R-loop-induced DNA damage during immunoglobulin class switch recombination

上级拨款的项目摘要/摘要 类切换重组(CSR)是B细胞切换抗体同型产生的遗传过程 通过DNA双链形成刺激的染色体内DNA定点重排 免疫球蛋白重链(IgH)基因的断裂(DSB)。复发性致癌易位涉及 IgH区分了许多人类淋巴系统恶性肿瘤；这些易位源于错误修复的DNA 在正常淋巴细胞发育过程中产生的DSB。DSB通常由非同源的 末端连接(NHEJ)和选择性末端连接(ALT-EJ)DNA修复途径。在CSR期间，DSB的形成是 高度调控涉及转录激活、蛋白质募集和染色质的复杂相互作用 重组。了解调节DSB形成和修复的因素对 淋巴肿大。R环是在CSR过程中在IgH形成的三条链RNA：DNA杂交结构。而R 环路在IGH促进了DSB的形成，它们在类开关重组中的作用仍然存在 未定义。确定持续R环如何阻碍CSR过程中的DNA修复，以及R环的作用 新陈代谢在抑制IgH的基因组不稳定性方面发挥了作用。我们培育的小鼠缺少两种与R相关的蛋白质 环去除：解开R环的解旋酶Senataxin(SETX-/-)；RNaseh2b是缺陷的 RNaseh2bf/fCD19cre(Rrnaseh2bf/fCD19cre)。 我们发现来自SETX-/-RNaseh2bf/f小鼠的B细胞擅长进行类开关重组，并且含有较高的 IGH中未修复的断裂和染色体融合水平。我们假设持久的R循环阻塞 免疫球蛋白重链上非同源末端连接的DNA修复 类切换重组，导致持续的、未修复的中断。我们将从功能上剖析 CSR过程中R环的异常形成对DNA修复和染色体融合的影响 SETX-/-、RNaseh2bf/f和SETX-/-RNaseh2bf/f细胞(目标1)。要定义持久化R循环对 NHEJ，我们将表征DNA修复蛋白在SETX-/-、Rrnaseh2bf/f和SETX-/-Rrnaseh2bf/f细胞中的募集 (目标2)。我们还将使用高通量全基因组来识别与IGH易位有关的基因组基因座 易位测序(HTGTS-Seq)。最后，我们将定义驱动频繁的 在SETX-/-RNaseh2bf/f细胞中观察到染色体融合(目标3)。我们的工作将定义持久的R循环 干扰类开关重组，导致未修复的断裂，并将揭开分子 IGH促进染色体融合的机制。我们是这个奖项的第二个年头，这个奖项将持续到 直到2024年7月31日。

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.