Transcriptional effects of CENP-A overexpression at single-cell resolution
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB41400 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:We performed bulk RNA-Seq to investigate global transcriptional changes upon overexpression of the centromeric H3 variant CenH3/CENP-A in p53 wild-type and defective cells, and after X-irradiation treatment. We established clonal MCF-10-2A TetOn-CENPA-FLAG-HA cell lines where CENP-A can be reversibly induced by Doxycycline (Dox) treatment. Upon CENP-A overexpression, these cells exhibit different radiosensitivity depending on p53 status. In order to profile global changes in expression, we compared MCF-10-2A TetOn-CENPA-FLAG-HA cells expressing either empty vector (p53-WT) or dominant-negative p53 (p53-DN) with 0X Dox (no Dox), 1X Dox (10 ng/ml), or 10X Dox (100 ng/ml) for 24h. At time 0, we irradiated one set of cells by X-ray generator (4gy) while a control set remained un-irradiated (0gy). 6h later, we extracted RNA for RNA-seq.
2021-03-01 | E-MTAB-9867 | biostudies-arrayexpress
Project description:We performed single-cell RNA-Seq to determine the effects of CENP-A overexpression and p53 status on cell fate over time. We established clonal MCF10-2A TetOn-CENPA-FLAG-HA cell lines where CENP-A can be reversibly induced by Doxycycline (Dox) treatment. We compared prolonged, continuous CENP-A overexpression (chronic induction) to acute CENP-A overexpression, as well as non-overexpressing conditions, in p53 wild-type and defective cells. We grew MCF10-2A TetOn-CENPA-FLAG-HA cells expressing either an empty control vector (p53-WT) or dominant-negative p53 (p53-DN) without Dox or with continuous exposure to Dox (10ng/ml, ++, chronic) for 69 days in parallel. Cells in the no Dox condition were split into two dishes at day 67 and Dox was added to one set on day 68 for 24h of CENP-A overexpression (10ng/ml, +, acute) while the other remained without Dox (-, control). We prepared samples in singlet according to the 10x Genomics Sample Preparation Demonstrated Protocol. In brief, we harvested cells by trypsinization, resuspended in typical media and mixed thoroughly by pipette, passed cells through a 40µm cell strainer, and counted cells by Beckman Automated Cell Counter. We resuspended cells in 1x PBS + 0.04% BSA, mixed, centrifuged gently, aspired supernatant and repeated wash two times. We passed cells through another cell strainer (40µm), counted again and diluted to a final concentration between 700 and 1200 cells/µl. We then proceeded directly to GEM generation and barcoding at the NGS platform, Institut Curie, using the Single-cell 3’ Reagent Kits v2 protocol with a targeted cell recovery of 2000 cells per sample, followed by post GEM-RT cleanup and cDNA amplification, then 3’ Gene Expression Library Construction, according to the manufacturer’s instructions. Sequencing was performed by the NGS platform with a NovaSeq 6000 sequencer.
2021-03-01 | E-MTAB-9861 | biostudies-arrayexpress
Project description:Single-cell RNA-Seq to study the effects of acute and chronic CENP-A overexpression depending on p53 status
| PRJEB41802 | ENA
Project description:Bulk RNA-Seq to study the effects of CENP-A overexpression, p53 status, and X- irradiation treatment in MCF10-2A cells
| PRJEB41399 | ENA
Project description:CENP-A, the centromeric histone H3 variant, is essential for the proper segregation of human chromosomes to daughter cells. High CENP-A expression leads to the misincorporation of CENP-A in non-centromeric chromatin and can promote an epithelial-mesenchymal transition (EMT). Here, we explored how CENP-A could promote EMT, considering its localization at EMT gene loci. For this, we used a MCF10-2A TetOn-CENPA-FLAG-HA cell line where high CENP-A expression can be induced by Doxycycline (Dox) treatment. This cell line is p53-defective (transduction with a dominant-negative (DN) p53 vector). Upon CENP-A overexpression, these cells progressively accumulate mesenchymal states. We performed bulk ChIP-seq 41 days post-induction to profile changes in CENP-A localization in cells expressing either basal CENP-A levels (non-induced, control with no Dox cultivated for 41 days), high CENP-A levels (induced for 41 days with 10 ng/ml Dox) or reversed from high to basal CENP-A levels (induction for 24 days followed by interruption for 17 days).
2026-04-24 | E-MTAB-16636 | biostudies-arrayexpress
Project description:CENP-A, the centromeric histone H3 variant, is essential for the proper segregation of human chromosomes to daughter cells. High CENP-A expression leads to the misincorporation of CENP-A in non-centromeric chromatin and can promote an epithelial-mesenchymal transition (EMT). Here, we explored how CENP-A could promote EMT, considering its localization at EMT gene loci. For this, we used a MCF10-2A TetOn-CENPA-FLAG-HA cell line where high CENP-A expression can be induced by Doxycycline (Dox) treatment. This cell line is p53-defective (transduction with a dominant-negative (DN) p53 vector). Upon CENP-A overexpression, these cells progressively accumulate mesenchymal states. We performed bulk ChIP-seq experiments at the same time points as our transcriptomic data (day 10 and 24). In order to profile changes in CENP-A localization, we compared cells expressing either basal CENP-A levels (non-induced, control with no Dox), or high CENP-A levels (induced with 10 ng/ml Dox).
2026-04-24 | E-MTAB-15579 | biostudies-arrayexpress
Project description:Centromeric localization of the evolutionarily conserved histone H3 variant, CENP-A. is essential for chromosomal stability. CENP-A overexpression (OE) causesd its mislocalization to non-centromeric regions resulting in chromosomal instability (CIN) in yeast, flies and human cells. CENP-A OE and mislocalization have been observed in cancers and correlates with poor prognosis. However, the molecular consequences of CENP-A OE on CIN and aneuploidy have not been defined. Here, we overexpressed YFP-CENP-A in a pseudodiploid DLD1 cell line and showed that CENP-A OE leads to its mislocalization and CIN due to defects in kinetochore integrity and kinetochore-microtubule attachments. CENP-A OE also leads to its mislocalization and CIN in a xenograft mouse model. Under these conditions, it contributes to aneuploidy with karyotypic heterogeneity in human cells and xenograft mouse model. In summary, our studies provide a molecular link between CENP-A OE and aneuploidy, and suggest that karyotypic heterogeneity may contribute to the aggressive phenotype of CENP-A overexpressing cancers.
2020-07-16 | GSE154460 | GEO
Project description:CENP-A, the centromeric histone H3 variant, is essential for the proper segregation of human chromosomes to daughter cells. High CENP-A expression leads to the misincorporation of CENP-A in non-centromeric chromatin and can promote an epithelial-mesenchymal transition (EMT). Here, we explored how CENP-A could promote EMT, considering both its impact on transcription and chromatin structure. For this, we used a MCF10-2A TetOn-CENPA-FLAG-HA cell line where high CENP-A expression can be induced by Doxycycline (Dox) treatment. This cell line is p53-defective (transduction with a dominant-negative (DN) p53 vector). Upon CENP-A overexpression, these cells progressively accumulate mesenchymal states. For the same nuclei, we jointly profiled single-nucleus RNA (snRNA) and single-nucleus ATAC (snATAC) data using 10X Multiome at days 10 and 24 (prior and after the mesenchymal states accumulation). In order to profile changes in transcription and cell states, we compared cells expressing either basal CENP-A levels (non-induced, control with no Dox), or high CENP-A levels (induced with 10 ng/ml Dox).
2026-04-24 | E-MTAB-15582 | biostudies-arrayexpress
Project description:CENP-A overexpression promotes aneuploidy and karyotypic heterogeneity
| PRJNA646383 | ENA
Project description:Bulk RNA-Seq to study the effects of CENP-A overexpression, p53 status, and X- irradiation treatment in MCF-10-2A cells
| PRJEB41819 | ENA