Figure 5. Activation-induced cytidine deaminase (AID) activity is required for persistent IgH breaks in Setx^-/-Rnaseh2b^f/f B cells.

(A) AID protein levels in WT B cells 72 hr post-stimulation to indicated isotypes (IgG1, LPS/IL-4/α-RP105; IgG3 with LPS/α-RP105; and α-RP105 alone). Actin served as a loading control. (B) Quantification of AID protein expression relative to Actin for three independent experiments, with AID expression in resting cells set as 1. Error bars show standard deviation. (C) Percent of cells undergoing class switch recombination (CSR) to IgG1 in B cells in response to α-RP105 stimulation. Error bars show standard deviation; statistical significance between each genotype was determined by one-way ANOVA (n = 3 mice/genotype). (D) Frequency of total spontaneous DNA damage under anti-RP105 treatment in vitro. Error bars show the standard deviation; statistical significance versus WT was determined by one-way ANOVA (n = 3 mice/genotype). (E) Frequency of total spontaneous DNA damage 72 hr post-stimulation with LPS/IL-4/α-RP105 in Aicda^-/-, Aicda^-/-Setx^-/-, Aicda^-/-Rnaseh2b^f/f, Aicda^-/-Setx^-/-Rnaseh2b^f/f, and Setx^-/-Rnaseh2b^f/f cells (n = 2 independent mice/genotype). (F) Frequency of spontaneous IgH damage in Aicda^-/-, Aicda^-/-Setx^-/-, Aicda^-/-Rnaseh2b^f/f, Aicda^-/-Setx^-/-Rnaseh2b^f/f, and Setx^-/-Rnaseh2b^f/f cells 72 hr after stimulation with LPS/IL-4/α-RP105. Error bars show the standard deviation; statistical significance versus WT was determined by one-way ANOVA.

Figure 5—figure supplement 1. Aicda expression, enrichment, and recruitment in cells lacking senataxin (SETX) and RNase H2.

(A) Activation-induced cytidine deaminase (AID) mRNA quantitation by RT-qPCR in resting cells, LPS/IL-4/α-RP105, LPS/α-RP105, and α-RP105-only stimulated cells. Error bars show the standard deviation from three independent experiments. (B) AID protein expression in WT, Setx^-/-, Rnaseh2b^f/f, Setx^-/-Rnaseh2b^f/f, and Aicda^-/- cells 72 hr post-stimulation to IgG1. Western blots were probed with an anti-AID antibody and anti-actin as loading control. Aicda^-/- cells were used as a negative control. (C) Quantification of AID protein expression versus WT related to B; error bars show standard deviation; statistical analysis was performed using multiple t-test (n = 3 mice/genotype). (D) Chromatin immunoprecipitation (ChIP) analysis for AID occupancy in Sμ and Sγ regions of primary B cells in response to LPS/IL-4/α-RP105 stimulation at 60 hr post-stimulation. Relative enrichment was calculated as ChIP/input. Error bars show standard deviation (n = 3 mice/genotype); statistical analysis versus Aicda^-/- control was performed using Student’s t-test, *p-value<0.05 compared to Aicda^-/-. (E) ChIP analysis for Pol ll (Ser5) occupancy in Sμ and Sγ regions of primary B cells in response to LPS/IL-4/α-RP105 stimulation. Relative enrichment was calculated as fold change relative to WT set to 1; error bars show standard deviation (n = 5 mice/genotype).