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Centromere sliding on a mammalian chromosome.

Purgato S, Belloni E, Piras FM, Zoli M, Badiale C, Cerutti F, Mazzagatti A, Perini G, Della Valle G, Nergadze SG, Sullivan KF, Raimondi E, Rocchi M, Giulotto E - Chromosoma (2014)

Bottom Line: Examination of the ten instances of chromosome 11 in the five individuals revealed seven distinct 'positional alleles', each one extending for about 80-160 kb, were found across a region of about 500 kb.Our results demonstrate that CENP-A binding domains are autonomous relative to the underlying DNA sequence and are characterized by positional instability causing the sliding of centromere position.We propose that this dynamic behaviour may be common in mammalian centromeres and may determine the establishment of epigenetic alleles.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Farmacia e Biotecnologie (FABIT), Università di Bologna, Bologna, Italy.

ABSTRACT
The centromere directs the segregation of chromosomes during mitosis and meiosis. It is a distinct genetic locus whose identity is established through epigenetic mechanisms that depend on the deposition of centromere-specific centromere protein A (CENP-A) nucleosomes. This important chromatin domain has so far escaped comprehensive molecular analysis due to its typical association with highly repetitive satellite DNA. In previous work, we discovered that the centromere of horse chromosome 11 is completely devoid of satellite DNA; this peculiar feature makes it a unique model to dissect the molecular architecture of mammalian centromeres. Here, we exploited this native satellite-free centromere to determine the precise localization of its functional domains in five individuals: We hybridized DNA purified from chromatin immunoprecipitated with an anti CENP-A antibody to a high resolution array (ChIP-on-chip) of the region containing the primary constriction of horse chromosome 11. Strikingly, each individual exhibited a different arrangement of CENP-A binding domains. We then analysed the organization of each domain using a single nucleotide polymorphism (SNP)-based approach and single molecule analysis on chromatin fibres. Examination of the ten instances of chromosome 11 in the five individuals revealed seven distinct 'positional alleles', each one extending for about 80-160 kb, were found across a region of about 500 kb. Our results demonstrate that CENP-A binding domains are autonomous relative to the underlying DNA sequence and are characterized by positional instability causing the sliding of centromere position. We propose that this dynamic behaviour may be common in mammalian centromeres and may determine the establishment of epigenetic alleles.

No MeSH data available.


Related in: MedlinePlus

Variable position of the centromere of horse chromosome 11. a DNA obtained by chromatin immunoprecipitation. Using an anti-CENP-A antibody, from five different horse fibroblast cultures was hybridized to a tiling array covering the centromere region. Results are presented as the log2 ratio of the hybridization signals obtained with immunoprecipitated DNA versus input DNA; x-axis, genomic coordinates on ECA11. Positions of informative SNPs are indicated as black dots (a single nucleotide of the SNP is enriched in immunoprecipitated DNA), red dots (both SNP alleles are present in immunoprecipitated DNA) and blue carats (SNPs shown in Fig. 3). b Peak positions are represented as boxes. Epiallele identification was obtained by combining ChIP-on-chip, SNP (Fig. 3) and fibre FISH (Fig. 4 and Supplementary Table 2) results. Sequence coordinates refer to the horse EquCab2.0 (2007) sequence assembly, as reported by the UCSC genome browser (http://genome.ucsc.edu). Alleles are designated by the letter of the horse they derive from, followed by ‘1’ or ‘2’ to distinguish the two variants. In HSF-D and HSF-E, where a single broad peak was identified by ChIP-on-chip while two distinct centromeric domains were identified by fibre-FISH (Fig. 4) and SNP analysis (Fig. 3 and Supplementary Table 2), dotted lines represent the region of overlap of the two binding domains in the reference sequence. Therefore, at least seven different centromeric domains can be identified: Ba/Ea, Bb, Ca, Cb, Da/Eb, Db/Ga, Gb
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Fig1: Variable position of the centromere of horse chromosome 11. a DNA obtained by chromatin immunoprecipitation. Using an anti-CENP-A antibody, from five different horse fibroblast cultures was hybridized to a tiling array covering the centromere region. Results are presented as the log2 ratio of the hybridization signals obtained with immunoprecipitated DNA versus input DNA; x-axis, genomic coordinates on ECA11. Positions of informative SNPs are indicated as black dots (a single nucleotide of the SNP is enriched in immunoprecipitated DNA), red dots (both SNP alleles are present in immunoprecipitated DNA) and blue carats (SNPs shown in Fig. 3). b Peak positions are represented as boxes. Epiallele identification was obtained by combining ChIP-on-chip, SNP (Fig. 3) and fibre FISH (Fig. 4 and Supplementary Table 2) results. Sequence coordinates refer to the horse EquCab2.0 (2007) sequence assembly, as reported by the UCSC genome browser (http://genome.ucsc.edu). Alleles are designated by the letter of the horse they derive from, followed by ‘1’ or ‘2’ to distinguish the two variants. In HSF-D and HSF-E, where a single broad peak was identified by ChIP-on-chip while two distinct centromeric domains were identified by fibre-FISH (Fig. 4) and SNP analysis (Fig. 3 and Supplementary Table 2), dotted lines represent the region of overlap of the two binding domains in the reference sequence. Therefore, at least seven different centromeric domains can be identified: Ba/Ea, Bb, Ca, Cb, Da/Eb, Db/Ga, Gb

Mentions: We established fibroblast cell lines from five horses (HSF-B, HSF-C, HSF-D, HSF-E and HSF-G). Using 17 microsatellite loci (Thermo Scientific Equine Genotypes Panel 1.1), we determined their likelihood of relation with the Familias 3.1.3 software, demonstrating that they were unrelated (see Materials and Methods). The unexpected observation of two CENP-A binding domains in the horse previously analysed (Wade et al. 2009) prompted us to extend the analysis to these five new individuals. Chromatin was immunoprecipitated with an antibody against CENP-A. DNA was then purified and hybridized to a 3.2 Mb tiling array (accession number: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE57986) spanning the centromeric region of horse chromosome 11 that we previously defined (Wade et al. 2009). The absence of satellite repeats at this locus (Wade et al. 2009) allowed us to position CENP-A-associated DNA (Fig. 1a). Strikingly, each individual exhibited a distinct arrangement of CENP-A binding domains. These were located across a region of approximately 500 kb, with some individuals (HSF-B, HSF-C and HSF-G) exhibiting two clearly defined peaks while others (HSF-D and HSF-E) showed one.Fig. 1


Centromere sliding on a mammalian chromosome.

Purgato S, Belloni E, Piras FM, Zoli M, Badiale C, Cerutti F, Mazzagatti A, Perini G, Della Valle G, Nergadze SG, Sullivan KF, Raimondi E, Rocchi M, Giulotto E - Chromosoma (2014)

Variable position of the centromere of horse chromosome 11. a DNA obtained by chromatin immunoprecipitation. Using an anti-CENP-A antibody, from five different horse fibroblast cultures was hybridized to a tiling array covering the centromere region. Results are presented as the log2 ratio of the hybridization signals obtained with immunoprecipitated DNA versus input DNA; x-axis, genomic coordinates on ECA11. Positions of informative SNPs are indicated as black dots (a single nucleotide of the SNP is enriched in immunoprecipitated DNA), red dots (both SNP alleles are present in immunoprecipitated DNA) and blue carats (SNPs shown in Fig. 3). b Peak positions are represented as boxes. Epiallele identification was obtained by combining ChIP-on-chip, SNP (Fig. 3) and fibre FISH (Fig. 4 and Supplementary Table 2) results. Sequence coordinates refer to the horse EquCab2.0 (2007) sequence assembly, as reported by the UCSC genome browser (http://genome.ucsc.edu). Alleles are designated by the letter of the horse they derive from, followed by ‘1’ or ‘2’ to distinguish the two variants. In HSF-D and HSF-E, where a single broad peak was identified by ChIP-on-chip while two distinct centromeric domains were identified by fibre-FISH (Fig. 4) and SNP analysis (Fig. 3 and Supplementary Table 2), dotted lines represent the region of overlap of the two binding domains in the reference sequence. Therefore, at least seven different centromeric domains can be identified: Ba/Ea, Bb, Ca, Cb, Da/Eb, Db/Ga, Gb
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig1: Variable position of the centromere of horse chromosome 11. a DNA obtained by chromatin immunoprecipitation. Using an anti-CENP-A antibody, from five different horse fibroblast cultures was hybridized to a tiling array covering the centromere region. Results are presented as the log2 ratio of the hybridization signals obtained with immunoprecipitated DNA versus input DNA; x-axis, genomic coordinates on ECA11. Positions of informative SNPs are indicated as black dots (a single nucleotide of the SNP is enriched in immunoprecipitated DNA), red dots (both SNP alleles are present in immunoprecipitated DNA) and blue carats (SNPs shown in Fig. 3). b Peak positions are represented as boxes. Epiallele identification was obtained by combining ChIP-on-chip, SNP (Fig. 3) and fibre FISH (Fig. 4 and Supplementary Table 2) results. Sequence coordinates refer to the horse EquCab2.0 (2007) sequence assembly, as reported by the UCSC genome browser (http://genome.ucsc.edu). Alleles are designated by the letter of the horse they derive from, followed by ‘1’ or ‘2’ to distinguish the two variants. In HSF-D and HSF-E, where a single broad peak was identified by ChIP-on-chip while two distinct centromeric domains were identified by fibre-FISH (Fig. 4) and SNP analysis (Fig. 3 and Supplementary Table 2), dotted lines represent the region of overlap of the two binding domains in the reference sequence. Therefore, at least seven different centromeric domains can be identified: Ba/Ea, Bb, Ca, Cb, Da/Eb, Db/Ga, Gb
Mentions: We established fibroblast cell lines from five horses (HSF-B, HSF-C, HSF-D, HSF-E and HSF-G). Using 17 microsatellite loci (Thermo Scientific Equine Genotypes Panel 1.1), we determined their likelihood of relation with the Familias 3.1.3 software, demonstrating that they were unrelated (see Materials and Methods). The unexpected observation of two CENP-A binding domains in the horse previously analysed (Wade et al. 2009) prompted us to extend the analysis to these five new individuals. Chromatin was immunoprecipitated with an antibody against CENP-A. DNA was then purified and hybridized to a 3.2 Mb tiling array (accession number: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE57986) spanning the centromeric region of horse chromosome 11 that we previously defined (Wade et al. 2009). The absence of satellite repeats at this locus (Wade et al. 2009) allowed us to position CENP-A-associated DNA (Fig. 1a). Strikingly, each individual exhibited a distinct arrangement of CENP-A binding domains. These were located across a region of approximately 500 kb, with some individuals (HSF-B, HSF-C and HSF-G) exhibiting two clearly defined peaks while others (HSF-D and HSF-E) showed one.Fig. 1

Bottom Line: Examination of the ten instances of chromosome 11 in the five individuals revealed seven distinct 'positional alleles', each one extending for about 80-160 kb, were found across a region of about 500 kb.Our results demonstrate that CENP-A binding domains are autonomous relative to the underlying DNA sequence and are characterized by positional instability causing the sliding of centromere position.We propose that this dynamic behaviour may be common in mammalian centromeres and may determine the establishment of epigenetic alleles.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Farmacia e Biotecnologie (FABIT), Università di Bologna, Bologna, Italy.

ABSTRACT
The centromere directs the segregation of chromosomes during mitosis and meiosis. It is a distinct genetic locus whose identity is established through epigenetic mechanisms that depend on the deposition of centromere-specific centromere protein A (CENP-A) nucleosomes. This important chromatin domain has so far escaped comprehensive molecular analysis due to its typical association with highly repetitive satellite DNA. In previous work, we discovered that the centromere of horse chromosome 11 is completely devoid of satellite DNA; this peculiar feature makes it a unique model to dissect the molecular architecture of mammalian centromeres. Here, we exploited this native satellite-free centromere to determine the precise localization of its functional domains in five individuals: We hybridized DNA purified from chromatin immunoprecipitated with an anti CENP-A antibody to a high resolution array (ChIP-on-chip) of the region containing the primary constriction of horse chromosome 11. Strikingly, each individual exhibited a different arrangement of CENP-A binding domains. We then analysed the organization of each domain using a single nucleotide polymorphism (SNP)-based approach and single molecule analysis on chromatin fibres. Examination of the ten instances of chromosome 11 in the five individuals revealed seven distinct 'positional alleles', each one extending for about 80-160 kb, were found across a region of about 500 kb. Our results demonstrate that CENP-A binding domains are autonomous relative to the underlying DNA sequence and are characterized by positional instability causing the sliding of centromere position. We propose that this dynamic behaviour may be common in mammalian centromeres and may determine the establishment of epigenetic alleles.

No MeSH data available.


Related in: MedlinePlus