A licensing step links AID to transcription elongation for mutagenesis in B cells

Abstract

Activation-induced deaminase (AID) mutates the immunoglobulin (Ig) genes to initiate somatic hypermutation (SHM) and class switch recombination (CSR) in B cells, thus underpinning antibody responses. AID mutates a few hundred other loci, but most AID-occupied genes are spared. The mechanisms underlying productive deamination versus non-productive AID targeting are unclear. Here we show that three clustered arginine residues define a functional AID domain required for SHM, CSR, and off-target activity in B cells without affecting AID deaminase activity or Escherichia coli mutagenesis. Both wt AID and mutants with single amino acid replacements in this domain broadly associate with Spt5 and chromatin and occupy the promoter of AID target genes. However, mutant AID fails to occupy the corresponding gene bodies and loses association with transcription elongation factors. Thus AID mutagenic activity is determined not by locus occupancy but by a licensing mechanism, which couples AID to transcription elongation.

AID–RNA polymerase II transcription-dependent deamination of IgV DNA

“…The Spt5 subunit of DSIF exhibits a peak genomic distribution that correlates strongly with genomic localization of AID (31,32). Reduction of Spt5 levels via knockdowns in CH12 cells show significantly reduced CSR (59), suggesting that Spt5 is required for CSR (32,59). In contrast, a reduction in Spt5 levels in Ramos B-cells leads to a slight increase in IgV SHM (51).…”

The endosomal sorting adaptor HD-PTP is required for ephrin-B:EphB signalling in cellular collapse and spinal motor axon guidance

“…BioID experiments were performed as described elsewhere, with modifications 77 . Briefly, Control and EphB2-OE Flp-In T-REx HEK293 cells (Invitrogen) (all cell lines can be found in Supplementary Table S3) were cultured in 15 cm plates (Corning) and treated with 1 µg/mL of tetracycline (Sigma Aldrich) for 18 h. The following day, the medium was removed and cells were incubated in serum-free medium for 6 h in the presence of 50 µM biotin (Sigma Aldrich) and pre-clustered ligand (Fc or eB2-Fc, 1.5 μg/mL, R&D Systems) or media.…”

“…Peptides were analysed by high-pressure liquid chromatography (HPLC) coupled to Orbitrap Velos Mass Spectrometer (Thermo Fisher Scientific) at the IRCM proteomics core facility. Peptide search, identification of proteins and mass spectrometry (MS) data analysis were carried out as described elsewhere 77 . The BioID-MS data was analysed using ProHits 78 .…”

The endosomal sorting adaptor HD-PTP is required for ephrin-B:EphB signalling in cell collapse and motor axon guidance

“…BioID experiments were performed as described elsewhere, with modifications (Methot et al, 2018). Briefly, Control and EphB2-OE Flp-In T-REx HEK293 cells (Invitrogen, Thermo Fisher Scientific, Waltham, MA) (all cell lines can be found in Supplementary Table 3) were cultured in 15 cm plates (Corning, New York) and treated with 1 µg/mL of tetracycline (Sigma Aldrich, St. Louis, MO) for 18 h. The following day, the medium was removed and cells were incubated in serum-free medium for 6 h in the presence of 50 µM biotin (Sigma Aldrich, St. Louis, MO) and pre-clustered ligand (Fc or eB2-Fc, 1.5 μ g/mL, R&D Systems, Minneapolis, MN) or media.…”

“…Peptides were analysed by high-pressure liquid chromatography (HPLC) coupled to Orbitrap Velos Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA) at the IRCM proteomics core facility. Peptide search, identification of proteins and mass spectrometry (MS) data analysis were carried out as described elsewhere (Methot et al, 2018). The BioID-MS data was analysed using ProHits (Liu et al, 2010).…”