Aims: PTEN-putative kinase 1 (PINK1)-Parkin-mediated mitophagy is crucial for the clearance of damaged mitochondria. However, the mechanisms underlying PINK1-Parkin-mediated mitophagy are not fully understood. The goal of this study is to identify new regulators and to elucidate the regulatory mechanisms of mitophagy. Results: Quantitative mitochondrial proteomic analysis revealed that 63 proteins showed increased levels and 36 proteins showed decreased levels in cells subjected to carbonyl cyanide m -chlorophenyl hydrazone (CCCP) treatment. Peroxiredoxin 6 (PRDX6 or Prx6), a unique member of the ubiquitous PRDX family, was recruited to depolarized mitochondria. Reactive oxygen species (ROS) generated by CCCP promoted PRDX6 accumulation and PINK1 stabilization in damaged mitochondria and induced mitophagy. In addition, depletion of PRDX6 resulted in the stabilization of PINK1, accumulation of autophagic marker, p62, translocation of Parkin to mitochondria, and lipidation of microtubule-associated protein 1 light chain 3. Furthermore, these events were blocked upon supplementation with antioxidant N -acetyl- l -cysteine or depletion of PINK1. Innovation: This is the first study to demonstrate that PRDX6 is the only member of the PRDX family that relocates to damaged mitochondria, where it plays a crucial role in the initial stage of mitophagy by controlling ROS homeostasis. Conclusion: ROS induce the recruitment of PRDX6 to mitochondria, where PRDX6 controls ROS homeostasis in the initial step of PINK1-Parkin-mediated mitophagy. Our study provides new insight into the initial regulatory mechanisms of mitophagy and reveals the protective role of PRDX6 in the clearance of damaged mitochondria. Antioxid. Redox Signal. 24, 486–501.

SHLD1 is part of the Shieldin (SHLD) complex, which acts downstream of 53BP1 to counteract DNA double-strand break (DSB) end resection and promote DNA repair via non-homologous end-joining (NHEJ). While 53BP1 is essential for immunoglobulin heavy chain class switch recombination (CSR), long-range V(D)J recombination and repair of RAG-induced DSBs in XLF-deficient cells, the function of SHLD during these processes remains elusive. Here we report that SHLD1 is dispensable for lymphocyte development and RAG-mediated V(D)J recombination, even in the absence of XLF. By contrast, SHLD1 is essential for restricting resection at AID-induced DSB ends in both NHEJ-proficient and NHEJ-deficient B cells, providing an end-protection mechanism that permits productive CSR by NHEJ and alternative end-joining. Finally, we show that this SHLD1 function is required for orientation-specific joining of AID-initiated DSBs. Our data thus suggest that 53BP1 promotes V(D)J recombination and CSR through two distinct mechanisms: SHLD-independent synapsis of V(D)J segments and switch regions within chromatin, and SHLD-dependent protection of AID-DSB ends against resection.

IgH class switch recombination (CSR) replaces Cμ constant region (C_H) exons with one of six downstream C_Hs by joining transcription-targeted double-strand breaks (DSBs) in the Cμ switch (S) region to DSBs in a downstream S region. Chromatin loop extrusion underlies fundamental CSR mechanisms including 3′IgH regulatory region (3′IgHRR)-mediated S region transcription, CSR center formation, and deletional CSR joining. There are 10 consecutive CTCF-binding elements (CBEs) downstream of the 3′IgHRR, termed the “3′IgH CBEs.” Prior studies showed that deletion of eight 3′IgH CBEs did not detectably affect CSR. Here, we report that deletion of all 3′IgH CBEs impacts, to varying degrees, germline transcription and CSR of upstream S regions, except that of Sγ1. Moreover, deletion of all 3′IgH CBEs rendered the 6-kb region just downstream highly transcribed and caused sequences within to be aligned with Sμ, broken, and joined to form aberrant CSR rearrangements. These findings implicate the 3′IgH CBEs as critical insulators for focusing loop extrusion-mediated 3′IgHRR transcriptional and CSR activities on upstream C_H locus targets. Significance B lymphocytes change antibody heavy chain (IgH) isotypes by a recombination/deletion process called IgH class switch recombination (CSR). CSR involves introduction of DNA breaks into a donor switch (S) region and also into one of six downstream S regions, with joining of the breaks changing antibody isotype. A chromatin super-anchor, of unknown function, is located just downstream of the IgH locus. We show that complete deletion of this super-anchor variably decreases CSR to most S regions and creates an ectopic S region downstream of IgH locus that undergoes aberrant CSR-driven chromosomal rearrangements. Based on these and other findings, we conclude that the super-anchor downstream of IgH is a critical insulator for focusing potentially dangerous CSR rearrangements to the IgH locus.

The diversification of B-cell receptor (BCR), as well as its secreted product, antibody, is a hallmark of adaptive immunity, which has more specific roles in fighting against pathogens. The antibody diversification is from recombination-activating gene (RAG)-initiated V(D)J recombination, activation-induced cytidine deaminase (AID)-initiated class switch recombination (CSR), and V(D)J exon somatic hypermutation (SHM). The proper repair of RAG- and AID-initiated DNA lesions and double-strand breaks (DSBs) is required for promoting antibody diversification, suppressing genomic instability, and oncogenic translocations. DNA damage response (DDR) factors and DSB end-joining factors are recruited to the RAG- and AID-initiated DNA lesions and DSBs to coordinately resolve them for generating productive recombination products during antibody diversification. Recently, cohesin-mediated loop extrusion is proposed to be the underlying mechanism of V(D)J recombination and CSR, which plays essential roles in promoting the orientation-biased deletional end-joining . Here, we will discuss the mechanism of DNA damage repair in antibody diversification.

The enhancer of zeste homolog 2 (EZH2) histone methyltransferase inhibitors tazemetostat and valemetostat recently have received approval for clinical use in follicular lymphoma and adult T-cell leukemia/lymphoma, respectively. In follicular lymphoma, the expression of activation-induced cytidine deaminase (AID) is responsible for increased mutational signatures and genomic instability. Because EZH2 inhibitors induce epigenetic and transcriptional changes in normal and lymphoma B cells, we studied whether these inhibitors could alter the pattern of AID-mediated chromosomal translocations. In this study, we showed that treatment with EZH2 inhibitors did not significantly change AID expression or AID-dependent chromosomal translocation frequency when used as monotherapy in either CH12F3 mouse B cells or MEC-1 human B cells. In contrast, when combined with phosphoinositide 3-kinase δ (PI3Kδ) inhibition, which enhances AID expression, EZH2 inhibition significantly increased the frequency of chromosomal translocations when compared with either EZH2 or PI3Kδ inhibition alone both in mouse CH12F3 cells and human MEC-1 cells. EZH2 inhibition also further enhanced translocation formation in mouse B cells that were DNA ligase IV (Ligase4) deficient. Mechanistically, EZH2 inhibition in B cells depletes the repressive histone modification H3 trimethylation at lysine 27 (H3K27me3) while concurrently enhancing the active histone modification H3 acetylation at lysine 27 (H3K27ac), thereby selectively increasing transcriptional activity and facilitating chromosomal translocation formation in the presence of high AID activity or Ligase4 deficiency. These findings highlight the impact of drugs that induce epigenetic changes to influence chromosomal translocations, and demonstrate the genetic safety of EZH2 inhibitors as monotherapy while highlighting the increased risk of genomic instability when used in cells prone to translocations, such as B cells with high AID levels or DNA repair deficiency.