Project Abstract/Summary for the parent grant Class switch recombination (CSR) is a genetic process where a B cell switches antibody isotype production through site-specific intra-chromosomal DNA rearrangement stimulated by the formation of DNA double-strand breaks (DSBs) at the immunoglobulin heavy chain (IgH) locus. Recurrent oncogenic translocations involving IgH distinguish many human lymphoid malignancies; these translocations originate from mis-repaired DNA DSBs generated during normal lymphocyte development. DSBs are normally repaired by the non-homologous end-joining (NHEJ) and alternative-end joining (alt-EJ) DNA repair pathways. During CSR, DSB formation is highly regulated involving a complex interplay of transcriptional activation, protein recruitment and chromatin reorganization. Understanding the factors regulating DSB formation and repair has a high impact on lymphomagenesis. R loops are three stranded RNA:DNA hybrid structures formed at IgH during CSR. While R loops are implicated in promoting DSB formation at IgH, their role in class switch recombination remains undefined. To determine how persistent R loops impede DNA repair during CSR, and the role R loop metabolism plays in suppressing genome instability at IgH. we bred mice lacking two proteins involved in R loop removal: the helicase Senataxin (Setx-/-) which unwinds R loops; and Rnaseh2b is defective for the RNase H2 nuclease that specifically digests the RNA component of R loops (Rrnaseh2bf/fCD19cre). We find that B cells from Setx-/-Rnaseh2bf/f mice are proficient at class switch recombination, and contain high levels of unrepaired breaks and chromosome fusions at IgH. We hypothesize that persistent R loops block efficient DNA repair by non-homologous end joining at the immunoglobulin heavy chain locus during class switch recombination, leading to persistent, unrepaired breaks. We will functionally dissect the consequences of aberrant R loop formation on DNA repair and chromosome fusions arising during CSR in Setx-/-, Rnaseh2bf/f, and Setx-/- Rnaseh2bf/f cells (Aim 1). To define the impact persistent R loops have on NHEJ, we will characterize DNA repair protein recruitment in Setx-/-, Rrnaseh2bf/f, and Setx-/- Rrnaseh2bf/f cells (Aim 2). We will also identify genomic loci involved in IgH translocations using high-throughput genome-wide translocation sequencing (HTGTS-Seq). Finally, we will define the molecular pathways driving the frequent chromosome fusions observed in Setx-/- Rnaseh2bf/f cells (Aim 3). Our work will define how persistent R loops interfere with class switch recombination, leading to unrepaired breaks, and will uncover the molecular mechanisms promoting chromosome fusions at IgH. We are in the second year of this award which will run until 7/31/2024.

Public health relevance  Defective repair of DNA damage causes the accumulation of mutations and genomic rearrangements  that predispose to cancer development. Setx and RNase H2 protect against immunoglobulin (Ig)  translocations driving many lymphoid cancers. Understanding the impact of defects in Senataxin and  RNase H2 on the formation of genomic mutations and rearrangements will provide novel and  important insights into the molecular events driving tumorigenesis.