Figure 1: IGH V genes possess upstream LINE-1 retrotransposons that promote somatic hypermutation.
(A-B) Genomic density distributions of upstream L1 elements for IGH V genes, all protein-coding genes, and known AID off-target genes in (A) human genome (hg38) and (B) mouse genome (mm39), with TSSs aligned at 0 bp.
(C) Schematic of the Ramos B cell mCherry reporter system with cell lines lacking (noL1) or possessing (yesL1) a nearby “old” L1 element upstream to the recombined VH gene.
(D) Representative flow cytometry panel assessing mCherry loss in noL1 and yesL1 cells after 7 days of 4-OHT treatment.
(E) Change in SHM frequency, measured by change in mCherry, normalized to the noL1 condition.
(F-G) Point mutation frequency across (F) the recombined VH(D)JH and (G) AID hotspot motifs, from noL1 and yesL1 cells.
(H) (Top) Representative SHM profiles of the recombined VH4–34 upstream region in noL1 and yesL1 cells, where the x-axis corresponds to PCR-amplified region per schematic, L1 element denoted by vertical dashes, L1 orientations indicated by black arrows, and purple arrows (0 bp) correspond to purple arrows in schematic below. (Bottom) Schematic of primer pairs used to assess mutation frequency of VH4–34 upstream regions. In noL1 cells, black primers amplify the L1-lacking region (see Methods). In yesL1 cells, black primers amplify the L1-lacking region on the non-recombined allele, while pink forward and black reverse primers amplify the L1-containing region on the recombined allele.
(I-J) Point mutation frequency across the indicated region upstream to the (I) recombined VH4–34 in noL1 and yesL1 cells, or (J) non-recombined and recombined VH4–34 in yesL1 cells only, as outlined in (H).
(K) AID ChIP-qPCR at the recombined VH(D)JH in noL1 and yesL1 cells, with schematic below indicating primer binding sites.
For (A-B), p-values were calculated by pairwise comparisons using the two-sample Kolmogorov-Smirnov (K-S) test. For all other plots, data are presented as mean ± SEM from at least three independent experiments, with p-values calculated by unpaired two-sided t-test in (E-F, I, K) or paired two-sided t-test in (J). ****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, p ≤ 0.05.