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1.
Nature ; 600(7888): 329-333, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34819671

RESUMO

Efficient humoral responses rely on DNA damage, mutagenesis and error-prone DNA repair. Diversification of B cell receptors through somatic hypermutation and class-switch recombination are initiated by cytidine deamination in DNA mediated by activation-induced cytidine deaminase (AID)1 and by the subsequent excision of the resulting uracils by uracil DNA glycosylase (UNG) and by mismatch repair proteins1-3. Although uracils arising in DNA are accurately repaired1-4, how these pathways are co-opted to generate mutations and double-strand DNA breaks in the context of somatic hypermutation and class-switch recombination is unknown1-3. Here we performed a genome-wide CRISPR-Cas9 knockout screen for genes involved in class-switch recombination and identified FAM72A, a protein that interacts with the nuclear isoform of UNG (UNG2)5 and is overexpressed in several cancers5. We show that the FAM72A-UNG2 interaction controls the levels of UNG2 and that class-switch recombination is defective in Fam72a-/- B cells due to the upregulation of UNG2. Moreover, we show that somatic hypermutation is reduced in Fam72a-/- B cells and that its pattern is skewed upon upregulation of UNG2. Our results are consistent with a model in which FAM72A interacts with UNG2 to control its physiological level by triggering its degradation, regulating the level of uracil excision and thus the balance between error-prone and error-free DNA repair. Our findings have potential implications for tumorigenesis, as reduced levels of UNG2 mediated by overexpression of Fam72a would shift the balance towards mutagenic DNA repair, rendering cells more prone to acquire mutations.


Assuntos
Linfócitos B , Reparo de Erro de Pareamento de DNA , Switching de Imunoglobulina , Região de Troca de Imunoglobulinas , Mutação , Hipermutação Somática de Imunoglobulina , Animais , Feminino , Masculino , Camundongos , Linfócitos B/metabolismo , Sistemas CRISPR-Cas/genética , Genoma/genética , Switching de Imunoglobulina/genética , Região de Troca de Imunoglobulinas/genética , Hipermutação Somática de Imunoglobulina/genética , Regulação para Cima , Uracila/metabolismo
2.
Genes Dev ; 30(19): 2152-2157, 2016 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-27798842

RESUMO

PAXX was identified recently as a novel nonhomologous end-joining DNA repair factor in human cells. To characterize its physiological roles, we generated Paxx-deficient mice. Like Xlf-/- mice, Paxx-/- mice are viable, grow normally, and are fertile but show mild radiosensitivity. Strikingly, while Paxx loss is epistatic with Ku80, Lig4, and Atm deficiency, Paxx/Xlf double-knockout mice display embryonic lethality associated with genomic instability, cell death in the central nervous system, and an almost complete block in lymphogenesis, phenotypes that closely resemble those of Xrcc4-/- and Lig4-/- mice. Thus, combined loss of Paxx and Xlf is synthetic-lethal in mammals.


Assuntos
Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário/genética , Mutações Sintéticas Letais/genética , Trissacarídeos/genética , Animais , Apoptose/genética , Proteínas de Ligação a DNA/metabolismo , Epistasia Genética , Instabilidade Genômica/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Tolerância a Radiação/genética , Trissacarídeos/metabolismo
3.
FASEB J ; 29(2): 455-63, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25376832

RESUMO

Within the bone marrow, the endosteal niche plays a crucial role in B-cell differentiation. Because spaceflight is associated with osteoporosis, we investigated whether changes in bone microstructure induced by a ground-based model of spaceflight, hind limb unloading (HU), could affect B lymphopoiesis. To this end, we analyzed both bone parameters and the frequency of early hematopoietic precursors and cells of the B lineage after 3, 6, 13, and 21 d of HU. We found that limb disuse leads to a decrease in both bone microstructure and the frequency of B-cell progenitors in the bone marrow. Although multipotent hematopoietic progenitors were not affected by HU, a decrease in B lymphopoiesis was observed as of the common lymphoid progenitor (CLP) stage with a major block at the progenitor B (pro-B) to precursor B (pre-B) cell transition (5- to 10-fold decrease). The modifications in B lymphopoiesis were similar to those observed in aged mice and, as with aging, decreased B-cell generation in HU mice was associated with reduced expression of B-cell transcription factors, early B-cell factor (EBF) and Pax5, and an alteration in STAT5-mediated IL-7 signaling. These findings demonstrate that mechanical unloading of hind limbs results in a decrease in early B-cell differentiation resembling age-related modifications in B lymphopoiesis.


Assuntos
Linfócitos B/citologia , Elevação dos Membros Posteriores/fisiologia , Linfopoese/fisiologia , Voo Espacial , Corticosteroides/metabolismo , Envelhecimento , Animais , Células da Medula Óssea/citologia , Remodelação Óssea , Diferenciação Celular , Linhagem da Célula , Citocinas/metabolismo , Células-Tronco Hematopoéticas/citologia , Imunoglobulinas/metabolismo , Interleucina-7/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Fator de Transcrição PAX5/metabolismo , Fator de Transcrição STAT5/metabolismo , Células-Tronco , Fatores de Tempo , Transativadores/metabolismo , Microtomografia por Raio-X
4.
J Cell Physiol ; 230(6): 1342-51, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25502698

RESUMO

Matrix proteins of the SIBLING family interact with bone cells, extracellular matrix and mineral and are thus in a key position to regulate the microenvironment of the bone tissue, including its hematopoietic component. In this respect, osteopontin (OPN) has been implicated in the hematopoietic stem cell (HSC) niche as negative regulator of the HSC function. We investigated the impact on hematopoietic regulation of the absence of the cognate bone sialoprotein (BSP). BSP knockout (-/-) mice display increased bone marrow cellularity, and an altered commitment of hematopoietic precursors to myeloid lineages, leading in particular to an increased frequency of monocyte/macrophage cells. The B cell pool is increased in -/- bone marrow, and its composition is shifted toward more mature lymphocyte stages. BSP-null mice display a decreased HSC fraction among LSK cells and a higher percentage of more committed progenitors as compared to +/+. The fraction of proliferating LSK progenitors is higher in -/- mice, and after PTH treatment the mutant HSC pool is lower than in +/+. Strikingly, circulating levels of OPN as well as its expression in the bone tissue are much higher in the -/-. Thus, a BSP-null bone microenvironment affects the hematopoietic system both quantitatively and qualitatively, in a manner in part opposite to the OPN knockout, suggesting that the effects might in part reflect the higher OPN expression in the absence of BSP.


Assuntos
Medula Óssea/metabolismo , Hematopoese/fisiologia , Sialoproteína de Ligação à Integrina/deficiência , Sialoproteína de Ligação à Integrina/metabolismo , Osteopontina/metabolismo , Animais , Osso e Ossos/metabolismo , Hematopoese/genética , Camundongos , Camundongos Nus , Osteogênese/fisiologia , Ativação Transcricional , Regulação para Cima
5.
Nat Commun ; 13(1): 3707, 2022 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-35764636

RESUMO

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.


Assuntos
Quebras de DNA de Cadeia Dupla , Recombinação V(D)J , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Switching de Imunoglobulina/genética , Recombinação V(D)J/genética
6.
Nat Commun ; 12(1): 5421, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34521823

RESUMO

MAD2L2 (REV7) plays an important role in DNA double-strand break repair. As a member of the shieldin complex, consisting of MAD2L2, SHLD1, SHLD2 and SHLD3, it controls DNA repair pathway choice by counteracting DNA end-resection. Here we investigated the requirements for shieldin complex assembly and activity. Besides a dimerization-surface, HORMA-domain protein MAD2L2 has the extraordinary ability to wrap its C-terminus around SHLD3, likely creating a very stable complex. We show that appropriate function of MAD2L2 within shieldin requires its dimerization, mediated by SHLD2 and accelerating MAD2L2-SHLD3 interaction. Dimerization-defective MAD2L2 impairs shieldin assembly and fails to promote NHEJ. Moreover, MAD2L2 dimerization, along with the presence of SHLD3, allows shieldin to interact with the TRIP13 ATPase, known to drive topological switches in HORMA-domain proteins. We find that appropriate levels of TRIP13 are important for proper shieldin (dis)assembly and activity in DNA repair. Together our data provide important insights in the dependencies for shieldin activity.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Proteínas de Ciclo Celular/genética , Reparo do DNA , Proteínas de Ligação a DNA/genética , DNA/genética , Proteínas Mad2/genética , ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Cisplatino/farmacologia , DNA/química , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Proteínas Mad2/química , Proteínas Mad2/metabolismo , Camundongos , Ftalazinas/farmacologia , Piperazinas/farmacologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
7.
Nat Commun ; 11(1): 5239, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33067475

RESUMO

The alternative non-homologous end-joining (NHEJ) pathway promotes DNA double-strand break (DSB) repair in cells deficient for NHEJ or homologous recombination, suggesting that it operates at all stages of the cell cycle. Here, we use an approach in which DNA breaks can be induced in G1 cells and their repair tracked, enabling us to show that joining of DSBs is not functional in G1-arrested XRCC4-deficient cells. Cell cycle entry into S-G2/M restores DSB repair by Pol θ-dependent and PARP1-independent alternative NHEJ with repair products bearing kilo-base long DNA end resection, micro-homologies and chromosome translocations. We identify a synthetic lethal interaction between XRCC4 and Pol θ under conditions of G1 DSBs, associated with accumulation of unresolved DNA ends in S-G2/M. Collectively, our results support the conclusion that the repair of G1 DSBs progressing to S-G2/M by alternative NHEJ drives genomic instability and represent an attractive target for future DNA repair-based cancer therapies.


Assuntos
Ciclo Celular , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fase G1 , Camundongos , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo
8.
Cell Rep ; 27(10): 2847-2858.e4, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-31167132

RESUMO

To reveal the relative contribution of the recombination activating gene (RAG)1/2 nuclease to lymphomagenesis, we conducted a genome-wide analysis of T cell lymphomas from p53-deficient mice expressing or lacking RAG2. We found that while p53-/- lymphoblastic T cells harbor primarily ectopic DNA deletions, Rag2-/-p53-/- T cell lymphomas display complex genomic rearrangements associated with amplification of the chromosomal location 9qA4-5.3. We show that this amplicon is generated by breakage-fusion-bridge during mitosis and arises distinctly in T cell lymphomas originating from an early progenitor stage. Notably, we report amplification of the corresponding syntenic region (11q23) in a subset of human leukemia leading to the overexpression of several cancer genes, including MLL/KMT2A. Our findings provide direct evidence that lymphocytes undergo malignant transformation through distinct genome architectural routes that are determined by both RAG-dependent and RAG-independent DNA damage and a block in cell development.


Assuntos
Dano ao DNA/genética , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica/genética , Linfoma de Células T/genética , Linfócitos T/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica/genética , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Linfoma de Células T/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , RNA-Seq , Linfócitos T/patologia , Translocação Genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
9.
Nat Commun ; 10(1): 4698, 2019 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-31619674

RESUMO

T helper 17 (Th17) cells have crucial functions in mucosal immunity and the pathogenesis of several chronic inflammatory diseases. The lineage-specific transcription factor, RORγt, encoded by the RORC gene modulates Th17 polarization and function, as well as thymocyte development. Here we define several regulatory elements at the human RORC locus in thymocytes and peripheral CD4+ T lymphocytes, with CRISPR/Cas9-guided deletion of these genomic segments supporting their role in RORγt expression. Mechanistically, T cell receptor stimulation induces cyclosporine A-sensitive histone modifications and P300/CBP acetylase recruitment at these elements in activated CD4+ T cells. Meanwhile, NFAT proteins bind to these regulatory elements and activate RORγt transcription in cooperation with NF-kB. Our data thus demonstrate that NFAT specifically regulate RORγt expression by binding to the RORC locus and promoting its permissive conformation.


Assuntos
Regulação da Expressão Gênica , Fatores de Transcrição NFATC/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Elementos Reguladores de Transcrição/genética , Células Th17/metabolismo , Timócitos/metabolismo , Ativação Transcricional , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Sistemas CRISPR-Cas , Linhagem da Célula , Citometria de Fluxo , Células HEK293 , Código das Histonas , Humanos , Células Jurkat , Células Th17/citologia , Timócitos/citologia , Fatores de Transcrição de p300-CBP/metabolismo
10.
Nat Commun ; 10(1): 5450, 2019 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-31772175

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

11.
Cell Death Differ ; 26(12): 2667-2681, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30996287

RESUMO

Poly (ADP-ribose) polymerase (PARP)-1 and PARP-2 regulate the function of various DNA-interacting proteins by transferring ADP-ribose emerging from catalytic cleavage of cellular ß-NAD+. Hence, mice lacking PARP-1 or PARP-2 show DNA perturbations ranging from altered DNA integrity to impaired DNA repair. These effects stem from the central role that PARP-1 and PARP-2 have on the cellular response to DNA damage. Failure to mount a proper response culminates in cell death. Accordingly, PARP inhibitors are emerging as promising drugs in cancer therapy. However, the full impact of these inhibitors on immunity, including B-cell antibody production, remains elusive. Given that mice carrying dual PARP-1 and PARP-2 deficiency develop early embryonic lethality, we crossed PARP-1-deficient mice with mice carrying a B-cell-conditional PARP-2 gene deletion. We found that the resulting dually PARP-1 and PARP-2-deficient mice had perturbed bone-marrow B-cell development as well as profound peripheral depletion of transitional and follicular but not marginal zone B-cells. Of note, bone-marrow B-cell progenitors and peripheral mature B-cells were conserved in mice carrying either PARP-1 or PARP-2 deficiency. In dually PARP-1 and PARP-2-deficient mice, B-cell lymphopenia was associated with increased DNA damage and accentuated death in actively proliferating B-cells. Moreover, dual PARP-1 and PARP-2 deficiency impaired antibody responses to T-independent carbohydrate but not to T-dependent protein antigens. Notwithstanding the pivotal role of PARP-1 and PARP-2 in DNA repair, combined PARP-1 and PARP-2 deficiency did not perturb the DNA-editing processes required for the generation of a protective antibody repertoire, including Ig V(D)J gene recombination and IgM-to-IgG class switching. These findings provide key information as to the potential impact of PARP inhibitors on humoral immunity, which will facilitate the development of safer PARP-targeting regimens against cancer.


Assuntos
Linfócitos B/citologia , Linfócitos B/metabolismo , Reparo do DNA , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Rearranjo Gênico , Genes de Imunoglobulinas , Humanos , Camundongos , Camundongos Knockout , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerases/deficiência , Poli(ADP-Ribose) Polimerases/genética
12.
BMC Genomics ; 9: 533, 2008 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-18992157

RESUMO

BACKGROUND: Tandemly Repeated DNA represents a large portion of the human genome, and accounts for a significant amount of copy number variation. Here we present a genome wide analysis of the largest tandem repeats found in the human genome sequence. RESULTS: Using Tandem Repeats Finder (TRF), tandem repeat arrays greater than 10 kb in total size were identified, and classified into simple sequence e.g. GAATG, classical satellites e.g. alpha satellite DNA, and locus specific VNTR arrays. Analysis of these large sequenced regions revealed that several "simple sequence" arrays actually showed complex domain and/or higher order repeat organization. Using additional methods, we further identified a total of 96 additional arrays with tandem repeat units greater than 2 kb (the detection limit of TRF), 53 of which contained genes or repeated exons. The overall size of an array of tandem 12 kb repeats which spanned a gap on chromosome 8 was found to be 600 kb to 1.7 Mbp in size, representing one of the largest non-centromeric arrays characterized. Several novel megasatellite tandem DNA families were observed that are characterized by repeating patterns of interspersed transposable elements that have expanded presumably by unequal crossing over. One of these families is found on 11 different chromosomes in >25 arrays, and represents one of the largest most widespread megasatellite DNA families. CONCLUSION: This study represents the most comprehensive genome wide analysis of large tandem repeats in the human genome, and will serve as an important resource towards understanding the organization and copy number variation of these complex DNA families.


Assuntos
Genoma Humano , Sequências de Repetição em Tandem/genética , Cromossomos Humanos/genética , DNA/química , DNA Satélite/genética , Dosagem de Genes , Variação Genética , Humanos , Hibridização in Situ Fluorescente , Retroelementos
13.
Nat Cell Biol ; 20(8): 954-965, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30022119

RESUMO

BRCA1 deficiencies cause breast, ovarian, prostate and other cancers, and render tumours hypersensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. To understand the resistance mechanisms, we conducted whole-genome CRISPR-Cas9 synthetic-viability/resistance screens in BRCA1-deficient breast cancer cells treated with PARP inhibitors. 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, 'Shieldin' (SHLD1/2), with FAM35A interacting with single-stranded DNA through its C-terminal oligonucleotide/oligosaccharide-binding fold region. We establish that Shieldin acts as the downstream effector of 53BP1/RIF1/MAD2L2 to promote DNA double-strand break (DSB) end-joining by restricting DSB resection and to counteract homologous recombination by antagonizing BRCA2/RAD51 loading in BRCA1-deficient cells. Notably, Shieldin inactivation further sensitizes 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.


Assuntos
Proteína BRCA1/genética , Neoplasias Ósseas/tratamento farmacológico , Neoplasias da Mama/tratamento farmacológico , Reparo do DNA por Junção de Extremidades , Resistencia a Medicamentos Antineoplásicos , Osteossarcoma/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas/metabolismo , Reparo de DNA por Recombinação , Animais , Proteína BRCA1/deficiência , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Cisplatino/farmacologia , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Células HEK293 , Humanos , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Camundongos , Complexos Multiproteicos , Osteossarcoma/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteínas/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Mech Ageing Dev ; 165(Pt A): 3-9, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-27863852

RESUMO

DNA double-strand breaks (DSBs) are commonly seen as lesions that threaten genome integrity and contribute to cancer and aging processes. However, in the context of antigen receptor gene assembly, known as V(D)J recombination, DSBs are obligatory intermediates that allow the establishment of genetic diversity and adaptive immunity. V(D)J recombination is initiated when the lymphoid-restricted recombination-activating genes RAG1 and RAG2 are expressed and form a site-specific endonuclease (the RAG nuclease or RAG recombinase). Here, we discuss the ability of the RAG nuclease to minimize the risks of genome disruption by coupling the breakage and repair steps of the V(D)J reaction. This implies that the RAG genes, derived from an ancient transposon, have undergone strong selective pressure to prohibit transposition in favor of promoting controlled DNA end joining in cis by the ubiquitous DNA damage response and DNA repair machineries. We also discuss the idea that, in addition to being essential for the rearrangement of antigen receptor genes, RAG-mediated DSBs could impact cellular processes and outcomes by affecting genetic and epigenetic programs.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica , Proteínas de Homeodomínio/metabolismo , Proteínas Nucleares/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Homeodomínio/genética , Humanos , Proteínas Nucleares/genética
15.
J Immunol Methods ; 451: 71-77, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28882611

RESUMO

Antigen receptor gene assembly is accomplished in developing lymphocytes by the V(D)J recombination reaction, which can be separated into two steps: DNA cleavage by the recombination-activating gene (RAG) nuclease and joining of DNA double strand breaks (DSBs) by components of the nonhomologous end joining (NHEJ) pathway. Deficiencies for NHEJ factors can result in immunodeficiency and a propensity to accumulate genomic instability, thus highlighting the importance of identifying all players in this process and deciphering their functions. Bcl2 transgenic v-Abl kinase-transformed pro-B cells provide a pseudo-physiological cellular system to study V(D)J recombination. Treatment of v-Abl/Bcl2 pro-B cells with the Abl kinase inhibitor Imatinib leads to G1 cell cycle arrest, the rapid induction of Rag1/2 gene expression and V(D)J recombination. In this system, the Bcl2 transgene alleviates Imatinib-induced apoptosis enabling the analysis of induced V(D)J recombination. Although powerful, the use of mouse models carrying the Bcl2 transgene for the generation of v-Abl pro-B cell lines is time and money consuming. Here, we describe a method for generating v-Abl/Bcl2 pro-B cell lines from wild type mice and for performing gene knock-out using episomal CRISPR/Cas9 targeting vectors. Using this approach, we generated distinct NHEJ-deficient pro-B cell lines and quantified V(D)J recombination levels in these cells. Furthermore, this methodology can be adapted to generate pro-B cell lines deficient for any gene suspected to play a role in V(D)J recombination, and more generally DSB repair.


Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Quebras de DNA de Cadeia Dupla , Edição de Genes/métodos , Células Precursoras de Linfócitos B/metabolismo , Reparo de DNA por Recombinação , Animais , Apoptose/efeitos dos fármacos , Proteínas Associadas a CRISPR/metabolismo , Linhagem Celular Transformada , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Genótipo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Mesilato de Imatinib/farmacologia , Camundongos Endogâmicos C57BL , Proteínas Oncogênicas v-abl/antagonistas & inibidores , Proteínas Oncogênicas v-abl/genética , Proteínas Oncogênicas v-abl/metabolismo , Fenótipo , Células Precursoras de Linfócitos B/efeitos dos fármacos , Células Precursoras de Linfócitos B/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Reparo de DNA por Recombinação/efeitos dos fármacos
16.
Nat Commun ; 7: 10529, 2016 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-26833222

RESUMO

XRCC4-like factor (XLF) functions in classical non-homologous end-joining (cNHEJ) but is dispensable for the repair of DNA double-strand breaks (DSBs) generated during V(D)J recombination. A long-standing hypothesis proposes that, in addition to its canonical nuclease activity, the RAG1/2 proteins participate in the DNA repair phase of V(D)J recombination. Here we show that in the context of RAG2 lacking the C-terminus domain (Rag2(c/c) mice), XLF deficiency leads to a profound lymphopenia associated with a severe defect in V(D)J recombination and, in the absence of p53, increased genomic instability at V(D)J sites. In addition, Rag2(c/c) XLF(-/-) p53(-/-) mice develop aggressive pro-B cell lymphomas bearing complex chromosomal translocations and gene amplifications involving Igh and c-myc/pvt1 loci. Our results reveal an unanticipated functional interplay between the RAG complex and XLF in repairing RAG-induced DSBs and maintaining genome integrity during antigen receptor gene assembly.


Assuntos
Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/fisiologia , Animais , Quebras de DNA , Proteínas de Ligação a DNA/genética , Instabilidade Genômica , Linfócitos/citologia , Linfócitos/fisiologia , Linfopenia/genética , Camundongos , Camundongos Knockout
17.
Cell Rep ; 16(11): 2967-2979, 2016 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-27601299

RESUMO

Paralog of XRCC4 and XLF (PAXX) is a member of the XRCC4 superfamily and plays a role in nonhomologous end-joining (NHEJ), a DNA repair pathway critical for lymphocyte antigen receptor gene assembly. Here, we find that the functions of PAXX and XLF in V(D)J recombination are masked by redundant joining activities. Thus, combined PAXX and XLF deficiency leads to an inability to join RAG-cleaved DNA ends. Additionally, we demonstrate that PAXX function in V(D)J recombination depends on its interaction with Ku. Importantly, we show that, unlike XLF, the role of PAXX during the repair of DNA breaks does not overlap with ATM and the RAG complex. Our findings illuminate the role of PAXX in V(D)J recombination and support a model in which PAXX and XLF function during NHEJ repair of DNA breaks, whereas XLF, the RAG complex, and the ATM-dependent DNA damage response promote end joining by stabilizing DNA ends.


Assuntos
Linfócitos B/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/química , Homologia de Sequência de Aminoácidos , Recombinação V(D)J/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sistemas CRISPR-Cas/genética , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Deleção de Genes , Edição de Genes , Rearranjo Gênico do Linfócito B , Imunoglobulinas/genética , Autoantígeno Ku/metabolismo , Modelos Biológicos , Proteínas Oncogênicas v-abl/metabolismo
19.
Aging Cell ; 9(3): 410-9, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20331442

RESUMO

Aging is accompanied by a reduction in the generation of B lymphocytes leading to impaired immune responses. In this study, we have investigated whether the decline in B lymphopoiesis is due to age-related defects in the hematopoietic stem cell compartment. The ability of hematopoietic stem cells from old mice to generate B cells, as measured in vitro, is decreased 2-5-fold, while myeloid potential remains unchanged. This age-related decrease in B-cell potential is more marked in common lymphoid progenitors (CLP) and was associated with reduced expression of the B-lineage specifying factors, EBF and Pax5. Notably, retrovirus-mediated expression of EBF complemented the age-related loss of B-cell potential in CLP isolated from old mice. Furthermore, transduction of CLP from old mice with a constitutively active form of STAT5 restored both EBF and Pax5 expression and increased B-cell potential. These results are consistent with a mechanism, whereby reduced expression of EBF with age decreases the frequency with which multipotent hematopoietic progenitors commit to a B-cell fate, without altering their potential to generate myeloid cells.


Assuntos
Envelhecimento , Linfócitos B/metabolismo , Diferenciação Celular , Células-Tronco Hematopoéticas/metabolismo , Transativadores/metabolismo , Animais , Linfócitos B/citologia , Linhagem da Célula , Regulação para Baixo , Células-Tronco Hematopoéticas/citologia , Camundongos , Camundongos Endogâmicos C57BL , Fator de Transcrição PAX5/genética , Fator de Transcrição PAX5/metabolismo , Fator de Transcrição STAT5/metabolismo , Transativadores/genética
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