Similar Datasets
Project description:The centromere is the genetic locus that organizes the proteinaceous kinetochore and is responsible for attachment of the chromosome to the spindle at mitosis and meiosis. In most eukaryotes, the centromere consists of highly repetitive DNA sequences that are occupied by nucleosomes containing the CenH3 histone variant, whereas in budding yeast, an ~120-bp Centromere DNA Element (CDE) that is sufficient for centromere function is occupied by a single right-handed CenH3 (Cse4) nucleosome. However, these in vivo observations are inconsistent with in vitro evidence for left-handed octameric CenH3 nucleosomes. To help resolve these inconsistencies, we characterized yeast centromeric chromatin at single base-pair resolution. Intact particles containing both Cse4 and H2A are precisely protected from micrococcal nuclease over the entire CDE of all 16 yeast centromeres in both solubilized chromatin and the insoluble kinetochore. Small DNA-binding proteins protect CDEI and CDEIII and delimit the centromeric nucleosome to the ~80-bp CDEII, only enough for a single DNA wrap. As expected for a tripartite organization of centromeric chromatin, loss of Cbf1 protein, which binds to CDEI, both reduces the size of the centromere-protected region and shifts its location towards CDEIII. Surprisingly, Cse4 overproduction caused genome-wide misincorporation of non-functional CenH3-containing nucleosomes that protect ~135 base pairs and are preferentially enriched at sites of high nucleosome turnover. Our detection of two forms of CenH3 nucleosomes in the yeast genome, a singly wrapped particle at the functional centromere and octamer-sized particles on chromosome arms, reconcile seemingly conflicting in vivo and in vitro observations. We used micrococcal nuclease mapping, chromatin immunoprecipitation and paired-end sequencing to determine the structure of yeast centromeres at single base-pair resolution.
2011-12-07 | E-GEOD-28298 | biostudies-arrayexpress
Project description:Centromeric chromatin in Koala
| PRJNA436016 | ENA
Project description:The centromere is the genetic locus that organizes the proteinaceous kinetochore and is responsible for attachment of the chromosome to the spindle at mitosis and meiosis. In most eukaryotes, the centromere consists of highly repetitive DNA sequences that are occupied by nucleosomes containing the CenH3 histone variant, whereas in budding yeast, an ~120-bp Centromere DNA Element (CDE) that is sufficient for centromere function is occupied by a single right-handed CenH3 (Cse4) nucleosome. However, these in vivo observations are inconsistent with in vitro evidence for left-handed octameric CenH3 nucleosomes. To help resolve these inconsistencies, we characterized yeast centromeric chromatin at single base-pair resolution. Intact particles containing both Cse4 and H2A are precisely protected from micrococcal nuclease over the entire CDE of all 16 yeast centromeres in both solubilized chromatin and the insoluble kinetochore. Small DNA-binding proteins protect CDEI and CDEIII and delimit the centromeric nucleosome to the ~80-bp CDEII, only enough for a single DNA wrap. As expected for a tripartite organization of centromeric chromatin, loss of Cbf1 protein, which binds to CDEI, both reduces the size of the centromere-protected region and shifts its location towards CDEIII. Surprisingly, Cse4 overproduction caused genome-wide misincorporation of non-functional CenH3-containing nucleosomes that protect ~135 base pairs and are preferentially enriched at sites of high nucleosome turnover. Our detection of two forms of CenH3 nucleosomes in the yeast genome, a singly wrapped particle at the functional centromere and octamer-sized particles on chromosome arms, reconcile seemingly conflicting in vivo and in vitro observations.
2011-12-07 | GSE28298 | GEO
Project description:Centromeres of most eukaryotes are multi-megabase arrays of satellite DNA that assemble proteinaceous kinetochores to facilitate faithful chromosome segregation. However, the nature of the chromatin landscape at centromeres remains unclear, in part due to the difficulty of isolating intact centromeric chromatin rendered insoluble by megadalton-size kinetochore protein complexes. To address this challenge we combined classical salt fractionation with chromatin immunoprecipitation to recover human centromeric chromatin under native conditions. We found that >85% of the total centromeric chromatin is insoluble under conditions typically used for native chromatin extraction. To map both soluble and insoluble chromatin in situ, we combined CUT&RUN, a targeted nuclease method, with salt fractionation. Using this approach, we found that the strength of Centromere Protein B (CENP-B) binding and the density of CENP-B motifs corresponds to the occupancy of Constitutive Centromere-Assocated Network (CCAN) complexes bound to α-satellite arrays. We also observed unexpected structural variations of CENP-A-containing complexes on different α-satellite dimeric units within highly homogenous arrays. Our results suggest that slight α-satellite sequence differences controls the structure and occupancy of the associated centromeric chromatin complex.
2018-01-31 | GSE104805 | GEO
Project description:CENP-A, a variant of histone H3, is incorporated into centromeric chromatin and plays a role during kinetochore establishment. In fission yeast, the localization of CENP-A is limited to a region spanning 10 to 20 kb of the core domain of the centromere. Here, we report a mutant (rpt3-1) in which this region is expanded to 40 to 70 kb. Likely due to abnormal distribution of CENP-A, this mutant exhibits chromosome instability and enhanced gene silencing. Interestingly, the rpt3+ gene encodes a subunit of the 19S proteasome, which localizes to the nuclear membrane. While Rpt3 associates with centromeric chromatin, the mutant protein has lost this localization. A loss of the cut8+ gene encoding an anchor of the proteasome to the nuclear membrane causes similar phenotypes as observed in the rpt3-1 mutant. Thus, we propose that the proteasome (or its subcomplex) associates with centromeric chromatin and regulates distribution of CENP-A. Chromosomal distributions of differentially expressed centromere protein A in wild-type and a proteasome mutant.
2014-04-07 | E-GEOD-44594 | biostudies-arrayexpress
Project description:Functional centromeres are essential for proper cell division. Centromeres are established largely by epigenetic processes resulting in incorporation of the histone H3 variant CENP-A. Here, we demonstrate the direct involvement of H2B monoubiquitination, mediated by RNF20 in humans or Brl1 in Schizosaccharomyces pombe, in centromeric chromatin maintenance. Monoubiquinated H2B (H2Bub1) is needed for this maintenance, promoting noncoding transcription, centromere integrity and accurate chromosomal segregation. A transient pulse of centromeric H2Bub1 leads to RNA polymerase II–mediated transcription of the centromere’s central domain, coupled to decreased H3 stability. H2Bub1-deficient cells have centromere cores that, despite their intact centromeric heterochromatin barriers, exhibit characteristics of heterochromatin, such as silencing histone modifications, reduced nucleosome turnover and reduced levels of transcription. In the H2Bub1-deficient cells, centromere functionality is hampered, thus resulting in unequal chromosome segregation. Therefore, centromeric H2Bub1 is essential for maintaining active centromeric chromatin.
2014-01-29 | GSE49874 | GEO
Project description:Functional centromeres are essential for proper cell division. Centromeres are established largely by epigenetic processes resulting in incorporation of the histone H3 variant CENP-A. Here, we demonstrate the direct involvement of H2B monoubiquitination, mediated by RNF20 in humans or Brl1 in Schizosaccharomyces pombe, in centromeric chromatin maintenance. Monoubiquinated H2B (H2Bub1) is needed for this maintenance, promoting noncoding transcription, centromere integrity and accurate chromosomal segregation. A transient pulse of centromeric H2Bub1 leads to RNA polymerase IIM-bM-^@M-^Smediated transcription of the centromereM-bM-^@M-^Ys central domain, coupled to decreased H3 stability. H2Bub1-deficient cells have centromere cores that, despite their intact centromeric heterochromatin barriers, exhibit characteristics of heterochromatin, such as silencing histone modifications, reduced nucleosome turnover and reduced levels of transcription. In the H2Bub1-deficient cells, centromere functionality is hampered, thus resulting in unequal chromosome segregation. Therefore, centromeric H2Bub1 is essential for maintaining active centromeric chromatin. ChIP: In total 17 samples; 15 ChIP DNA files (7 different conditions) 2 Input files. Cnp1_WT_input.bar (antibody against Cnp1 in strains Hu303, Hu29 (WT) (Cnp1_Hu303.CEL, AS_10.CEL) vs input (JW_1.CEL, JW_2_2.CEL)); Cnp1_Hu2640_WT.bar (antibody against Cnp1 in strain Hu2640 (htb1K119R) (Cnp1_Hu2640_A.CEL, Cnp1_Hu2640_B.CEL) vs strains Hu303, Hu29 (WT) (Cnp1_Hu303.CEL, AS_10.CEL)); Cnp1_Hu2640_input.bar (antibody against Cnp1 in strain Hu2640 (htb1K119R) (Cnp1_Hu2640_A.CEL, Cnp1_Hu2640_B.CEL) vs input (JW_1.CEL, JW_2_2.CEL)); H2Bub1_Hu303_input.bar (antibody against H2Bub1 in strain Hu303 (WT) (H2Bub1_Hu303_A.CEL, H2Bub1_Hu303_B.CEL) vs input (JW_1.CEL, JW_2_2.CEL)); H3_WT_input.bar (antibody against H3 in strains Hu303, Hu29 (WT) (H3_Hu303.CEL, As_12.CEL) vs input (JW_1.CEL, JW_2_2.CEL); H3_Hu2640_WT.bar (antibody against H3 in strain Hu2640 (htb1K119R) (H3_Hu2640_A.CEL, H3_Hu2640_B.CEL, H3_Hu2640_C.CEL) vs strains Hu303, Hu29 (WT) (H3_Hu303.CEL, AS_12.CEL)); H3_Hu2640_input.bar (antibody against H3 in strain Hu2640 (htb1K119R) (H3_Hu2640_A.CEL, H3_Hu2640_B.CEL, H3_Hu2640_C.CEL) vs input (JW_1.CEL, JW_2_2.CEL)); H3K9me2_Hu303_input.bar (antibody against H3K9me2 in strains Hu303, Hu29 (WT) (H3K9me2_Hu303.CEL, AS_6.CEL) vs input (JW_1.CEL, JW_2_2.CEL)); H3k9me2_Hu2640_Hu303.bar (antibody against H3K9me2 in strain Hu2640 (htb1K119R) (H3K9me2_Hu2640_A.CEL, H3K9me2_Hu2640_B.CEL) vs strain Hu303, Hu29 (WT) (H3K9me2_Hu303.CEL, AS_6.CEL)); H3K9me2_Hu2640_input.bar (antibody against H3K9me2 in strain Hu2640 (htb1K119R) (H3K9me2_Hu2640_A.CEL, H3K9me2_Hu2640_B.CEL) vs input (JW_1.CEL, JW_2_2.CEL)). RNA: 4 RNA samples: 2 replicates of WT RNA (RNA_Hu303_A.CEL, RNA_Hu303_B.CEL) and 2 replicates of htb1-K119R RNA (RNA_Hu2640_A.CEL, RNA_Hu2640_B.CEL).
2014-01-29 | E-GEOD-49874 | biostudies-arrayexpress
Project description:In most plants, centromeric DNA contains highly repetitive sequences, including tandem repeats and retrotransposons; however, the roles of these sequences in the structure and function of the centromere are unclear. Here, we found that retrotransposon-derived back-spliced RNA can bind to the centromere through R-loops and regulate the formation of centromeric chromatin loops. Multiple RNA sequences from centromeric retrotransposons (CRMs) were enriched in maize (Zea mays) centromeres and back spliced RNA from CRM1 were detected. We identified three types of circular CRM1 RNAs with the same back-splicing site based on the back-spliced sequences. These circular RNAs bound to the centromere through R-loops. Two R-loop sites inside a single circular RNA promoted the formation of chromatin loops in CRM1 regions. When RNAi was used to target the back-spliced site of the circular CRM1 RNAs, the levels of R-loops and chromatin loops formed by these circular RNAs decreased, while the levels of R-loops produced by linear RNAs with similar binding sites increased. Linear RNAs with only one R-loop site could not promote chromatin loop formation. Higher levels of R-loops and lower levels of chromatin loops in the CRM1 regions of RNAi plants led to a reduced localization of the centromeric H3 variant (CENH3). Our work reveals that centromeric chromatin organization by circular CRM1 RNAs via R-loops and chromatin loops. R-loops are integral components of centromeric chromatin. Proper centromere structure is essential for CENH3 localization. CRM1 elements may have helped to build a suitable chromatin environment during centromere evolution.
2020-01-01 | GSE137701 | GEO
Project description:We report the application of single-molecule-based sequencing technology for high-throughput profiling of histone H3 trimethylation in rice endosperm. By obtaining about four hundred million bases of sequence from rice chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps of rice endosperm. We find that the presence of H3K27me3 in either upstream or downstream of a gene is predominately associated with repression of the gene, while its absence is mainly associated with high gene expression. Examination of Histone H3 lysine 27 trimethylation in rice endosperm.
2011-12-06 | E-GEOD-27048 | biostudies-arrayexpress
Project description:We report the application of single-molecule-based sequencing technology for high-throughput profiling of histone modifications in yeast. By obtaining bases of sequence from chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps of saccharomuces cerevisiae.We find that H3T11 phosphorylationlysine is widely distributed in gene promoter region and chromosome telomere region
2020-03-12 | GSE146821 | GEO