Extended Data Fig. 10 |. In vivo somatic hypermutation exploration in Dis3^C/C B cells.

a. B splenocytes from one pair of AID^cre/+ Dis3^C/+ and AID^cre/+ Dis3^C/C mice were stimulated. GFP expression was examined after 4 days. In ex vivo culture conditions AID^cre/+ Dis3^C/C cells do not encounter selection and maintain numbers of GFP⁺ cells. b. RT-qPCR quantification of Dis3 mRNA expression in AID^cre/+ Dis3^C/C Peyer’s patches B cells. B220⁺ GL7⁺ and B220⁺ GL7⁻ cells were sorted to show the specific deletion of the Dis3^COIN allele in activated B cells (one experiment performed in triplicate, mean is shown +/− s.e.m., two-tailed unpaired t-test). c. RNA-seq showing increased ncRNAs overlapping J_H genes in the absence of DIS3 activity during B cell stimulation. Top. RNA-seq tracks encompassing the Eμ intronic enhancer and J_H genes. Bottom. RNA-seq tracks showing increased sense transcription (red) and DNA:RNA hybrid accumulation (black) downstream of the J_H4 gene in the absence of DIS3 activity (2 RNA-sequencing and 3 DRIP-sequencing) d. Top: Schematic of wt recombined VDJ genes in the context of CBEs. Bottom: V_HB1–8^KI alleles have different configuration, conserving the major CTCF-binding region IGCR1 (IGCR1 is deleted from the functional allele in physiological conditions). e. DNA mutation analyses of GC (B220⁺ GL7⁺) B cells from V_HB1–8^KI/KI AID^cre/+ Dis3^C/+ (n = 3) mice. Tail DNA from the same animals was used as control (n = 2). AID hot-spots are indicated, and C or G mutations refer to the sense DNA strand. f. Quantification of C and G mutation frequencies in GC B cells. 3 pairs of V_HB1–8^KI/KI AID^cre/+ Dis3^C/+ and V_HB1–8^KI/KI AID^cre/+ Dis3^C/C mice were used, analyses were performed at the J_H gene from the V_HB1–8 allele. χ² two-tailed proportions tests were used to compare the number of C to T mutations relatively to the total C sequenced, and the number of G to A mutations compared to the total G sequenced.