Role of R-loop in antibody class switching and B cell lymphomagenesis

2021 Fiscal Year Research-status Report

• Project/Area Number: 21K06015
• Research Institution: Kyoto University
• Principal Investigator: Begum NasimAra 京都大学, 医学研究科, 特定准教授 (80362507)
• Project Period (FY): 2021-04-01 – 2024-03-31
• Keywords: B Cells / CSR / R-loop / AID / Genomic Instability

Outline of Annual Research Achievements

Aim-To understand the role of the R-loop in AID-induced DNA break/repair during CSR.Targets-(1) Identify novel R-loop regulating factor(s) involved in CSR. (2) Examine DNA break and repair under the condition that promotes or suppresses R-loop. (3) Evaluate the effect of R-loop regulation on CSR and IgH/cMyc translocation.

Applying a candidate gene screening approach for RNA binding proteins, we successfully (1) identified a candidate protein, HNRNPU, as a critical regulator of the R-loop. Depletion of HNRNPU elevates R-loop formation at IgH and cMyc loci. (2) Knockdown of HNRNPU did not affect (2) DNA break but impaired regular repair of the CSR junctions. Evidence indicates that HNRNPU loss inhibits C-NHEJ-mediated repair essential to CSR. (3) AID-induced CSR and chromosomal translocation were strongly impaired upon HNRNPU depletion. As knockdown of R-loop resolving RNase H1 showed a similar phenotype, we predict a potential link between R-loop and choice of DNA repair pathway.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

As we successfully identified R-loop regulatory candidate proteins, we can proceed for the functional dissection. For the next step, we are undertaking necessary steps as follows_

(1) Construction of many HNRNPU and RNase H1mutants has been completed. (2) Using Coral Hue (MBL) system, analyzing the protein-protein interaction. (3) Performing the R-loop protein complex immunoprecipitation by S9.6 antibody. (4) Trying to generate an HNRNPU knockout B cell line (not yet successful). (5) Developing suitable conditions to study HNRNPU and RNaseH1 in the primary B cells.

Strategy for Future Research Activity

Loss of HNRNPU inhibits not only AID-induced antibody isotype switching but also the IgH/cMyc translocation, responsible for lymphoid malignancy. Therefore, we plan to investigate the underlying mechanism of (A) locus-specific R-loop regulation and (B) C-NHEJ-mediated DNA repair/recombination by HNRNPU.
(i) As R-loop/DNA:Hybrid formation at the target locus is the critical factor, we will compare R-loop associated complex before and after HNRNPU depletion. In parallel, HNRNPU IP will also be conducted.
(ii) Gene expression and ChIP analyses well identify the possible cause of the C-NHEJ defect. (iii) If we find that the RNA binding domain is critical for CSR, R-loop, and Genomic stability regulation, we will analyze HNRNPU bound RNAs.

Research Products

• [Journal Article] Phf5a regulates DNA repair in class switch recombination via p400 and histone H2A variant deposition (2021)
  • Author(s): Begum, N. A., Haque, F., Stanlie, A., Husain, A., Mondal, S., Nakata, M., Taniguchi, T., Taniguchi, H., and Honjo, T.
  • Journal Title: EMBO J Volume: 40:e106393 Pages: 1-21
  • DOI: 10.15252/embj.2020106393
  • Peer Reviewed / Int'l Joint Research