CFP1 controls H3K4me3-mediated chromatin architecture to promote v(D)j recombination in early B cell development Free

V(D)J recombination is a fundamental process in B lymphocyte development that generates a diverse immunoglobulin (Ig) repertoire through stepwise assembly of variable (V), diversity (D), and joining (J) gene segments. This highly regulated process is initiated by the RAG1/2 recombinase complex and is tightly linked to chromatin structure and epigenetic modifications. Notably, the RAG2 PHD domain recognizes H3K4me3, a histone mark enriched at recombination centers and regulatory elements within the Ig heavy chain (IgH) locus. In parallel, long-range chromatin interactions, mediated by the intronic enhancer iEμ and distal Pax5-activated intergenic repeats (PAIRs), promote locus contraction to bring distal V_H segments into spatial proximity with the DJ_H region. While PAIRs and iEμ are both enriched for H3K4me3, the precise epigenetic mechanisms that coordinate this histone mark with chromatin topology and recombination efficiency remain poorly understood. CFP1 (CXXC finger protein 1), a DNA-binding subunit of the SETD1 H3K4 methyltransferase complex, is essential for embryogenesis, germ cell development, and early hematopoiesis. However, its role in B lineage commitment and IgH recombination has not been defined. We hypothesized that CFP1 regulates early B cell development by orchestrating an H3K4me3-rich chromatin landscape that supports RAG2 recruitment, antisense transcription at regulatory elements, and IgH locus contraction.

To evaluate this hypothesis, we generated mice with conditional deletion of Cfp1 in the B cell lineage using Mb1-Cre. Loss of CFP1 resulted in a profound developmental arrest at the pro-B cell stage, accompanied by a dramatic reduction in V_H-to-DJ_H recombination. Semi-quantitative PCR and V(D)J sequencing revealed a near-complete loss of distal V_H gene usage, with residual recombination restricted to D-proximal V_H segments. Consequently, complementarity-determining region 3 (CDR3) diversity was markedly reduced. CUT&Tag profiling demonstrated a genome-wide decrease in H3K4me3 deposition, with pronounced loss at the recombination center and multiple PAIR elements, including PAIR4 and PAIR6. ChIP-Seq confirmed a significant reduction of RAG2 occupancy at these regulatory regions in CFP1-deficient pro-B cells. Furthermore, strand-specific RNA-Seq revealed markedly diminished antisense transcription at PAIR elements, which has been implicated in promoting locus accessibility and recombination readiness. Using high-resolution 4C-Seq with iEμ as bait, we observed a profound reduction in long-range chromatin contacts between iEμ and distal V_H or PAIR elements, indicating loss of IgH locus contraction and impaired spatial organization of the recombination substrate.

Collectively, these findings define CFP1 as a critical epigenetic regulator of early B cell development. By promoting H3K4me3 deposition at recombination centers and distal regulatory elements, CFP1 facilitates RAG2 recruitment, antisense transcription, and locus contraction, all of which are essential for effective V(D)J recombination involving distal V_H segments. In the absence of CFP1, this coordinated chromatin program is disrupted, leading to recombination failure, skewed IgH repertoire, and developmental arrest. Our study uncovers a previously unrecognized role for CFP1 in regulating chromatin topology and antigen receptor recombination. These findings have broader implications for understanding the epigenetic regulation of lymphocyte development and provide insight into the mechanisms underlying primary immunodeficiencies or B-cell malignancies such as pediatric leukemia, where recombination defects or impaired repertoire diversity contribute to pathogenesis.