Long noncoding RNA CCTT recruits CENP-C to centromeres by directly binding to centromeric DNA [SHAPE-Map]
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PROVIDER: PRJNA628992 | ENA |
REPOSITORIES: ENA
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Project description:To investigate the exact locations of CCTT-chromatin interaction on a genome-wide scale, we modified previously reported ChIRP-seq (chromatin isolation by RNA purification followed by deep-sequencing) with a crosslinker 4’- aminomethyltrioxalen (AMT, a psoralen derivative), which allows fixation of nucleic acid interaction by ultraviolet light without crosslinking proteins. We designed 8 complementary DNA oligonucleotides that tiled the Alu-depleted part of CCTT. Affinity-purified CCTT-binding DNAs were sequenced and mapped to the HuRef genome hg38, which contains human α-satellite sequence models in each centromeric region. In contrast with 5.38% of input reads mapping to α-satellite sequence, 16.89% of CCTT-binding reads were enriched at α-satellites. Peaks bound by lnc-CCTT were identified by MACS2 algorithm, which compared the reads in CCTT-captured samples with that in input ones. CCTT-binding peaks mapped across the centromeric regions of all 23 reference centromeres. Next, we validated the enrichment of centromeric peaks located at all chromosomes via ChIRP-qPCR. To validate our ChIRP-seq results, we selected representatives of three major subpopulations: multimapping peaks in one specific chromosome and several chromosomes, as well as single-copy peaks. We performed ChIRP-qPCR and found abundant CCTT-binding centromeric peaks throughout all 23 chromosomes. Importantly, CCTT ChIRP-seq profile was highly correlated with the previously reported ChIP-seq profiles of CENP-C (Pearson correlation R = 0.82), both of which showed very strong, extensive signals across entire centromeric regions in HeLa cells.
2022-04-20 | GSE149532 | GEO
Project description:To identify potential lncRNAs that associate with CENP-C, RNA immunoprecipitation (RIP) using the CENP-C and control IgG antibody was performed in the whole-cell lysates of HeLa cells, followed by Illumina short-read sequencing (RIP-seq). We found three CENP-C-binding lncRNAs that meet the enrichment criteria (fold change > 2, p < 0.01), including AGAP2-AS1, GS1-124K5.4, and AC124789.1 (lnc-CCTT) . Consistent with the results of RIP-seq, RIP-qPCR assay in whole-cell lysates showed that all three lncRNAs could bind to CENP-C, while lnc-CCTT showing the most robust association of CENP-C. ). It is well-established that the localization of lncRNAs within the cell is the primary determinant of their molecular functions. RT-qPCR (quantitative PCR with reverse transcription) assay of subcellular fractionation and RNA fluorescence in situ hybridization (RNA-FISH) revealed that AGAP2-AS1 and GS1-124K5.4 dominantly distributed in the cytoplasm, whereas lnc-CCTT, specifically and almost exclusively, localized to the nucleus. Thus, lnc-CCTT appears to be a prime interacting RNA of CENP-C at centromere.
2022-04-20 | GSE149528 | GEO
Project description:Long noncoding RNA CCTT recruits CENP-C to centromeres by directly binding to centromeric DNA
| PRJNA628989 | ENA
Project description:To fine-map the position of lnc-CCTT that directly interact with CENP-C, we performed irCLIP-seq (infrared crosslingking and immunoprecipitation followed by high throughput RNA sequencing), which utilize ultraviolet (UV) light to induce zero-length covalent bonds between RNA and the directly attached protein and an infrared-dye-conjugated and biotinylated ligation adaptor to isolate RNA fragments. irCLIP-seq identified a possible CENP-C binding region of lnc-CCTT ranging from nucleotides 127-177nt.
2022-04-20 | GSE149530 | GEO
Project description:Long noncoding RNA CCTT recruits CENP-C to centromeres by directly binding to centromeric DNA [RIP-Seq]
| PRJNA628993 | ENA
Project description:Long noncoding RNA CCTT recruits CENP-C to centromeres by directly binding to centromeric DNA [ChIRP-Seq]
| PRJNA628994 | ENA
Project description:Long noncoding RNA CCTT recruits CENP-C to centromeres by directly binding to centromeric DNA [irCLIP-Seq]
| PRJNA628995 | ENA
Project description:To refine the authentic CENP-C binding sites of lnc-CCTT and globally map lnc-CCTT secondary structure, we also performed SHAPE-MaP (selective 2’-hydroxyl acylation analyzes by primer extension and mutational profiling), which uses hydroxyl-selective electrophiles to modify the 2’-hydroxyl groups of unbound single-stranded nucleotides, in HeLa cells both ex vivo and in vivo. Lnc-CCTT secondary structure was modeled by combination SHAPE data from cell-free ex vivo with pairing probabilities. As expected, nucleotides 43-79 nt, a determinant for RNA-DNA triplex formation, exhibited a continuous single-strandedness, which may be prone to binding DNA. More importantly, only nucleotides 118-177 nt, which was folded into a stem-loop structure in the secondary structure, showed a significant reduced SHAPE reactivities in cell when comparing to cell-free state, suggesting this region could be attributed to interaction with protein components.
2022-04-20 | GSE149529 | GEO
Project description:Centromeres are specialized chromatin regions marked by the presence of nucleosomes containing the centromere-specific histone H3 variant CENP-A, which is essential for chromosome segregation. Assembly and disassembly of nucleosomes is intimately linked to DNA topology and DNA topoisomerases have previously been implicated in the dynamics of canonical H3 nucleosomes. Here we show that Schizosaccharomyces pombe Top3 and its partner Rqh1 are involved in controlling the levels of CENP-ACnp1 at centromeres. Both top3 and rqh1 mutants display defects in chromosome segregation. Using chromatin immunoprecipitation and tiling microarrays we show that Top3 unlike Top1 and Top2 is highly enriched at centromeric central domains, demonstrating that Top3 is the major topoisomerase in this region. Moreover, centromeric Top3 occupancy positively correlates with CENP-ACnp1 occupancy. Intriguingly, both top3 and rqh1 mutants display increased relative enrichment of CENP-ACnp1 at centromeric central domains. Thus, Top3 and Rqh1 normally limit the levels of CENP-ACnp1 in this region. This new role is independent of the established function of Top3 and Rqh1 in homologous recombination downstream of Rad51. Therefore, we hypothesize that the Top3-Rqh1 complex has an important role in controlling centromere DNA topology which in turn affects the dynamics of CENP-ACnp1 nucleosomes. For transcription: Total RNA from top3-105 mutant and WT control cells after 8 hours at 36C in biological duplicates. For Top3-myc chromatin immunoprecipitation: DNA immunoprecipitated with mouse anti-Myc using chromatin extracts from cells expressing Top3-Myc from the endogenous locus at 30C in biological duplicates normalized to input DNA from wild type cells at 30C in biological duplicates. For CENP-A/Cnp1 chromatin immunoprecipitation: DNA immunoprecipitated with anti-Cnp1 serum using chromatin extracts from top3-105 mutant and wild type control cells after 8 hours at 36C in in biological duplicates normalized to input DNA from each strain.
2013-03-14 | E-GEOD-44206 | biostudies-arrayexpress
Project description:Centromeres are specialized chromatin regions marked by the presence of nucleosomes containing the centromere-specific histone H3 variant CENP-A, which is essential for chromosome segregation. Assembly and disassembly of nucleosomes is intimately linked to DNA topology and DNA topoisomerases have previously been implicated in the dynamics of canonical H3 nucleosomes. Here we show that Schizosaccharomyces pombe Top3 and its partner Rqh1 are involved in controlling the levels of CENP-ACnp1 at centromeres. Both top3 and rqh1 mutants display defects in chromosome segregation. Using chromatin immunoprecipitation and tiling microarrays we show that Top3 unlike Top1 and Top2 is highly enriched at centromeric central domains, demonstrating that Top3 is the major topoisomerase in this region. Moreover, centromeric Top3 occupancy positively correlates with CENP-ACnp1 occupancy. Intriguingly, both top3 and rqh1 mutants display increased relative enrichment of CENP-ACnp1 at centromeric central domains. Thus, Top3 and Rqh1 normally limit the levels of CENP-ACnp1 in this region. This new role is independent of the established function of Top3 and Rqh1 in homologous recombination downstream of Rad51. Therefore, we hypothesize that the Top3-Rqh1 complex has an important role in controlling centromere DNA topology which in turn affects the dynamics of CENP-ACnp1 nucleosomes.
2013-03-14 | GSE44206 | GEO