E3 Ubiquitin Ligases RNF20 and RNF40 Are Required for Double-Stranded Break (DSB) Repair: Evidence for Monoubiquitination of Histone H2B Lysine 120 as a Novel Axis of DSB Signaling and Repair

Abstract

Histone posttranslational modifications play fundamental roles in the regulation of double-stranded DNA break (DSB) repair. RNF20/RNF40-mediated monoubiquitination of histone H2B on lysine 120 (H2Bub) has been suggested as a potential mediator of DSB repair, although the nature and function of this posttranslational modification remain enigmatic. In this report, we demonstrate that RNF20 and RNF40 are required for DSB repair leading to homologous recombination (HR) and class switch recombination, a process driven by nonhomologous end joining (NHEJ), in mouse B cells. These findings suggest a role for RNF20 and RNF40 in DSB repair proximal to NHEJ/HR pathway choice and likely in the signaling of DSBs. We found that DSBs led to a global increase in H2Bub but not the transcription-associated posttranslational modifications H3K4me3 and H3K79me2. We also found that H2AX phosphorylation was dispensable for H2Bub and that ATM and ATR jointly regulate ionizing radiation (IR)-induced H2Bub. Together, our results suggest that RNF20, RNF40, and H2Bub may represent a novel pathway for DSB sensing and repair.

Keywords: B cell; H2BK120; RNF20; RNF40; class switch recombination; double-stranded DNA repair; histone ubiquitination; homologous recombination; nonhomologous recombination.

FIG 1

RNF20 and RNF40 are required for class switch recombination (CSR). (A) Quantification of Rnf20 and Rnf40 mRNAs in shRNF20 and shRNF40 CH12 cells, respectively, by qPCR. Values were calculated using the Pfaffl method, normalizing expression to that in shGFP CH12 cells. (B) Detection of H2Bub expression in shRNF20 and shRNF40 CH12 cells by Western blotting. (C) Quantification of Rnf20 and Rnf40 mRNAs in shRNF20 and shRNF40 WEHI-279 cells, respectively, by qPCR. Values were calculated using the Pfaffl method, normalizing expression to that in shGFP WEHI-279 cells. (D) Quantification of CSR to IgA in control, shRNF20, and shRNF40 CH12 cells as measured by flow cytometry 2 days after cytokine stimulation with αCD40, IL-4, and TGF-β (CIT). (E) Quantification of Aicda mRNA in shRNF20 and shRNF40 CH12 cells at steady state or 1 day post-CIT. (F) AID protein in shRNF20 and shRNF40 CH12 cells at steady state or 1 day post-CIT. The white line indicates stitching of different lanes from the same blot. (G) Densitometry analysis of AID protein levels relative to H2B in control, shRNF20, and shRNF40 CH12 cells at steady state or 1 day post-CIT. AU, arbitrary units. (H and I) Quantification of germ line transcription of switch regions μ (Sμ) (H) and α (Sα) (I) at steady state or 1 day post-CIT by qPCR. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 2

RNF20 and RNF40 knockdown does not grossly affect transcription of DSB repair genes at steady state or after IR. mRNA levels of RNF20 (Rnf20) (A), RNF40 (Rnf40) (B), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Gapdh) (C), β-actin (Bact) (D), p53 (tp53) (E), p21 (tp21) (F), MDM2 (Mdm2) (G), ATM (Atm) (H), DNA-PKcs (Prkdc) (I), 53BP1 (Trp53bp1) (J), and BRCA1 (Brca1) (K) in shGFP, shRNF20, and shRNF40 CH12 cells at steady state or 4 h after 8 Gy IR as measured by qPCR.

FIG 3

RNF20 and RNF40 are required for Cas9-mediated switching and homologous recombination. (A) Schematic of the mouse immunoglobulin heavy chain (Igh) locus and Cas9/sgRNA targeting for CSR. Constant regions Cμ, Cγ, and Cα are preceded by repetitive switch regions Sμ, Sγ, and Sα, respectively. sgRNAs (depicted as black bars) were designed to target outside these switch regions. Recombination between DSBs generated at S′μ and S′γ results in class switching to IgG1 (left), while recombination between DSBs generated at S′μ and S′α results in class switching to IgA (right). The schematic is not to scale. (B) Quantification of Cas9-mediated switching to IgA in Aicda^−/− CH12 cells and representative flow plots. (C to E) Cas9-mediated switching to IgG1 in CH12 cells and representative flow plots (C), Cas9-mediated switching to IgA in WEHI-279 cells and representative flow plots (D), and I-SceI-mediated homologous recombination in DR-GFP CH12 cells and representative flow plots (E) following control, RNF20, or RNF40 knockdown as measured by flow cytometry at 3 days posttransfection. FSC, forward scatter. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 4

RNF20 and RNF40 knockdown does not significantly perturb cellular proliferation dynamics. (A and B) Proliferation of shGFP, shRNF20, and shRNF40 CH12 cells in the steady state (A) and after stimulation with CIT (B). (C) Representative flow cytometry plots depicting cell cycle phases of shGFP, shRNF20, and shRNF40 CH12 cells in the steady state and after CIT stimulation. EdU^lo PI^lo cells (lower left gate) are in G₀/G₁ phase, EdU^hi cells (upper gate) are in S phase, and EdU^lo PI^hi cells (lower right gate) are in G₂/M phase. (D and E) Quantification of shGFP, shRNF20, and shRNF40 CH12 cells in G₀/G₁, S, and G₂/M phases in the steady state (D) and after CIT stimulation (E). Comparisons are not statistically significant unless specifically denoted.

FIG 5

Ionizing radiation (IR) induces a dramatic increase in H2Bub but not other transcription-associated histone PTMs. (A) H2Bub, H3K4me3, and H3K79me2 expression in the nuclear fraction of wild-type CH12 cells at various time points after 8 Gy IR detected by Western blotting. (B) H2Bub, H3K4me3, and H3K79me2 expression in the nuclear fraction of shGFP, shRNF20, and shRNF40 CH12 cells at steady state. (C to E) Densitometry analysis of H2Bub (C), H3K4me3 (D), and H3K79me2 (E) in shGFP, shRNF20, and shRNF40 CH12 cells at steady state. (F to J) Same as panels A to E but instead normalized to histone H3 levels. ns, not significant; *, P < 0.05.

FIG 6

Camptothecin and etoposide induce a dramatic increase in H2Bub but not H3K4me3 or H3K79me2. (A) H2Bub, H3K4me3, and H3K79me2 expression in wild-type CH12 cells pulsed with 5 μM camptothecin (CPT) for 1 h. (B to D) Densitometry analysis of H2Bub (B), H3K4me3 (C), and H3K79me2 (D) at the indicated time points after camptothecin treatment relative to β-actin. (E) H2Bub, H3K4me3, and H3K79me2 expression in wild-type CH12 cells pulsed with 25 μM etoposide (ETP) for 1 h. (F to H) Densitometry analysis of H2Bub (F), H3K4me3 (G), and H3K79me2 (H) at the indicated time points after etoposide treatment relative to β-actin.

FIG 7

IR-induced γH2AX and H2Bub can arise independently of each other. (A) γH2AX and H2Bub expression in shGFP, shRNF20, and shRNF40 CH12 cells at various time points after 8 Gy IR detected by Western blotting. “−,” steady state. (B and C) Densitometry analysis of H2Bub (B) and γH2AX (C) protein levels in shGFP, shRNF20, and shRNF40 CH12 cells at the indicated time points relative to β-actin. (D) Schematic illustrating the sgRNA targeting strategy for CRISPR/Cas9-mediated H2afx gene knockout in CH12 cells. (E) RT-PCR of H2afx and Gapdh mRNAs from H2afx^+/+ and H2afx^−/− CH12 cells. (F) AID-mediated CSR to IgA in two H2afx^+/+ and four H2afx^−/− CH12 clones. Cells were harvested and analyzed by flow cytometry 2 days after cytokine stimulation with αCD40, IL-4, and TGF-β. (G) γH2AX and H2Bub expression in representative H2afx^+/+ and H2afx^−/− CH12 clones at various time points after 8 Gy IR detected by Western blotting. “−,” steady state. (H and I) Densitometry analysis of γH2AX (H) and H2Bub (I) protein levels from two H2afx^+/+ and four H2afx^−/− CH12 clones at the indicated time points relative to β-actin. Statistical comparisons between control and experimental groups were conducted using 2-way analysis of variance (ANOVA). Comparisons are not statistically significant unless specifically denoted. **, P < 0.01; ****, P < 0.0001.

FIG 8

IR induces RNF20/RNF40-mediated H2B ubiquitination but not acetylation. (A) H2Bub and H2Bac protein levels in shGFP, shRNF20, and shRNF40 CH12 cells at steady state detected by Western blotting. (B and C) Densitometry analysis of H2Bub (B) and H2Bac (C) protein levels in shGFP, shRNF20, and shRNF40 CH12 cells at steady state relative to β-actin. (D) H2Bub and H2Bac protein levels in shGFP, shRNF20, and shRNF40 CH12 cells at the indicated time points after IR. “−,” steady state. (E and F) Densitometry analysis of H2Bub (E) and H2Bac (F) protein levels in shGFP, shRNF20, and shRNF40 CH12 cells at the indicated time points after IR relative to β-actin. ns, not significant; *, P < 0.05.

FIG 9

ATM and DNA-PKcs are dispensable for IR-induced H2Bub kinetics. (A) Sequenced Prkdc alleles showing Cas9-mediated deletions spanning the start codon. Amino acid residues and position numbers are indicated above the Prkdc nucleotide (nt) sequence. The Prkdc sgRNA target sequence (data not shown) is also indicated. (B) Quantification of Prkdc mRNA in wild-type (WT) and Prkdc^−/− CH12 cells by qPCR. (C) AID-mediated CSR to IgA in wild-type, Prkdc^−/−, Ku86^−/−, and LigIV^−/− CH12 cells. Cells were harvested and analyzed by flow cytometry 2 days after cytokine stimulation with αCD40, IL-4, and TGF-β (CIT). (D and E) Proliferation of wild-type, Prkdc^−/−, Ku86^−/−, and LigIV^−/− CH12 cells in the steady state (D) and after stimulation with CIT (E). (F) H2Bub, γH2AX, and pKap1 expression at various time points after 8 Gy IR in four treatment groups of CH12 cells (DMSO treated, ATM inhibited, DNA-PKcs deficient, and ATM inhibited plus DNA-PKcs deficient) detected by Western blotting. “−,” steady state. (G to I) Densitometry analysis of pKap1 (G), γH2AX (H), and H2Bub (I) protein levels at the indicated time points relative to β-actin. (J) H2Bub and pKap1 expression at various time points after 8 Gy IR in DMSO- and ATMi-treated WEHI-279 cells. “−,” steady state. (K) Densitometry analysis of H2Bub protein levels at the indicated time points after 8 Gy IR relative to H2B. (L) H2Bub and pKap1 expression at various time points after 2 Gy IR in DMSO- and ATMi-treated WEHI-279 cells. (M) Densitometry analysis of H2Bub protein levels at the indicated time points after 2 Gy IR relative to H2B. Statistical comparisons between control and experimental groups were conducted using 2-way ANOVA. Comparisons are not statistically significant unless specifically denoted. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 10

Simultaneous inhibition of ATM and ATR inhibits IR-induced H2Bub. (A) H2Bub, pChk1, and pKap1 protein levels in H₂O- and caffeine-treated CH12 cells at the indicated time points after IR. (B to D) Densitometry analysis of pChk1 (B), pKap1 (C), and H2Bub (D) protein levels at various time points after IR relative to H2B. (E) H2Bub, pChk1, and pKap1 protein levels in DMSO-, ATRi-, and ATRi-plus-ATMi-treated CH12 cells at the indicated time points after IR. (F to H) Densitometry analysis of pChk1 (F), pKap1 (G), and H2Bub (H) protein levels at various time points after IR relative to H2B. Statistical comparisons between control and experimental groups were conducted using 2-way ANOVA. Comparisons are not statistically significant unless specifically denoted. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.