA sequences in most eukaryotic species. Several rice centromeres have been fully sequenced, making rice an excellent model for centromere research. We conducted genome-wide mapping of cytosine methylation using methylcytosine immunoprecipitation combined with Illumina sequencing. The DNA sequences in the core domains of rice Cen4, Cen5, and Cen8 showed elevated methylation levels compared to the sequences in the pericentromeric regions. In addition, elevated methylation levels were associated with the DNA sequences in the CENH3-binding subdomains compared to the sequences in the flanking H3 subdomains. In contrast, the centromeric domain of Cen11, which is composed exclusively of centromeric satellite DNA, is hypomethylated compared to the pericentromeric domains. Thus, the DNA sequences associated with functional centromeres can be either hypomethylated or hypermethylated. The methylation patterns of centromeric DNA appear to be correlated with the composition of the associated DNA sequences. We propose that both hypomethylation and hypermethylation of CENH3-associated DNA sequences can serve as epigenetic marks to distinguish where CENH3 deposition will occur within the surrounding H3 chromatin.
Project description:The centromere is defined by the presence of a centromere-specific histone H3 variant, CENH3. Establishment and maintenance of the centromeric chromatin (CEN chromatin) is determined by poorly understood epigenetic mechanisms. Interestingly, CEN chromatin in several eukaryotes showed euchromatic characteristics although being embedded within pericentromeric heterochromatin. Specifically, H3K4me2 appeared to be a unique histone modification mark associated with animal centromeres. We developed a genomic tiling array for four fully sequenced rice centromeres. A ChIP-chip approach was used to study the patterns of several euchromatic histone modification marks, including H3K4me2, H3K4me3, H3K36me3, and H3K4K9a, associated with rice centromeres. We demonstrate that the CENH3 subdomains within the four centromeres are depleted with the four histone H3 marks. The vast majority of the four histone marks were associated with the genes located in the H3 subdomains within the centromeric cores. Genes in the centromeres showed similar histone modification patterns as those located outside of the centromeres. Thus, the euchromatic characteristics of rice CEN chromatin are trademarks of the transcribed sequences embedded in the H3 subdomains of the centromeres. We propose that the constitutively expressed genes located in rice centromeres may provide a barrier for loading of CENH3 into the H3 subdomains. The separation of CENH3 into the H3 subdomains is favorable for the three dimensional structure and its associated function of rice centromeres.
Project description:The centromere is defined by the presence of a centromere-specific histone H3 variant, CENH3. Establishment and maintenance of the centromeric chromatin (CEN chromatin) is determined by poorly understood epigenetic mechanisms. Interestingly, CEN chromatin in several eukaryotes showed euchromatic characteristics although being embedded within pericentromeric heterochromatin. Specifically, H3K4me2 appeared to be a unique histone modification mark associated with animal centromeres. We developed a genomic tiling array for four fully sequenced rice centromeres. A ChIP-chip approach was used to study the patterns of several euchromatic histone modification marks, including H3K4me2, H3K4me3, H3K36me3, and H3K4K9a, associated with rice centromeres. We demonstrate that the CENH3 subdomains within the four centromeres are depleted with the four histone H3 marks. The vast majority of the four histone marks were associated with the genes located in the H3 subdomains within the centromeric cores. Genes in the centromeres showed similar histone modification patterns as those located outside of the centromeres. Thus, the euchromatic characteristics of rice CEN chromatin are trademarks of the transcribed sequences embedded in the H3 subdomains of the centromeres. We propose that the constitutively expressed genes located in rice centromeres may provide a barrier for loading of CENH3 into the H3 subdomains. The separation of CENH3 into the H3 subdomains is favorable for the three dimensional structure and its associated function of rice centromeres. We developed a genomic tiling array that covers four rice centromeres (Cen4, Cen5, Cen7, and Cen8) using the NimbleGen 3x720K array based on the NimbleGen HD2 platform. We used four antibodies (H3K4me3, H3K4me2, H3K36me3, H3K4K9ac) to perform ChIP-chip experiments. ChIP was conducted using leaf tissue from two-week old rice seedlings. We have 3 biological replicates for each antibody and 6 technological replicates of hybridization with position exchange on the array. Thus, each histone modification included 18 hybridization experiments.
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.