SHLD1 is dispensable for 53BP1-dependent V(D)J recombination but critical for productive class switch recombination
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE202567 | GEO | 2022/05/11
REPOSITORIES: GEO
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Project description:IgH class switch recombination (CSR) in B lymphocytes switches IgH constant regions to change antibody functions. CSR is initiated by DNA double strand breaks (DSBs) within a donor IgH switch (S) region and a downstream acceptor S region. CSR is completed by fusing donor and acceptor S region DSB ends by classical non-homologous end-joining (C-NHEJ) and, in its absence, by alternative end-joining (A-EJ) that is more biased to use longer junctional micro-homologies (MHs). Deficiency for DSB response (DSBR) factors, including ATM and 53BP1, variably impair CSR end-joining, with 53BP1 deficiency having the greatest impact. However, studies of potential impact of DSBR factor deficiencies on MH-mediated CSR end-joining have been technically limited. We now use a robust DSB joining assay to elucidate impacts of deficiencies for DSBR factors on CSR and chromosomal translocation junctions in primary mouse B cells and CH12F3 B lymphoma cells. Compared to wild-type, CSR and c-Myc to S region translocation junctions in the absence of 53BP1, and to a lesser extent other DSBR factors, have increased MH-utilization; indeed, 53BP1-deficient MH-profiles resemble those associated with C-NHEJ deficiency. Yet, translocation junctions between c-Myc DSB and general DSBs genome-wide are not MH-biased in ATM-deficient versus wild-type CH12F3 cells and less biased in 53BP1- and C-NHEJ-deficient cells than CSR junctions or c-Myc to S region translocation junctions. We discuss potential roles of DSBR factors in suppressing increased MH-mediated DSB end-joining and features of S regions that may render their DSBs prone to MH-biased end-joining in the absence of DSBR factors.
2018-01-08 | GSE106922 | GEO
Project description:Classical non-homologous end-joining (C-NHEJ) is a major mammalian DNA double strand break (DSB) repair pathway. Core C-NHEJ factors, such as XRCC4, are required for joining DSB intermediates of the G1 phase-specific V(D)J recombination reaction in progenitor lymphocytes. Core factors also contribute to joining DSBs in cycling mature B-lineage cells, including DSBs generated during antibody class switch recombination (CSR) and DSBs generated by ionizing radiation (IR). The XLF C-NHEJ factor is dispensable for V(D)J recombination in normal cells, but, due to functional redundancy, is absolutely required for this process in cells deficient for the ATM DSB response factor. The recently identified PAralogue of XRCC4 and XLF (PAXX) factor has homology to these two proteins and variably contributes to IR-induced DSB repair in human and chicken cells. We now report that PAXX is dispensable for joining V(D)J recombination DSBs in G1-arrested mouse pro-B cell lines, dispensable for joining CSR-associated DSBs in a cycling mouse B cell line, and dispensable for normal IR-resistance in both G1-arrested and cycling pro-B lines. However, we find that combined deficiency for PAXX and XLF in G1-arrested pro-B lines abrogates DSB joining during V(D)J recombination and sensitizes the cells to IR exposure. Thus, PAXX provides core C-NHEJ factor-associated functions in the absence of XLF and vice versa in G1-arrested Pro-B cell lines. Finally, we also find that PAXX-deficiency has no impact on V(D)J recombination DSB joining in ATM-deficient pro-B lines. We discuss implications of these findings with respect to potential PAXX and XLF functions in C-NHEJ. Examination of CSR Switch mu-to-alpha junctions from mu bait DSBs using LAM-HTGTS and Illumina Miseq. Two clones of PAXX-/- and XLF-/- and 1 clone of Ligase4-/- were derived from the parental CH12F3 line; the second Ligase4-/- clone was acquired from Keifei Yu. Two clones of XLF-/-PAXX-/-CH12 cells were derived from one of the PAXX-/- clones. Three biological replicates were analyzed for each clone.
2016-08-19 | E-GEOD-84102 | biostudies-arrayexpress
Project description:BRCA1 deficiencies cause breast, ovarian and other cancers, and render tumours hypersensitive to PARP-inhibitors. To understand resistance mechanisms, we conducted whole-genome CRISPR-Cas9 synthetic-viability/resistance screens in BRCA1-deficient breast cancer cells treated with PARP-inhibitors. Thus, we identified two previously uncharacterized proteins, C20orf196 and FAM35A, whose inactivation confers strong PARP-inhibitor resistance. Mechanistically, we show that C20orf196 and FAM35A form a complex, termed “Shieldin” (SHLD1/2), with FAM35A interacting with single-stranded DNA via its C-terminal OB-fold region. We establish that Shieldin promotes DNA double-strand break (DSB) end-joining, acting as the downstream effector of 53BP1/RIF1/MAD2L2 to restrict DSB resection and counteract homologous recombination in BRCA1-deficient cells by antagonising BRCA2/RAD51 loading. Notably, Shieldin inactivation further sensitises BRCA1-deficient cells to cisplatin, suggesting how defining the SHLD1/2 status of BRCA1-deficient tumours might aid patient stratification and yield new treatment opportunities. Highlighting this potential, we document reduced SHLD1/2 expression in human breast cancers displaying intrinsic or acquired PARP-inhibitor resistance.
2018-05-31 | PXD009830 | Pride
Project description:The 53BP1-RIF1 pathway antagonizes resection of DNA broken ends and confers PARP inhibitor sensitivity on BRCA1-mutated tumors. However, it is unclear how this pathway suppresses initiation of resection. Here, we identify ASF1 as a partner of RIF1 via an interacting manner similar to its interactions with histone chaperones CAF-1 and HIRA. ASF1 is recruited to distal chromatin flanking DNA breaks by 53BP1-RIF1 and promotes non-homologous end joining (NHEJ) using its histone chaperone activity. Epistasis analysis shows that ASF1 acts in the same NHEJ pathway as RIF1, but via a parallel pathway with the shieldin complex, which suppresses resection after initiation. Moreover, defects in end resection and homologous recombination (HR) in BRCA1-deficient cells are largely suppressed by ASF1 deficiency. Mechanistically, ASF1 compacts adjacent chromatin by heterochromatinization to protect broken DNA ends from BRCA1-mediated resection. Taken together, our findings identified a RIF1-ASF1 histone chaperone complex that promotes changes in high-order chromatin structure to stimulate the NHEJ pathway for DSB repair.
2022-02-04 | PXD031311 | Pride
Project description:During B cell development, RAG endonuclease cleaves immunoglobulin heavy chain (IgH) V, D, and J gene segments and orchestrates their fusion as deletional events that assemble a V(D)J exon in the same transcriptional orientation as adjacent Cμ constant region exons. In mice, six additional sets of constant region exons (CHs) lie 100-200kb downstream in the same transcriptional orientation as V(D)J and Cμ exons. Long repetitive switch (S) regions precede Cμ and downstream CHs. In mature B cells, class switch recombination (CSR) generates different antibody classes by replacing Cμ with a downstream CH. Activation Induced Cytidine Deaminase (AID) initiates CSR by promoting deamination lesions within Sμ and a downstream acceptor S region; these lesions are converted into DNA double strand breaks (DSBs) by general DNA repair factors. Productive CSR must occur in a deletional orientation by joining the upstream end of an Sμ DSB to the downstream end of an acceptor S region DSB. However, the relative frequency of deletional to inversional CSR junctions had not been measured. Thus, whether orientation-specific joining is a programmed mechanistic feature of CSR as it is for V(D)J recombination and, if so, how this is achieved was unknown. To address this question, we adapted high throughput genome wide translocation sequencing (HTGTS) into a highly sensitive DSB end-joining assay and applied it to endogenous AID-initiated S region DSBs. We find that CSR indeed is programmed to occur in a productive deletional orientation and does so via an unprecedented mechanism that involves in cis IgH organizational features in combination with frequent S region DSBs initiated by AID. We further implicate ATM-dependent DSB response (DSBR) factors in enforcing this mechanism and provide a solution to the enigma of why CSR is so reliant on the 53BP1 DSBR factor. We performed high-throughput genome-wide translocation sequencing (HTGTS) with different B cell genotypes that induces either I-SceI or AID-initiated DSBs as bait to study their joining pattern to AID-initiated S region breaks Please note that the 'ΔSγ1_2xI/ΔSµ_2xI-3' raw data files were analyzed twice with different reference assemblies to address specific points, and associated with both 'ΔSγ1_2xI_ΔSµ_2xI-3'.txt and Sγ1_2xI_ΔSµ_2xI-3'_trans-Sm.txt processed data files. As for the reference genome mm9_129_IgHC, it is a custom built generated based on mm9 (Bl6 line based mouse genome) and the partial genome sequence of another mouse line 129sv. It is not currently available in any public database however can be easily obtained either from the authors or self-built using information provided in the associated manuscript.
2015-08-26 | E-GEOD-71005 | biostudies-arrayexpress
Project description:During B cell development, RAG endonuclease cleaves immunoglobulin heavy chain (IgH) V, D, and J gene segments and orchestrates their fusion as deletional events that assemble a V(D)J exon in the same transcriptional orientation as adjacent Cμ constant region exons. In mice, six additional sets of constant region exons (CHs) lie 100-200kb downstream in the same transcriptional orientation as V(D)J and Cμ exons. Long repetitive switch (S) regions precede Cμ and downstream CHs. In mature B cells, class switch recombination (CSR) generates different antibody classes by replacing Cμ with a downstream CH. Activation Induced Cytidine Deaminase (AID) initiates CSR by promoting deamination lesions within Sμ and a downstream acceptor S region; these lesions are converted into DNA double strand breaks (DSBs) by general DNA repair factors. Productive CSR must occur in a deletional orientation by joining the upstream end of an Sμ DSB to the downstream end of an acceptor S region DSB. However, the relative frequency of deletional to inversional CSR junctions had not been measured. Thus, whether orientation-specific joining is a programmed mechanistic feature of CSR as it is for V(D)J recombination and, if so, how this is achieved was unknown. To address this question, we adapted high throughput genome wide translocation sequencing (HTGTS) into a highly sensitive DSB end-joining assay and applied it to endogenous AID-initiated S region DSBs. We find that CSR indeed is programmed to occur in a productive deletional orientation and does so via an unprecedented mechanism that involves in cis IgH organizational features in combination with frequent S region DSBs initiated by AID. We further implicate ATM-dependent DSB response (DSBR) factors in enforcing this mechanism and provide a solution to the enigma of why CSR is so reliant on the 53BP1 DSBR factor.
2015-08-26 | GSE71005 | GEO
Project description:Classical non-homologous end-joining (C-NHEJ) is a major mammalian DNA double strand break (DSB) repair pathway. Core C-NHEJ factors, such as XRCC4, are required for joining DSB intermediates of the G1 phase-specific V(D)J recombination reaction in progenitor lymphocytes. Core factors also contribute to joining DSBs in cycling mature B-lineage cells, including DSBs generated during antibody class switch recombination (CSR) and DSBs generated by ionizing radiation (IR). The XLF C-NHEJ factor is dispensable for V(D)J recombination in normal cells, but, due to functional redundancy, is absolutely required for this process in cells deficient for the ATM DSB response factor. The recently identified PAralogue of XRCC4 and XLF (PAXX) factor has homology to these two proteins and variably contributes to IR-induced DSB repair in human and chicken cells. We now report that PAXX is dispensable for joining V(D)J recombination DSBs in G1-arrested mouse pro-B cell lines, dispensable for joining CSR-associated DSBs in a cycling mouse B cell line, and dispensable for normal IR-resistance in both G1-arrested and cycling pro-B lines. However, we find that combined deficiency for PAXX and XLF in G1-arrested pro-B lines abrogates DSB joining during V(D)J recombination and sensitizes the cells to IR exposure. Thus, PAXX provides core C-NHEJ factor-associated functions in the absence of XLF and vice versa in G1-arrested Pro-B cell lines. Finally, we also find that PAXX-deficiency has no impact on V(D)J recombination DSB joining in ATM-deficient pro-B lines. We discuss implications of these findings with respect to potential PAXX and XLF functions in C-NHEJ.
2016-08-19 | GSE84102 | GEO
Project description:53BP1 regulates DNA end-joining in lymphocytes, diversifying immune antigen receptors. This involves nucleosome-bound 53BP1 at DNA double-stranded breaks (DSBs) recruiting RIF1 and shieldin, a poorly understood DNA-binding complex. The 53BP1-RIF1-shieldin axis is pathological in BRCA1-mutated cancers, blocking homologous recombination (HR) and driving illegitimate non-homologous end-joining (NHEJ). However, how this axis regulates DNA end-joining and HR suppression remains unresolved. We investigated shieldin and its interplay with CST, a complex recently implicated in 53BP1-dependent activities. Immunophenotypically, mice lacking shieldin or CST are equivalent, with class-switch recombination co-reliant on both complexes. DNA damage-responsive signalling promotes shieldin-CST interaction, demonstrating shieldin as a DSB-specific CST adaptor. Furthermore, DNA polymerase ζ functions downstream of shieldin, establishing DNA fill-in synthesis as the physiological function of shieldin-CST. Lastly, 53BP1 suppresses HR and promotes NHEJ in BRCA1-deficient cells independently of shieldin. These findings showcase the resilience of the 53BP1 pathway, achieved through the collaboration of chromatin-bound 53BP1 complexes and DNA end-processing effector proteins.
2024-08-27 | PXD045534 | Pride
Project description:The Shieldin complex, consisting of SHLD1, SHLD2, SHLD3 and REV7, shields double strand DNA breaks (DSBs) from nucleolytic resection. The end-protecting activity of Shieldin promotes productive non-homologous end joining (NHEJ) in G1 but can threaten genome integrity during S-phase by blocking homologous recombination (HR). Curiously, the penultimate Shieldin component, SHLD1 is one of the least abundant mammalian proteins. Here, we report that the transcription factors THAP1, YY1 and HCF1 bind directly to the SHLD1 promoter, where they cooperatively maintain the low basal expression of SHLD1. Functionally, this transcriptional network ensures that SHLD1 protein levels are kept in check to enable a proper balance between end protection and end resection during physiological DSB repair. In the context of BRCA1 deficiency, loss of THAP1 dependent SHLD1 expression confers cross resistance to PARP inhibitor and cisplatin, and shorter progression free survival in ovarian cancer patients. In contrast, loss of THAP1 in BRCA2 deficient cells increases genome instability and correlates with improved responses to chemotherapy. Pathogenic THAP1 mutations are causatively linked to adult-onset torsion dystonia type 6 (DYT6) movement disorder, but the critical disease targets are unknown. We further demonstrate that murine models of Thap1-associated dystonia show reduced Shld1 expression concomitant with elevated levels of unresolved DNA damage in the brain. In summary, our study provides the first example of a transcriptional network that directly controls DSB repair choice and reveals a previously unsuspected link between DNA damage and dystonia.
2021-04-13 | GSE154728 | GEO
Project description:The Shieldin complex, consisting of SHLD1, SHLD2, SHLD3 and REV7, shields double strand DNA breaks (DSBs) from nucleolytic resection. The end-protecting activity of Shieldin promotes productive non-homologous end joining (NHEJ) in G1 but can threaten genome integrity during S-phase by blocking homologous recombination (HR). Curiously, the penultimate Shieldin component, SHLD1 is one of the least abundant mammalian proteins. Here, we report that the transcription factors THAP1, YY1 and HCF1 bind directly to the SHLD1 promoter, where they cooperatively maintain the low basal expression of SHLD1. Functionally, this transcriptional network ensures that SHLD1 protein levels are kept in check to enable a proper balance between end protection and end resection during physiological DSB repair. In the context of BRCA1 deficiency, loss of THAP1 dependent SHLD1 expression confers cross resistance to PARP inhibitor and cisplatin, and shorter progression free survival in ovarian cancer patients. In contrast, loss of THAP1 in BRCA2 deficient cells increases genome instability and correlates with improved responses to chemotherapy. Pathogenic THAP1 mutations are causatively linked to adult-onset torsion dystonia type 6 (DYT6) movement disorder, but the critical disease targets are unknown. We further demonstrate that murine models of Thap1-associated dystonia show reduced Shld1 expression concomitant with elevated levels of unresolved DNA damage in the brain. In summary, our study provides the first example of a transcriptional network that directly controls DSB repair choice and reveals a previously unsuspected link between DNA damage and dystonia.
2021-04-13 | GSE154726 | GEO