CTCF-mediated cis-regulatory chromatin insulation enforces a central B-cell tolerance checkpoint [RNA-seq]

The generation of a diverse and self-tolerant B cell repertoire is essential for adaptive immunity and is achieved through V(D)J recombination. In mice, Ig? is the dominant light chain, whereas Ig? rearrangement typically occurs in response to nonproductive or autoreactive Ig? recombination, a process termed receptor editing. Recombination at the RS element deletes the Ig? constant exon, silencing the locus and enabling Ig? expression. However, the epigenetic regulatory framework that orchestrates and governs receptor editing remains poorly defined. Here, we identify a CTCF-binding insulator element (CBE) within the 3' Ig? super-enhancer (3'-SE?) that regulates receptor editing and directs the ?-to-? switch required for Ig?? B-cell development. Mechanistically, loss of this CBE activates an insulated enhancer within the 3'-SE?, causing aberrant V? rearrangements and altered chromatin interactions through disrupted loop extrusion dynamics. Notably, loss of this CBE in mice leads to increased autoantibody production by ten weeks of age, demonstrating that CBE-mediated chromatin architecture shapes B cell fate by constraining autoreactive potential. Collectively, our findings define a novel CTCF-dependent cis- regulatory insulation checkpoint that connects chromatin loop extrusion to antigen- driven receptor editing, thereby enforcing B-cell tolerance. Overall design: Genome-wide RNA profiling was performed on ex vivo small pre-B cells isolated from 6–12-week-old C57BL/6 WT and ?rsCBE mice.