Extended Data Fig. 2: Dependence of V_H-DJ_H recombination on chromatin looping and V_H gene orientation.

a, ChIP-seq analysis of CTCF binding in the distal 890-kb region in short-term-cultured pro-B cells from Igh^{890-inv/890-inv} and Igh^+/+ mice. Vertical bars indicated CTCF peaks defined by ‘peak calling’. The ChIP-seq data of the Igh^890-inv allele were aligned on the wild-type Igh sequences. One of two experiments per genotype is shown. b, Igh positions of the 3C-seq viewpoints, which are referred to by the nearest V_H gene, the hypersensitive site 5 (HS5) in the 3′CBE region or the CBE array insertion (Supplementary Table 1c). c, Interactions from the 5′ (V_H1-86; left) and 3′ (HS5; right) viewpoints in short-term-cultured pro-B cells from Igh^{890-inv/890-inv}Rag2^−/− and Igh^+/+Rag2^−/− mice. The 3C-seq reads along the Igh locus (top two panels) are shown as RPM values with the respective Q values, which were calculated by the r3Cseq program based on two 3C-seq experiments per genotype. The bottom panel shows the fold-change of the RPM values for interaction differences of >2-fold (dashed line) between experimental (Exp) Igh^{890-inv/890-inv}Rag2^−/− and control (Ctrl) Igh^+/+Rag2^−/− pro-B cells. d, Schematic summarizing the loop formation at the Igh^890-inv allele in pro-B cells. The CBEs with their orientation are indicated by red and blue arrowheads, V_H genes by grey arrows and loops by arches. The new loop ___domain is indicated in red. e, Schematic of the Igh^∆890, Igh^V8-8 and Igh^V8-8-inv alleles. The Igh^V8-8 and Igh^V8-8-inv alleles were generated by insertion of the V_H8-8 gene with 500 bp of its 5′ and 3′ flanking sequences (lacking any CBE) in the forward or reverse orientation at the deletion point (117,126,667 / 116,237,220; mm9, Chr. 12) of the Igh^∆890 allele (Methods). The distances from the 3′ end of the V_H8-8 gene to the next CBEs and V_H genes are indicated. The sequence of the inserted V_H8-8 with its flanking DNA sequences is shown in Supplementary Table 1b. f, RNA-seq profile of the Igh locus in immature B cells from the bone marrow of Igh^V8-8/∆890, Igh^{V8-8-inv/∆890} and Igh^+/∆890 mice. The V_H8-8 gene position and Igh annotation are indicated. The data of one of two RNA-seq experiments per genotype is shown. g, V_H8-8 mRNA expression in immature B cells of the indicated genotypes is shown as mean TPM value. Each dot corresponds to one experiment.

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