C‐terminal deletion‐induced condensation sequesters AID from IgH targets in immunodeficiency

Abstract In activated B cells, activation‐induced cytidine deaminase (AID) generates programmed DNA lesions required for antibody class switch recombination (CSR), which may also threaten genome integrity. AID dynamically shuttles between cytoplasm and nucleus, and the majority stays in the cytoplasm due to active nuclear export mediated by its C‐terminal peptide. In immunodeficient‐patient cells expressing mutant AID lacking its C‐terminus, a catalytically active AID‐delC protein accumulates in the nucleus but nevertheless fails to support CSR. To resolve this apparent paradox, we dissected the function of AID‐delC proteins in the CSR process and found that they cannot efficiently target antibody genes. We demonstrate that AID‐delC proteins form condensates both in vivo and in vitro, dependent on its N‐terminus and on a surface arginine‐rich patch. Co‐expression of AID‐delC and wild‐type AID leads to an unbalanced nuclear AID‐delC/AID ratio, with AID‐delC proteins able to trap wild‐type AID in condensates, resulting in a dominant‐negative phenotype that could contribute to immunodeficiency. The co‐condensation model of mutant and wild‐type proteins could be an alternative explanation for the dominant‐negative effect in genetic disorders.

5'-FAM-labeled product on urea gel (A) CSR levels to IgG1 in LPS/IL4-stimulated Aicda -/splenic primary B cells complemented with retroviral-expressed AID variants. Left, representative flow cytometry plots at Day 3 after stimulation. Co-expressed GFP indicates productively infected cells. Middle, bar plot shows the mean with SD of three biological replicates. Right, effect of AID and AID DC proteins on the cell proliferation. GFP + indicates the retroviral-infected cell population and the relative proportion of GFP + cells at various time points post-infection was used to evaluate the cell viability. The mean with SD of three biological replicates is shown. One-way ANOVA followed by Dunnett's multiple comparisons test was performed. ****, p<0.0001.
(C) Representative western blots show the retroviral-expression of AID and AID R190X proteins in Aicda -/splenic primary B cells (top) and Aicda -/-CH12F3 cells (middle and bottom). Coexpressed GFP in Aicda -/splenic B cells was used as an internal control. For Aicda -/-CH12F3 cells, representative western blots with three repeats are shown (middle). Serial diluted CH12F3 cell samples were loaded on Tricine-SDS-PAGE, and protein levels of AID and AID R190X were accessed semi-quantitatively (bottom). AID proteins were detected with a custom anti-AID N terminus (7-24 aa) antibody. The arrows indicate AID and AID R190X bands respectively, while the asterisk indicates an unspecific band. EV, empty vector control. Of note, AID and AID R190X proteins display a bigger difference when they are separated on Tris-Glycine-SDS-PAGE, even with only a 9-aa difference, while they are not well-separated on Tricine-SDS-PAGE. Without specifically noted, Tris-Glycine-SDS-PAGE was used in this study.
(D) The indicated subcellular fractions of AID and AID R190X from retroviral-infected Aicda -/-CH12F3 cells were analyzed by western blot. b-tubulin was used as a marker for the cytoplasmic fraction, and PARP1 was used as a nuclear marker. The arrows indicate AID and AID R190X bands respectively, while the asterisks indicate unspecific bands.
(E) MBP-tagged AID variants purified from Expi293F cells are shown by Coomassie blue staining of SDS-PAGE. White arrows indicate the bands of MBP-fusion AID variants.
(F) The in vitro deamination activity of AID variants on branched DNA substrate. Left, a schematic illustration of in vitro deamination assay. Each reaction was performed with 0.5 µM DNA substrate. Middle, representative urea gel for visualization of the 5' FAM-labeled DNA substrate and product in the deamination reaction. Right, the percentage of deaminated DNA products in the indicated reaction is summarized in line plot as mean with SD of three biological replicates. Figure S2. AID C-terminal truncation mutants fail to initiate breaks at the IgH locus.   (F) Growth curves of CH12F3-miniS cells with the indicated AID expression are presented as mean with SD of three biological replicates. AID variants were retroviral-expressed with an AID-IRES-Puro cassette, and the corresponding cells were cultured in the presence of 1 µg/mL puromycin. The mean with SD of three replicates is shown. The survival data were fitted to a mixed-effects model using l-mer function from the R package 'lme4', with "Day" and "genotype" as fixed-effects parameters, and "cell number from each repeat" as the random effect parameter. The significance of the fixed effects parameters is obtained by the t-test. ****, p<0.0001; **, p<0.01; ns, p>0.05.

Appendix
(G) Representative western blot showed expression levels of Flag-tagged dSpCas9 and MCPtagged AID variants in the indicated Aicda -/-CH12F3-miniS cells. White arrows indicate MCP or MCP-tagged AID variant bands respectively, while asterisks indicate unspecific bands.   (C) 5% HEX abolishes optoAID DC condensates. Pre-assembled optoAID DC condensates were treated with 5% HEX for 15 s. Left, representative images for indicated optoAID DC before (top) and after (bottom) HEX treatment. Scale bar, 5 µm. Right, the dissolving speed of pre-assembled optoAID DC droplets in 5% HEX treatment is shown by line plots. Mean with SD of three independent biological replicates at indicated time points.

Figure S4
(D) Representative FRAP images of pre-assembled optoAID DC droplets in HEK293T cells.  (D) Co-condensation of wild-type AID and AID R190X in the nucleus of U2OS cells. Wild-type AID-GFP (green) and mCherry-tagged AID variants (red) were co-transfected in U2OS cells. Left, representative fluorescence images are shown. Scale bar, 5 µm. Right, quantification of cotransfected U2OS cells with puncta in nuclei is shown by bar plots and the total observed cell number of each genotype is indicated in parentheses.
(E) Wild-type AID protein can be selectively trapped into AID DC condensates. AID-GFP fusion protein (green) was co-expressed with the optoDroplet system (red), and representative images after triple blue light activation are shown. Scale bar, 5 µm.

AID DC condensation.
(A) Schematic illustration of AID mutant protein domain architectures and their abilities to form optoDroplet upon light-activation are summarized on the right panel. An IDR prediction by using IUPred2A is listed in the lower panel. IUPred2A score is shown on the y-axis, and amino acid position is shown on the x-axis. "+++": strong capacity with a cutoff of 90% cells showing optoDroplet puncta formation. "++": a lower capacity with 50-90% of cells showing puncta formation. "+": capacity with less than 50% of cells showing puncta formation. "-": empty vector control level.
(B) Representative optoDroplet images are shown for the indicated AID mutants before (top) and after (bottom) three times of blue light activation. AID DC mutants, including AID V186X , AID R190X , and AID cry , formed optoDroplet. While the AID DNDC variants (AID R190XDN12 and AID cryDN7 ) and AID DC-4R (AID R190X-4R and AID cry-4R ) were unable to form optoDroplet. Scale bar, 5 µm.
(C) The optoDroplet capacity that was assessed based on the dynamics of optoDroplet formation is shown for the indicated AID cry mutants. Top, the procedure of optoDroplet assay is illustrated with indicated light stimulation (blue arrow) and image acquisition (black arrow) time points. Bottom, the percentage of cells with optoDroplet formation at each time point is shown by line plots as mean with SEM, and the total acquired cell number of each genotype is indicated in parentheses. Significance assessment between AID cry and AID cry-4R is shown at each time point. Unpaired two-tailed Student's t-test was performed. ****, p<0.0001; **, p<0.01.

Appendix Figure S8. Cell proliferation and CSR levels of B cells in the presence of different forms of AID.
(A) Representative images showing the subcellular localization of indicated AID variants in nucleofected Aicda -/-CH12F3 cells. The nucleolus is indicated by mCherry-FBL. White dashed line depicts the shape of the nucleus based on Hoechst staining. Scale bar, 5 µm.
(B) Left, CSR levels to IgA in Aicda -/-CH12F3 cells complemented with retroviral-expressed AID mutants. The mean with SD of three biological replicates in the bar plot. Right, effect of AID mutants on the cell viability is indicated by the percentage of GFP + cells. One-way ANOVA followed by Dunnett's multiple comparisons test was performed. ****, p<0.0001; ***, p<0.001.        (E) Representative differential interference contrast (DIC) images of AID cry condensation are shown after cutting MBP tag off with PreScission enzyme. Twenty micromoles of MBP-AID cry protein were used, and 10% PEG8000 was added to induce droplet formation. Scale bar, 10 µm.

AID F T S W S P C Y D C A R H V A D F L R G N P N L S L R I F T A R L Y F C E D R K A E P E G L R R L A G V Q I A I M T K D Y Y C W N T F V E N H E R T F K F H R F AIDR190X F T S W S P C Y D C A R H V A D F L R G N P N L S L R I F T A R L Y F C E D R K A E P E G L R R L A G V Q I A I M T K D Y Y C W N T F V E N H E R T F K F H R F AIDcryCTT F T S W S P C Y D C A R H V A D F L R G N P N L S L R I F T A R L Y F C E D R K A E P E G L R R L A G V Q I A I M T K D Y Y C W N T F V E N H E R T F K Y A E E AIDcry F T S W S P C Y D C A R H V A D F L R G N P N L S L R I F T A R L Y F C E D R K A E P E G L R R L A G V Q I A I M T K D Y Y C W N T F V E N H E R T F K Y A E E AIDv F T S W S P C Y D C A R H V A D F L R G N P N L S L R I F T A R L Y F C E D R K A E P E G L R R L A G V Q I A I M T K D Y Y C W N T F V E N H E R T F K F H R F AIDvd15 F T S W S P C Y D C A R H V A D F L R G N P N L S L R I F T A R L Y F C E D R K A E P E G L R R L A G V Q I A I M T K D Y Y C W N T F V E N H E R T F K
(F) The two-phase mixtures of MBP-AID cry or AID cry were centrifuged to isolate the dilute phase (supernatant, S) and dense phase (pellet, P). Proteins were separated by SDS-PAGE staining with Coomassie blue. White arrows indicate the specific bands.
(G) In vitro droplet formation of MBP-GFP-AID cry at different protein concentrations in the presence of 10% PEG-8000. Left, representative images of MBP-GFP-AID cry condensates at the indicated protein concentrations. Scale bar, 10 µm. Right, sizes of MBP-GFP-AID cry droplets at the indicated concentrations are plotted as box plots with all points listed and mean indicated by "+" (n=5 technical replicates). Values between lower quartile and upper quartile are represented by box ranges, a horizontal line within the box represents the median, and whisker extends from the minimum value to the maximum value. Unpaired two-tailed Student's t-test was performed. ****, p<0.0001.
(I) MBP-GFP-AID cry droplet formation at different concentrations of NaCl in the presence of 10% PEG-8000. The protein concentration of MBP-GFP-AID cry is 5 µM. Left, representative images at the indicated salt concentrations are shown. Scale bar, 10 µm. Right, size distribution of MBP-GFP-AID cry droplets at the indicated salt concentrations are shown in box plots with all points listed and mean indicated by "+" (n=5 technical replicates). One-way ANOVA followed by Dunnett's multiple comparisons was performed. ****, p<0.0001. (B) Left, CSR levels in the indicated CH12F3 cells without and with the treatment of 4-hydroxy tamoxifen (4-OHT). The dominant-negative effect is accessed by comparing the CSR levels without (-) and with (+) 4-OHT induced ER fusion protein activation. Bar plot shows the mean with SD of three technical replicates. Right, cell viability is indicated by the percentage of the GFP + population as mean with SD at various time points post-infection. Paired two-tailed t-test was performed. *, p<0.05; ns, p>0.05.
(C) Representative fluorescence images at the indicated time points were shown for the trapping of MBP-GFP-AID by MBP-mCherry-AID cry condensates. MBP-mCherry-AID cry condensation was induced by 10% PEG8000 first, and then MBP-GFP-AID was added into the two-phase mixture. Scale bar, 5 µm.
(D) Single-cell quantitative RT-PCR analysis of Aicda and Gapdh in splenic germinal center B cells. AU, arbitrary units. For each gene, mean Ct of four 10-cell wells (Ct 10c ) was subjected to estimate Ct of a single cell. For individual cells, relative mRNA level was determined by 2^(Ct 10c + log210 -Ct). Each symbol represents the mRNA level in one cell (n = 90).