Necessity of HuR/ELAVL1 for activation-induced cytidine deaminase-dependent decrease in topoisomerase 1 in antibody diversification

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Figure Legends

Fig. 1. The screening of the major AU-rich element binding proteins identified HuR/ELAVL1 as

(A) The knockdown effect of the major ARE binding factors, Brf1, Brf2, hnRNPd, Khsrp, Zfp36, and

HuR on CSR to IgA. CRISPR interference was used for knockdown except for HuR. CSR to IgA at

24 hours after the start of CIT stimulation was evaluated. The mean ± SD values calculated from three

stimulation samples.

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independent experiments are shown. CIT, cytokine cocktail of CD40L, IL-4, and TGF#. NS, no-

(B) Expression level of each molecule after knockdown shown in (A). The q-PCR signal was

normalized by #2M while Gapdh was used for HuR and the control sample (=1).

(C) AID mRNA expression in the cells shown in (A) measured by qPCR.

(D) !- and $-germline transcripts (GLT) of the switch regions in the cells shown in (A).

Fig. 2. HuR positively contributes to IgH gene diversification by regulating the DNA cleavage

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frequency.

(A) HuR expression detected by western blot in HuR knockout (KO) cell lines #101 and #111 stably

transfected by the 3XFLAG-tagged HuR (HuR+) or empty vectors (HuR–) with or without stimulation

by CIT. AID expression is also shown. $tubulin is a loading control. Wild-type cell lysate is from the

wild-type CH12 cells. no stim., without stimulation.

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(B) IgA% (the mean ± SD) at 24 (top) and 48 (bottom) hours from the start of CIT stimulation in the

#101 and #111 cell lines with or without replacement of HuR. P-values in CSR evaluation were

calculated by student t-test. *, P < 0.05; **, P < 0.01.

(C) Representative flowcytometric patterns of IgA (left) or IgG3 (right) expression of the selected

clones #22B (HuR–) and #11A (HuR+). IgA% and IgG3% were detected at 72 hours from the start of

stimulation. no stim., without stimulation. CI, stimulation with CD40L and IL-4. SSC, side scattered.

(D-E) The mean ± SD of IgA% (D) and IgG3% (E) of HuR– (22B) and HuR+ (11A) cells at the

indicated time points of the five to six independent experiments. ***, P < 0.001.

(F) The analyzed region mutated by AID-dependent hypermutation (G).

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the factor required in CSR.

(G) Mutation frequency of HuR– and HuR+ cells. P values were calculated by Fisher’s exact test. **,

P < 0.01; ***, P < 0.001; n.s., not significant.

(H) DNA break assay using biotinylated d-UTP (Bio-dUTP) labeling by terminal deoxynucleotidyl

transferase. HuR+ and HuR– cells with or without CIT stimulation were used. CIT-stimulated AIDdescribed in Supplementary Table 1.

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Fig. 3. Reduced efficiencies of CSR, SHM, and DNA cleavage in HuR-deficient cells are not due

to the defect of cell cycle progression or oxidative stress responses.

(A-C) Cell cycle analysis with CellTrace Violet dye (CTV) using the monoclonal #22B HuR– and

#11A HuR+ CH12 cells.

(A) Time course of the cell proliferation assay and CIT stimulation. The cells were incubated with

CTV in their culture medium following CIT stimulation while aphidicolin samples were unstimulated.

(B) IgA% in the cells used in the CTV cell proliferation assay. The mean ± SD is shown.

(C) Representative histogram of HuR– (blue) and HuR+ (red) cells labelled by CTV. Whole cells

(left) or switched (IgA+) (right) populations are shown. HuR– without stimulation was incubated with

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aphidicolin to monitor the cell division. Inset shows the result of HuR–, non-stimulated cells

incubated with (green) or without (magenta) aphidicolin. no stim., not stimulated.

(D-F) The effects of reactive oxygen species (ROS) scavenger drugs on CSR in HuR– and HuR+ cells

derived from #111 HuR-KO clones.

(D) Time course for IgA% and dead cell% analyses with two ROS scavenger drugs, N-Acetyl-L-

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Cysteine (L-NAC) and chloro[[2,2'-[1,2-ethanediylbis[(nitrilo-κN)methylidyne]]bis[6methoxyphenolato-κO]]]-manganese (EUK-134).

(E-F) Analysis of CSR to IgA (top) and dead cells (bottom) exposed to the serial concentration of LNAC (E, 0 - 50 mM) or EUK-134 (F, 0 - 10 mM) in HuR– and HuR+ cells. Gray triangles show the

increment of the concentration of the drugs.

(G) Detection of the inclusion of Intron (Int) 10 of dihydrolipoamide S-succinyltransferase (Dlst)

mRNA evaluated by qPCR with the two primer sets covering exon-intron boundaries in #22B, HuR–

and #11A, HuR+ cells. The signals from the specific primers were normalized by the signals of

Gapdh mRNA. The mean ± SD of each primer set of three independent experiments. The arrows

show the primers amplifying Exon (Ex) 10- Int 10 transcripts, and the triangles show the primers

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knockout cells and HuR+ cells without labeling were used as negative control. The primers are

amplifying Int 10-Ex 11 transcripts. *, P < 0.05; **, P < 0.01; n.s., not significant; no stim., without

stimulation.

(H) Conventional RT-PCR analysis amplifying Ex 10-Ex 11 evaluating alternative splicing in Dlst

mRNA. Exon 10b (Ex 10b) is an unusual cryptic exon described by Diaz-Muñoz et al. (31).

non-stimulated HuR– and HuR+ cells out of three experiments. GAPDH (I) and #&actin (J) are

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loading controls.

Fig. 4. AID-dependent repression of Top1 is eliminated in HuR-deficient cells.

(A-B) Top1 protein change by CIT or CI stimulation in #22B HuR– and #11A HuR+ cells. The

proteins were extracted by PBS with TritonX-100 (A) or RIPA (B) buffer. GAPDH is the loading

control.

(C) Top1 mRNA expression in #22B HuR– and #11A HuR+ cells with or without CIT or CI

stimulation analyzed by q-PCR. The mean ± SD of Top1 mRNA signals normalized by Gapdh mRNA

is shown.

(D) Mouse Top1 mRNA structure and the position of the eight ATTTA motifs. The primer set

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position (a ~ h) used for q-PCR in RNA-IP experiments (E-H) are shown by the bars.

(E-F) RNA-IP analysis using #11A HuR+ cells and anti-HuR antibody.

(E) Western blot analysis showing the immunoprecipitation (IP) efficiency.

(F) Enrichment of Top1, Gapdh, or c-Myc mRNA to HuR protein evaluated by q-PCR. The mean ±

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SD values of the three independent experiments are shown.

(G-H) RNA-IP analysis using wildtype and AID knockout mice spleen B cells and anti-HuR antibody

after stimulation with LPS and IL-4 for four days.

(G) Western blot analysis showing the immunoprecipitation (IP) efficiency.

(H) Enrichment of Top1, Gapdh, or c-Myc mRNA by HuR protein evaluated by q-PCR. The mean ±

SD values of the two independent experiments are shown.

(I-N) Polysome analysis comparing CIT-stimulated and unstimulated #22B HuR– and #11A HuR+

cells.

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(I-J) Representative western blot images of Dlst (I) and c-Myc (J) protein expression in stimulated or

(I-J) Optical density (OD) was measured at 256 nm in the fractions (#1-#90) obtained by

ultracentrifuging of the 10-45% sucrose gradient in CIT-stimulated or unstimulated HuR– (I) or HuR+

cells (J).

(K-N) RNA analysis of the pooled samples collected from 10 serial fractions. Top1 mRNA in HuR–

of the sucrose concentration of the pooled samples.

partially in HuR-deficient cells.

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Fig. 5. Knockdown of Top1 rescues the impairment of CSR to IgG3 completely and CSR to IgA

(A) Time course of Top1 knockdown experiments.

(B) Western blot analysis for Top1 protein expression after repeated siTop1. Actin was used as a

loading control. 1.6 or 3.2 105 cells/lane were loaded in siTop1, and 0.8 or 1.6

loaded in siControl.

105 cells/lane were

(C) Representative flowcytometric pattern of CSR to IgA (left two rows) and IgG3 (right two rows).

HuR– or HuR+ cells stimulated with CI or CIT for 72 hours transfected with or without 3 mM siTop1.

(D-E) The mean ± SD of IgA% (D) and IgG3% (E) from six and three independent experiments,

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respectively, observed from days 1 to 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant.

Fig. 6. HuR is required for decrease in Top1, DNA cleavage, and CSR. When amount of Top1

protein is decreased by the repeated knockdown of Top1, HuR-deficient cells show “rescue” of CSR

efficiency to the level of HuR-proficient cells in CSR to IgG3. The reason for this rescue may be that

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the equally minimized amount of Top1 protein in both HuR-deficient and -proficient cells achieved

similarly sufficient levels of altered non-B DNA structure and DNA cleavage.

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(K) or HuR+ cells (L). b2M mRNA in HuR– (M) or HuR+ cells (N). Grey triangles show the change

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