Figure 5: Transcription of VH Upstream L1s Recruits SHM Activity to the Recombined VH(D)JH.
(A) Percent GFP-positive cells, 2 days post-transfection with L1-IRES-GFP reporter constructs, treated with or without 5-AZA-dC. For construct labels, s = sense, as = antisense, Δ = truncated.
(B-C) RT-qPCR of recombined VH4–34 (B) sense RNA levels and (C) antisense RNA levels, from noL1 and yesL1 reporter cells, possessing or lacking HUSH subunit MPP8. Control comparison cell lines included SUDHL10 and HEK293T, possessing or lacking a VH4–34 upstream L1. All conditions normalized to ACTB and relative to SUDHL10 (noL1) condition.
(D) GRO-seq signal of nascent sense and antisense transcription from the recombined VH4–34 promoter (pink) or from AID off-target gene promoters (gray) in Ramos cells.
(E) Illumina RNA-seq read distributions for steady-state sense and antisense transcription from all recombined VH genes, aligned at TSSs (0 bp) from WT mouse GC B cells.
(F) Schematic of CRISPR-Cas9-mediated deletion of the recombined VH4–34 bidirectional promoter in noL1 and yesL1 reporter cells.
(G) RT-qPCR of VH4–34 sense and antisense RNA levels from noL1 and yesL1 reporter cells, possessing or lacking recombined VH4–34 promoter activity. All conditions normalized to ACTB and relative to SUDHL10 (noL1) condition.
(H) Change in SHM frequency, measured by change in mCherry, of promoter-intact and promoter-dead cell lines, normalized to the unedited noL1 condition.
For all plots, data are presented as mean ± SEM from at least three independent experiments, with p-values calculated by one-way ANOVA with Tukey’s multiple comparisons test in (A-C, H), where “a” and “b” groups are significantly different from each other, but not significantly different within groups. ****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01; ns, not significant.