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Single-cell RNA-Seq to study the effects of acute and chronic CENP-A overexpression depending on p53 status

ABSTRACT: Single-cell RNA-Seq to study the effects of acute and chronic CENP-A overexpression depending on p53 status

PROVIDER: PRJEB41802 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR4969410_1.fastq.gz	Fastqsanger.gz
	ERR4969410_2.fastq.gz	Fastqsanger.gz
	ERR4969420_1.fastq.gz	Fastqsanger.gz
	ERR4969420_2.fastq.gz	Fastqsanger.gz
	ERR4969411_1.fastq.gz	Fastqsanger.gz

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Single-cell RNA-Seq to study the effects of acute and chronic CENP-A overexpression depending on p53 status

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

Bulk RNA-Seq to study the effects of CENP-A overexpression, p53 status, and X- irradiation treatment in MCF-10-2A cells

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

CENP-A overexpression promotes distinct fates in human cells, depending on p53 status.

Project description:Tumour evolution is driven by both genetic and epigenetic changes. CENP-A, the centromeric histone H3 variant, is an epigenetic mark that directly perturbs genetic stability and chromatin when overexpressed. Although CENP-A overexpression is a common feature of many cancers, how this impacts cell fate and response to therapy remains unclear. Here, we established a tunable system of inducible and reversible CENP-A overexpression combined with a switch in p53 status in human cell lines. Through clonogenic survival assays, single-cell RNA-sequencing and cell trajectory analysis, we uncover the tumour suppressor p53 as a key determinant of how CENP-A impacts cell state, cell identity and therapeutic response. If p53 is functional, CENP-A overexpression promotes senescence and radiosensitivity. Surprisingly, when we inactivate p53, CENP-A overexpression instead promotes epithelial-mesenchymal transition, an essential process in mammalian development but also a precursor for tumour cell invasion and metastasis. Thus, we uncover an unanticipated function of CENP-A overexpression to promote cell fate reprogramming, with important implications for development and tumour evolution.

| S-EPMC7997993 | biostudies-literature

Transcriptional effects of CENP-A overexpression at single-cell resolution

Project description:Single-cell RNA-Seq to study the effects of acute and chronic CENP-A overexpression depending on p53 status

| PRJEB41400 | ENA

CENP-A overexpression promotes aneuploidy and karyotypic heterogeneity

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

Bulk RNA-Seq to study the effects of CENP-A overexpression, p53 status, and X- irradiation treatment in MCF-10-2A cells

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

CENP-T and CENP-C bridge adjacent CENP-A nucleosomes on young alpha-satellite dimers

Project description:We find that CENP-T acts as a bridge between two well-positioned CENP-A nucleosomes that are present on young alpha-satellite dimers that dominate functional human centromeres. CENP-T is centered over the CENP-B box, where it interacts with the CENP-B/CENP-C complex. Upon cross-linking, the entire CENP-A/CENP-C/CENP-T-containing complex is recovered as a nuclease-protected particle over an alpha-satellite dimer that comprises the fundamental unit of kinetochore chromatin. Our work reveals that CENP-A/CENP-C and CENP-T branches of kinetochore assembly are physically integrated.

2016-05-24 | GSE69839 | GEO

Transcriptional effects of CENP-A overexpression in bulk cell populations

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

Gene expression patterns associated with p53 status in breast cancer

Project description:Breast cancer subtypes identified in genomic studies have different underlying genetic defects. Mutations in the tumor suppressor p53 occur more frequently in estrogen receptor (ER) negative, basal-like and HER2-amplified tumors than in luminal, ER positive tumors. Thus, because p53 mutation status is tightly linked to other characteristics of prognostic importance, it is difficult to identify p53's independent prognostic effects. The relation between p53 status and subtype can be better studied by combining data from primary tumors with data from isogenic cell line pairs (with and without p53 function). In this study, the p53-dependent gene expression signatures of four cell lines (MCF-7, ZR-75-1, and two immortalized human mammary epithelial cell lines) were identified by comparing p53-RNAi transduced cell lines to their parent cell lines. Cell lines were treated with vehicle only or doxorubicin to identify p53 responses in both non-induced and induced states. Each cell line displayed unique patterns of gene expression, but cell type specific trends were evident. A common gene expression signature associated with p53 loss across all four cell lines was identified. This signature showed overlap with the signature of p53 loss in primary breast tumors and predicted relapse-free survival and overall survival in independent test data sets. Keywords: untreated x treated

2007-02-20 | GSE3178 | GEO

Comparison of gene expression in tumor ovarian surface epithelial cells with different p53 status

Project description:The impact of specific p53 mutations on ovarian tumor development and response to therapeutic treatment remain limited. Here, using transgenic mouse models of epithelial ovarian cancer (EOC), we demonstrated that the Trp53R172H mutation promotes EOC progression compared to wild-type p53, but with different consequences between heterozygous and homozygous mutation status. EOC expressing heterozygous Trp53R172H mutation has enhanced responsiveness to steroid hormones and at late stage developed mucinous cystadenocarcinoma. These findings open new realms for exploring the interaction between p53 and steroid receptor, and the allelic status of p53 in EOC development and treatment.

2016-02-03 | GSE65206 | GEO

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