PHF2 regulates homology-directed DNA repair by controlling the resection of DNA double strand breaks

Post-translational histone modifications and chromatin remodelling play a critical role controlling the integrity of the genome. Here, we identify histone lysine demethylase PHF2 as a novel regulator of the DNA damage response by regulating DNA damage-induced focus formation of 53BP1 and BRCA1, critical factors in the pathway choice for DNA double strand break repair. PHF2 knockdown leads to impaired BRCA1 focus formation and delays the resolution of 53BP1 foci. Moreover, irradiation-induced RPA phosphorylation and focus formation, as well as localization of CtIP, required for DNA end resection, to sites of DNA lesions are affected by depletion of PHF2. These results are indicative of a defective resection of double strand breaks and thereby an impaired homologous recombination upon PHF2 depletion. In accordance with these data, Rad51 focus formation and homology-directed double strand break repair is inhibited in cells depleted for PHF2. Importantly, we demonstrate that PHF2 knockdown decreases CtIP and BRCA1 protein and mRNA levels, an effect that is dependent on the demethylase activity of PHF2. Furthermore, PHF2-depleted cells display genome instability and are mildly sensitive to the inhibition of PARP. Together these results demonstrate that PHF2 promotes DNA repair by homologous recombination by controlling CtIP-dependent resection of double strand breaks.España Ministerio de Ciencia e Innovacion SAF2016-80626-REspaña, Fundación Canaria Instituto de Investigación Sanitaria de Canarias (FIISC) [PIFUN16/18

Loss of ZBTB24 impairs nonhomologous end-joining and class-switch recombination in patients with ICF syndrome

The autosomal recessive immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is a genetically heterogeneous disorder. Despite the identification of the underlying gene defects, it is unclear how mutations in any of the four known ICF genes cause a primary immunodeficiency. Here we demonstrate that loss of ZBTB24 in B cells from mice and ICF2 patients affects nonhomologous end-joining (NHEJ) during immunoglobulin class-switch recombination and consequently impairs immunoglobulin production and isotype balance. Mechanistically, we found that ZBTB24 associates with poly(ADP-ribose) polymerase 1 (PARP1) and stimulates its auto-poly(ADP-ribosyl)ation. The zinc-finger in ZBTB24 binds PARP1-associated poly(ADP-ribose) chains and mediates the PARP1-dependent recruitment of ZBTB24 to DNA breaks. Moreover, through its association with poly(ADP-ribose) chains, ZBTB24 protects them from degradation by poly(ADP-ribose) glycohydrolase (PARG). This facilitates the poly(ADP-ribose)-dependent assembly of the LIG4/XRCC4 complex at DNA breaks, thereby promoting error-free NHEJ. Thus, we uncover ZBTB24 as a regulator of PARP1-dependent NHEJ and class-switch recombination, providing a molecular basis for the immunodeficiency in ICF2 syndrome

Hematopoiesis and Lymphocyte Development:An Introduction

The production of blood and immune cells is a continuous process throughout life and essential for the existence of any individual human being. This process is referred to as HEMATOPOIESIS (from the Greek hematos, blood, and poiein, to generate) and includes the generation of three major cell lineages: (1) ERYTHROCYTES (red cells) that transport oxygen, (2) THROMBOCYTES (platelets) that mediate blood clotting, and (3) LEUKOCYTES (white blood cells) that function in host defense and consist of various types of GRANULOCYTES, LYMPHOCYTES, and MONOCYTES. The major site of HEMATOPOIESIS is the BONE MARROW, but it can occur at other (extramedullary) locations as well.</p

Hematopoiesis and Lymphocyte Development:An Introduction

The production of blood and immune cells is a continuous process throughout life and essential for the existence of any individual human being. This process is referred to as HEMATOPOIESIS (from the Greek hematos, blood, and poiein, to generate) and includes the generation of three major cell lineages: (1) ERYTHROCYTES (red cells) that transport oxygen, (2) THROMBOCYTES (platelets) that mediate blood clotting, and (3) LEUKOCYTES (white blood cells) that function in host defense and consist of various types of GRANULOCYTES, LYMPHOCYTES, and MONOCYTES. The major site of HEMATOPOIESIS is the BONE MARROW, but it can occur at other (extramedullary) locations as well.</p

XPC–PARP complexes engage the chromatin remodeler ALC1 to catalyze global genome DNA damage repair

Cells employ global genome nucleotide excision repair (GGR) to eliminate a broad spectrum of DNA lesions, including those induced by UV light. The lesion-recognition factor XPC initiates repair of helix-destabilizing DNA lesions, but binds poorly to lesions such as CPDs that do not destabilize DNA. How difficult-to-repair lesions are detected in chromatin is unknown. Here, we identify the poly-(ADP-ribose) polymerases PARP1 and PARP2 as constitutive interactors of XPC. Their interaction results in the XPC-stimulated synthesis of poly-(ADP-ribose) (PAR) by PARP1 at UV lesions, which in turn enables the recruitment and activation of the PAR-regulated chromatin remodeler ALC1. PARP2, on the other hand, modulates the retention of ALC1 at DNA damage sites. Notably, ALC1 mediates chromatin expansion at UV-induced DNA lesions, leading to the timely clearing of CPD lesions. Thus, we reveal how chromatin containing difficult-to-repair DNA lesions is primed for repair, providing insight into mechanisms of chromatin plasticity during GGR.German Research Foundation 213249687 - SFB 1064, 325871075 - SFB 1309Dutch Cancer Society KWF-YIG 11367European Research Council 310913Netherlands Organization for Scientific Research 711.018.007Institute of Basic Science IBS-R022-A1Natural Sciences and Engineering Research Council of Canada RGPIN-2016-05868, RGPAS-492875-2016Israel Cancer Research Fund Research Career Development Award 3013004741Dutch Research Council ENW-M (OCENW.KLEIN.090), ALW.016.161.320, VI.C.182.052National Cancer Insitute P01- CA092584Israel Cancer Association 20210078Israel Science Foundation 1710/1

Increased ID2 Levels in Adult Precursor B Cells as Compared with Children Is Associated with Impaired Ig Locus Contraction and Decreased Bone Marrow Output

Precursor B cell production from bone marrow in mice and humans declines with age. Because the mechanisms behind are still unknown, we studied five precursor B cell subsets (ProB, PreBI, PreBII large, PreBII small, immature B) and their differentiation-stage characteristic gene expression profiles in healthy individual toddlers and middle-aged adults. Notably, the composition of the precursor B cell compartment did not change with age. The expression levels of several transcripts encoding V(D)J recombination factors were decreased in adults as compared with children: RAG1 expression was significantly reduced in ProB cells, and DNA-PKcs, Ku80, and XRCC4 were decreased in PreBI cells. In contrast, TdTwas 3-fold upregulated in immature B cells of adults. Still, N-nucleotides, P-nucleotides, and deletions were similar for IGH and IGK junctions between children and adults. PreBII large cells in adults, but not in children, showed highly upregulated expression of the differentiation inhibitor, inhibitor of DNA binding 2 (ID2), in absence of changes in expression of the ID2-binding partner E2A. Further, we identified impaired Ig locus contraction in adult precursor B cells as a likely mechanism by which ID2-mediated blocking of E2A function results in reduced bone marrow B cell output in adults. The reduced B cell production was not compensated by increased proliferation in adult immature B cells, despite increased Ki67 expression. These findings demonstrate distinct regulatory mechanisms in B cell differentiation between adults and children with a central role for transcriptional regulation of ID2

The Fanconi anemia core complex promotes CtIP-dependent end-resection to drive homologous recombination at DNA double-strand breaks

Homologous Recombination (HR) is a high-fidelity repair mechanism of DNA Double-Strand Breaks (DSBs), which are induced by irradiation, genotoxic chemicals or physiological DNA damaging processes. DSBs are also generated as intermediates during the repair of interstrand crosslinks (ICLs). In this context, the Fanconi anemia (FA) core complex, which is effectively recruited to ICLs, promotes HR-mediated DSB-repair. However, whether the FA core complex also promotes HR at ICL-independent DSBs remains controversial. Here, we identified the FA core complex members FANCL and Ube2T as HR-promoting factors in a CRISPR/Cas9-based screen with cells carrying the DSB-repair reporter DSB-Spectrum. Using isogenic cell-line models, we validated the HR-function of FANCL and Ube2T, and demonstrated a similar function for their ubiquitination-substrate FANCD2. We further show that FANCL and Ube2T are directly recruited to DSBs and are required for the accumulation of FANCD2 at these break sites. Mechanistically, we demonstrate that FANCL ubiquitin ligase activity is required for the accumulation of the nuclease CtIP at DSBs, and consequently for optimal end-resection and Rad51 loading. CtIP overexpression rescues HR in FANCL-deficient cells, validating that FANCL primarily regulates HR by promoting CtIP recruitment. Together, these data demonstrate that the FA core complex and FANCD2 have a dual genome maintenance function by promoting repair of DSBs as well as the repair of ICLs.</p

Zinc finger protein ZNF384 is an adaptor of Ku to DNA during classical non-homologous end-joining

International audienceDNA double-strand breaks (DSBs) are among the most deleterious types of DNA damage as they can lead to mutations and chromosomal rearrangements, which underlie cancer development. Classical non-homologous end-joining (cNHEJ) is the dominant pathway for DSB repair in human cells, involving the DNA-binding proteins XRCC6 (Ku70) and XRCC5 (Ku80). Other DNA-binding proteins such as Zinc Finger (ZnF) domain-containing proteins have also been implicated in DNA repair, but their role in cNHEJ remained elusive. Here we show that ZNF384, a member of the C2H2 family of ZnF proteins, binds DNA ends in vitro and is recruited to DSBs in vivo. ZNF384 recruitment requires the poly(ADP-ribosyl) polymerase 1 (PARP1)-dependent expansion of damaged chromatin, followed by binding of its C2H2 motifs to the exposed DNA. Moreover, ZNF384 interacts with Ku70/Ku80 via its N-terminus, thereby promoting Ku70/Ku80 assembly and the accrual of downstream cNHEJ factors, including APLF and XRCC4/LIG4, for efficient repair at DSBs. Altogether, our data suggest that ZNF384 acts as a ’Ku-adaptor’ that binds damaged DNA and Ku70/Ku80 to facilitate the build-up of a cNHEJ repairosome, highlighting a role for ZNF384 in DSB repair and genome maintenance