CTCF ChIP-seq in AC16 cell: A Conserved Chromatin Structural Program Underlies Injury-Induced Cardiomyocyte Proliferation
Because adult mammalian cardiomyocytes cannot reactivate their proliferative machinery, the heart does not recover effectively after injury. In contrast, during the early neonatal period, cardiomyocytes retain a transient regenerative capacity. Myocardial injury performed before postnatal day 3 triggers robust cardiomyocyte proliferation and complete recovery within weeks, and apical resection during the first postnatal days leads to full regeneration without scar formation. In pigs, apical resection at day 1 followed by myocardial infarction at day 28 induces strong cardiomyocyte proliferation and complete functional recovery by day 56. To investigate the molecular programs that underlie this transient regenerative competence, we reanalyzed mouse and pig snRNA-seq datasets using a cell cycle–specific autoencoder (CSA) and a chromatin structural factor–specific autoencoder (CSF). Both frameworks consistently identified a distinct cardiomyocyte population (CM1) enriched for mitotic markers after injury. Gene Ontology analysis revealed coordinated activation of cell-cycle pathways together with chromatin organization and remodeling processes, suggesting that cell-cycle re-entry requires extensive epigenetic restructuring. Cross-species comparison identified a hallmark of chromatin structural regulators associated to cardiomytocyte proliferation. Among these factors, CTCF emerged as a central regulator. Its expression declined after birth but was robustly reactivated in injured mouse and pig hearts. Functional assays demonstrated that CTCF overexpression promotes cardiomyocyte-like cell proliferation, and induce targeted redistribution of CTCF binding at key cell-cycle regulatory loci. Together, these findings identify conserved modulation of chromatin structural factors as a key mechanism underlying cardiomyocyte cell-cycle reactivation and position CTCF as a prominent regulator within this architectural program during cardiac regeneration. Overall design: Plamids containing seqences to overexpress CTCF (CTCF-OE) and empty (control) were transfected into AC16 cell lines. The cells were harvested three days after transfection. Three biological replicates of approximately 1×106 AC16 cardiomyocyte-like cells transfected with either empty pcDNA vector or pcDNA-CTCF were crosslinked with disuccinimidyl glutarate (DSG; 30 min at room temperature), followed by fixation with 1% formaldehyde for 10 min at room temperature and quenching with 125 mM glycine for 5 minutes. Cells were lysed, and chromatin was sonicated for 60 min using the Pixul platform (Active Motif), under the following parameters: Pulse (N):50, PRF (kHz): 1.00, Process Time: 60 minutes, Burst Rate (Hz): 20. Chromatin was incubated overnight at 4 °C with Protein G/A Dynabeads (Invitrogen, #10003D) pre-bound to 1.5 µg of anti-CTCF antibody (Active Motifs, #91285). Bead-bound complexes were extensively washed and resuspended in TT buffer (10 mM Tris-HCl, pH 8.0, 0.05% Tween-20). On-bead library preparation was performed using the NEBNext Ultra II DNA Library Prep Kit (NEB, #E7645L). Libraries were barcoded using NEXTFLEX Unique Dual Index Barcodes, PCR-amplified for 14 cycles, with Solexa 1GA/1GB primers, and size-selected (200–500 bp) prior to sequencing on an Illumina NovaSeq SP100 platform.For processing, the CTCF ChIP-seq data were aligned via Bowtie genome with Human / GRCh38 no-alt analysis reference genome (https://benlangmead.github.io/aws-indexes/bowtie). Peak calling was done via Macs2 program (https://pypi.org/project/MACS2/).