biostudies-arrayexpressCharlène Renaud-PageotHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16636CENP-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).biostudies-arrayexpressSample Collection - MCF-10-2A TetOn-CENPA-FLAG-HA cells stably expressing a dominant-negative p53 vector (p53-DN) were cultured for 41 days in the absence (no Dox), presence of Dox (10 ng/mL) or with interrupted induction with Dox (10ng/mL Dox for 24 days followed by 17 days without Dox) to induce basal or high CENP-A expression. Culture medium (+/- Dox) was renewed every 2-3 days. We harvested cells at day 41 post-induction.Library Construction - Quality and concentration were assessed using a TapeStation 4200 (Agilent) and a Qubit 3.0 fluorometer (Invitrogen). Sequencing libraries were prepared with the Illumina TruSeq ChIP kit.Nucleic Acid Extraction - For each IP, 5 million cells were harvested and processed in aliquots of 1 million cells per tube by resuspending cell pellets in 100 µL ice-cold lysis buffer for a 4-min incubation at room temperature. We prepared antibody-coupled Protein A Dynabeads by blocking for 3h at room temperature in 1 mL blocking buffer, washing once with PBS-T and incubating for 1h at room temperature in 200 µL PBS-T with 5 µg anti-H3.3 (Active Motif, 91191), 10 µg anti-CENP-A (Cell Signaling, 2186), or 10 µg control IgG (Abcam, ab46540) antibodies. After a wash in binding buffer, we added the antibody-coupled beads to the chromatin and incubated overnight at 4°C on a rotating wheel. The next day, beads were recovered using a magnetic rack and sequentially washed with appropriate buffers. Beads were treated with RNase A and proteinase K in the presence of 2% SDS. DNA was purified using AMPure XP beads (Beckman Coulter, A63880) as per the manufacturer’s protocol and eluted in nuclease-free water.Sequencing - Sequencing was perfomred on the Illumina NextSeq 2000 at the Institut Curie Next Generation Sequencing (NGS) platform.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesMetabolomicsUnknownTranscriptomicsGenomicsProteomicsNextSeq 2000ChIP-seqHomo sapiensNon-centromeric CENP-A epigenetically regulates epithelial-mesenchymal plasticity and heterogeneity in human cellsCharlène Renaud-Pageot, Daniele Capocefalo, Sébastien Lemaire, Audrey Forest, Laura Cantini, Geneviève AlmouzniCharlène Renaud-PageotfalseCENP-A ChIP-seq of MCF10-2A cells to study CENP-A localization following reversal from high to basal CENP-A expressionCENP-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).2026-04-24T00:00:00Z2026-04-24T16:03:38.921Z2026-02-11T11:22:32.791ZE-MTAB-16636ERP188991EFO_0002944EFO_0004170EFO_0002692EFO_0005518EFO_0004184