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1.
Curr Biol ; 30(3): 408-420.e5, 2020 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-31902729

RESUMO

Meiotic sex chromosome inactivation (MSCI) is an essential event in the mammalian male germline. MSCI is directed by a DNA damage response (DDR) pathway centered on the phosphorylation of histone variant H2AX at serine 139 (termed γH2AX). The failure to initiate MSCI is linked to complete meiotic arrest and elimination of germ cells; however, the mechanisms underlying this arrest and elimination remain unknown. To address this question, we established a new separation-of-function mouse model for H2ax that shows specific and complete defects in MSCI. The genetic change is a point mutation in which another H2AX amino acid residue important in the DDR, tyrosine 142 (Y142), is converted to alanine (H2ax-Y142A). In H2ax-Y142A meiosis, the establishment of DDR signals on the chromosome-wide domain of the sex chromosomes is impaired. The initiation of MSCI is required for stage progression, which enables crossover formation, suggesting that the establishment of MSCI permits the timely progression of male meiosis. Our results suggest that normal meiotic progression requires the removal of ATR-mediated DDR signaling from autosomes. We propose a novel biological function for MSCI: the initiation of MSCI sequesters DDR factors from autosomes to the sex chromosomes at the onset of the pachytene stage, and the subsequent formation of an isolated XY nuclear compartment-the XY body-sequesters DDR factors to permit meiotic progression from the mid-pachytene stage onward. VIDEO ABSTRACT.

2.
Acta Neuropathol ; 139(1): 157-174, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31664505

RESUMO

In Neurofibromatosis type 1, NF1 gene mutations in Schwann cells (SC) drive benign plexiform neurofibroma (PNF), and no additional SC changes explain patient-to-patient variability in tumor number. Evidence from twin studies suggests that variable expressivity might be caused by unidentified modifier genes. Whole exome sequencing of SC and fibroblast DNA from the same resected PNFs confirmed biallelic SC NF1 mutations; non-NF1 somatic SC variants were variable and present at low read number. We identified frequent germline variants as possible neurofibroma modifier genes. Genes harboring variants were validated in two additional cohorts of NF1 patients and by variant burden test. Genes including CUBN, CELSR2, COL14A1, ATR and ATM also showed decreased gene expression in some neurofibromas. ATM-relevant DNA repair defects were also present in a subset of neurofibromas with ATM variants, and in some neurofibroma SC. Heterozygous ATM G2023R or homozygous S707P variants reduced ATM protein expression in heterologous cells. In mice, genetic Atm heterozygosity promoted Schwann cell precursor self-renewal and increased tumor formation in vivo, suggesting that ATM variants contribute to neurofibroma initiation. We identify germline variants, rare in the general population, overrepresented in NF1 patients with neurofibromas. ATM and other identified genes are candidate modifiers of PNF pathogenesis.

3.
Artigo em Inglês | MEDLINE | ID: mdl-31275435

RESUMO

XRCC2 is one of five somatic RAD51 paralogs, all of which have Walker A and B ATPase motifs. Each of the paralogs, including XRCC2, has a function in DNA double-strand break repair by homologous recombination (HR). However, their individual roles are not as well understood as that of RAD51 itself. The XRCC2 protein forms a complex (BCDX2) with three other RAD51 paralogs, RAD51B, RAD51C and RAD51D. It is believed that the BCDX2 complex mediates HR downstream of BRCA2 but upstream of RAD51, as XRCC2 is involved in the assembly of RAD51 into DNA damage foci. XRCC2 can bind DNA and, along with RAD51D, can promote homologous pairing in vitro. Consistent with its role in HR, XRCC2-deficient cells have increased levels of spontaneous chromosome instability, and exhibit hypersensitivity to DNA interstrand crosslinking agents such as mitomycin C and cisplatin as well as ionizing radiation, alkylating agents and aldehydes. XRCC2 also functions in promoting DNA replication and chromosome segregation. Biallelic mutation of XRCC2 (FANCU) causes the FA-U subtype of FA, while heterozygosity for deleterious mutations in XRCC2 may be associated with an increased breast cancer risk. XRCC2 appears to function 'downstream' in the FA pathway, since it is not required for FANCD2 monoubiquitination, which is the central step in the FA pathway. Clinically, the only known FA-U patient in the world exhibits severe congenital abnormalities, but had not developed, by seven years of age, the bone marrow failure and cancer that are often seen in patients from other FA complementation groups.

4.
Nat Commun ; 10(1): 2866, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253785

RESUMO

Precise genome editing/correction of DNA double-strand breaks (DSBs) induced by CRISPR-Cas9 by homology-dependent repair (HDR) is limited by the competing error-prone non-homologous end-joining (NHEJ) DNA repair pathway. Here, we define a safer and efficient system that promotes HDR-based precise genome editing, while reducing NHEJ locally, only at CRISPR-Cas9-induced DSBs. We fused a dominant-negative mutant of 53BP1, DN1S, to Cas9 nucleases, and the resulting Cas9-DN1S fusion proteins significantly block NHEJ events specifically at Cas9 cut sites and improve HDR frequency; HDR frequency reached 86% in K562 cells. Cas9-DN1S protein maintains this effect in different human cell types, including leukocyte adhesion deficiency (LAD) patient-derived immortalized B lymphocytes, where nearly 70% of alleles were repaired by HDR and 7% by NHEJ. Our CRISPR-Cas9-DN1S system is clinically relevant to improve the efficiencies of precise gene correction/insertion, significantly reducing error-prone NHEJ events at the nuclease cleavage site, while avoiding the unwanted effects of global NHEJ inhibition.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Reparo do DNA , Edição de Genes/métodos , Reparo de DNA por Recombinação/fisiologia , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Humanos , Mutagênese Insercional , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética
5.
Leukemia ; 33(11): 2585-2598, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30967619

RESUMO

SETD2, an epigenetic tumor suppressor, is frequently mutated in MLL-rearranged (MLLr) leukemia and relapsed acute leukemia (AL). To clarify the impact of SETD2 mutations on chemotherapy sensitivity in MLLr leukemia, two loss-of-function (LOF) Setd2-mutant alleles (Setd2F2478L/WT or Setd2Ex6-KO/WT) were generated and introduced, respectively, to the Mll-Af9 knock-in leukemia mouse model. Both alleles cooperated with Mll-Af9 to accelerate leukemia development that resulted in resistance to standard Cytarabine-based chemotherapy. Mechanistically, Setd2-mutant leukemic cells showed downregulated signaling related to cell cycle progression, S, and G2/M checkpoint regulation. Thus, after Cytarabine treatment, Setd2-mutant leukemic cells exit from the S phase and progress to the G2/M phase. Importantly, S and G2/M cell cycle checkpoint inhibition could resensitize the Mll-Af9/Setd2 double-mutant cells to standard chemotherapy by causing DNA replication collapse, mitotic catastrophe, and increased cell death. These findings demonstrate that LOF SETD2 mutations confer chemoresistance on AL to DNA-damaging treatment by S and G2/M checkpoint defects. The combination of S and G2/M checkpoint inhibition with chemotherapy can be explored as a promising therapeutic strategy by exploiting their unique vulnerability and resensitizing chemoresistant AL with SETD2 or SETD2-like epigenetic mutations.

6.
J Pers Med ; 9(1)2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30832243

RESUMO

Breast cancer screening modalities and guidelines continue to evolve and are increasingly based on risk factors, including genetic risk and a personal or family history of cancer. Here, we review genetic testing of high-penetrance hereditary breast and ovarian cancer genes, including BRCA1 and BRCA2, for the purpose of identifying high-risk individuals who would benefit from earlier screening and more sensitive methods such as magnetic resonance imaging. We also consider risk-based screening in the general population, including whether every woman should be genetically tested for high-risk genes and the potential use of polygenic risk scores. In addition to enabling early detection, the results of genetic screens of breast cancer susceptibility genes can be utilized to guide decision-making about when to elect prophylactic surgeries that reduce cancer risk and the choice of therapeutic options. Variants of uncertain significance, especially missense variants, are being identified during panel testing for hereditary breast and ovarian cancer. A finding of a variant of uncertain significance does not provide a basis for increased cancer surveillance or prophylactic procedures. Given that variant classification is often challenging, we also consider the role of multifactorial statistical analyses by large consortia and functional tests for this purpose.

7.
Nucleic Acids Res ; 47(7): 3503-3520, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30715513

RESUMO

The primary function of the UBE2T ubiquitin conjugase is in the monoubiquitination of the FANCI-FANCD2 heterodimer, a central step in the Fanconi anemia (FA) pathway. Genetic inactivation of UBE2T is responsible for the phenotypes of FANCT patients; however, a FANCT patient carrying a maternal duplication and a paternal deletion in the UBE2T loci displayed normal peripheral blood counts and UBE2T protein levels in B-lymphoblast cell lines. To test whether reversion by recombination between UBE2T AluYa5 elements could have occurred in the patient's hematopoietic stem cells despite the defects in homologous recombination (HR) in FA cells, we constructed HeLa cell lines containing the UBE2T AluYa5 elements and neighboring intervening sequences flanked by fluorescent reporter genes. Introduction of a DNA double strand break in the model UBE2T locus in vivo promoted single strand annealing (SSA) between proximal Alu elements and deletion of the intervening color marker gene, recapitulating the reversion of the UBE2T duplication in the FA patient. To test whether UBE2T null cells retain HR activity, the UBE2T genes were knocked out in HeLa cells and U2OS cells. CRISPR/Cas9-mediated genetic knockout of UBE2T only partially reduced HR, demonstrating that UBE2T-independent pathways can compensate for the recombination defect in UBE2T/FANCT null cells.


Assuntos
Elementos Alu/genética , Anemia de Fanconi/genética , Recombinação Homóloga/genética , Enzimas de Conjugação de Ubiquitina/genética , Sistemas CRISPR-Cas/genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Anemia de Fanconi/patologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Deleção de Genes , Duplicação Gênica/genética , Células HeLa , Células-Tronco Hematopoéticas/metabolismo , Humanos , Herança Materna/genética , Herança Paterna/genética
8.
Hepatology ; 69(6): 2608-2622, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30693543

RESUMO

Replication fork stability during DNA replication is vital for maintenance of genomic stability and suppression of cancer development in mammals. ATR (ataxia-telangiectasia mutated [ATM] and RAD3-related) is a master regulatory kinase that activates the replication stress response to overcome replication barriers. Although many downstream effectors of ATR have been established, the upstream regulators of ATR and the effect of such regulation on liver cancer remain unclear. The ubiquitin conjugase BRUCE (BIR Repeat containing Ubiquitin-Conjugating Enzyme) is a guardian of chromosome integrity and activator of ATM signaling, which promotes DNA double-strand break repair through homologous recombination. Here we demonstrate the functions for BRUCE in ATR activation in vitro and liver tumor suppression in vivo. BRUCE is recruited to induced DNA damage sites. Depletion of BRUCE inhibited multiple ATR-dependent signaling events during replication stress, including activation of ATR itself, phosphorylation of its downstream targets CHK1 and RPA, and the mono-ubiquitination of FANCD2. Consequently, BRUCE deficiency resulted in stalled DNA replication forks and increased firing of new replication origins. The in vivo impact of BRUCE loss on liver tumorigenesis was determined using the hepatocellular carcinoma model induced by genotoxin diethylnitrosamine. Liver-specific knockout of murine Bruce impaired ATR activation and exacerbated inflammation, fibrosis and hepatocellular carcinoma, which exhibited a trabecular architecture, closely resembling human hepatocellular carcinoma (HCC). In humans, the clinical relevance of BRUCE down-regulation in liver disease was found in hepatitis, cirrhosis, and HCC specimens, and deleterious somatic mutations of the Bruce gene was found in human hepatocellular carcinoma in the Cancer Genome Atlas database. Conclusion: These findings establish a BRUCE-ATR signaling axis in accurate DNA replication and suppression of liver cancer in mice and humans and provides a clinically relevant HCC mouse model.

9.
Haematologica ; 103(7): 1110-1123, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29650642

RESUMO

SET domain containing 2 (Setd2), encoding a histone methyltransferase, is associated with many hematopoietic diseases when mutated. By generating a novel exon 6 conditional knockout mouse model, we describe an essential role of Setd2 in maintaining the adult hematopoietic stem cells. Loss of Setd2 results in leukopenia, anemia, and increased platelets accompanied by hypocellularity, erythroid dysplasia, and mild fibrosis in bone marrow. Setd2 knockout mice show significantly decreased hematopoietic stem and progenitor cells except for erythroid progenitors. Setd2 knockout hematopoietic stem cells fail to establish long-term bone marrow reconstitution after transplantation because of the loss of quiescence, increased apoptosis, and reduced multiple-lineage terminal differentiation potential. Bioinformatic analysis revealed that the hematopoietic stem cells exit from quiescence and commit to differentiation, which lead to hematopoietic stem cell exhaustion. Mechanistically, we attribute an important Setd2 function in murine adult hematopoietic stem cells to the inhibition of the Nsd1/2/3 transcriptional complex, which recruits super elongation complex and controls RNA polymerase II elongation on a subset of target genes, including Myc Our results reveal a critical role of Setd2 in regulating quiescence and differentiation of hematopoietic stem cells through restricting the NSDs/SEC mediated RNA polymerase II elongation.


Assuntos
Diferenciação Celular/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Histona-Lisina N-Metiltransferase/genética , RNA Polimerase II/metabolismo , Fase de Repouso do Ciclo Celular/genética , Alelos , Animais , Apoptose/genética , Biomarcadores , Biópsia , Linhagem da Célula/genética , Proliferação de Células , Autorrenovação Celular/genética , Técnicas de Silenciamento de Genes , Hematopoese , Histona-Lisina N-Metiltransferase/metabolismo , Imuno-Histoquímica , Imunofenotipagem , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Elongação Traducional da Cadeia Peptídica , Fosforilação
10.
PLoS Genet ; 14(2): e1007233, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29462142

RESUMO

The sex chromosomes are enriched with germline genes that are activated during the late stages of spermatogenesis. Due to meiotic sex chromosome inactivation (MSCI), these sex chromosome-linked genes must escape silencing for activation in spermatids, thereby ensuring their functions for male reproduction. RNF8, a DNA damage response protein, and SCML2, a germline-specific Polycomb protein, are two major, known regulators of this process. Here, we show that RNF8 and SCML2 cooperate to regulate ubiquitination during meiosis, an early step to establish active histone modifications for subsequent gene activation. Double mutants of Rnf8 and Scml2 revealed that RNF8-dependent monoubiquitination of histone H2A at Lysine 119 (H2AK119ub) is deubiquitinated by SCML2, demonstrating interplay between RNF8 and SCML2 in ubiquitin regulation. Additionally, we identify distinct functions of RNF8 and SCML2 in the regulation of ubiquitination: SCML2 deubiquitinates RNF8-independent H2AK119ub but does not deubiquitinate RNF8-dependent polyubiquitination. RNF8-dependent polyubiquitination is required for the establishment of H3K27 acetylation, a marker of active enhancers, while persistent H2AK119ub inhibits establishment of H3K27 acetylation. Following the deposition of H3K27 acetylation, H3K4 dimethylation is established as an active mark on poised promoters. Together, we propose a model whereby regulation of ubiquitin leads to the organization of poised enhancers and promoters during meiosis, which induce subsequent gene activation from the otherwise silent sex chromosomes in postmeiotic spermatids.


Assuntos
Histonas/metabolismo , Proteínas do Grupo Polycomb/fisiologia , Cromossomos Sexuais/genética , Ativação Transcricional/genética , Ubiquitina-Proteína Ligases/fisiologia , Ubiquitinação/genética , Acetilação , Animais , Feminino , Masculino , Meiose/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cromossomos Sexuais/metabolismo , Espermátides/fisiologia , Espermatogênese/genética
11.
Hum Mol Genet ; 27(7): 1136-1149, 2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29360988

RESUMO

The continuity of life depends on mechanisms in the germline that ensure the integrity of the genome. The DNA damage response/checkpoint kinases ATM and ATR are essential signaling factors in the germline. However, it remains unknown how a downstream transducer, Checkpoint Kinase 1 (CHEK1 or CHK1), mediates signaling in the male germline. Here, we show that CHEK1 has distinct functions in both the mitotic and meiotic phases of the male germline in mice. In the mitotic phase, CHEK1 is required for the resumption of prospermatogonia proliferation after birth and the maintenance of spermatogonia. In the meiotic phase, we uncovered two functions for CHEK1: one is the stage-specific attenuation of DNA damage signaling on autosomes, and the other is coordination of meiotic stage progression. On autosomes, the loss of CHEK1 delays the removal of DNA damage signaling that manifests as phosphorylation of histone variant H2AX at serine 139 (γH2AX). Importantly, CHEK1 does not have a direct function in meiotic sex chromosome inactivation (MSCI), an essential event in male meiosis, in which ATR is a key regulator. Thus, the functions of ATR and CHEK1 are uncoupled in MSCI, in contrast to their roles in DNA damage signaling in somatic cells. Our study reveals stage-specific functions for CHEK1 that ensure the integrity of the male germline.


Assuntos
Quinase 1 do Ponto de Checagem/metabolismo , Dano ao DNA , Meiose , Transdução de Sinais , Espermatogônias/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quinase 1 do Ponto de Checagem/genética , Masculino , Camundongos , Camundongos Knockout , Espermatogônias/citologia
12.
Atlas Genet Cytogenet Oncol Haematol ; 22(12): 484-490, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31413733

RESUMO

PALB2 (Partner and Localizer of BRCA2) was first identified as a BRCA2-interacting protein. Subsequently, PALB2 has been recognized as a cog in the cellular machinery for DNA repair by homologous recombination (HR). PALB2 also mediates S and G2 DNA damage checkpoints, and has an apparent function in protecting transcriptionally active genes from genotoxic stress. PALB2 also interacts with, is localized by, and functions downstream of BRCA1. Further, PALB2 interacts with other essential effectors of HR, including RAD51 and RAD51C, as well as BRCA2. Consistent with its function in HR and its interaction with key HR proteins, PALB2-deficient cells are hypersensitive to ionizing radiation and DNA interstrand crosslinking agents such as mitomycin C and cisplatin. Mechanistically, PALB2 is required for HR by mediating the recruitment of BRCA2 and the RAD51 recombinase to sites of DNA damage. Similar to bi-allelic loss-of-function mutations of BRCA1, BRCA2, RAD51 and RAD51C, bi-allelic mutations in PALB2 cause Fanconi anemia (FA), a rare childhood disorder which is associated with progressive bone marrow failure, congenital anomalies, and a predisposition to leukemia and solid tumors. Due to their close functional relationship, bi-allelic mutations of PALB2 and BRCA2 cause particularly severe forms of FA, called FANCN and FANCD1, both characterized by severe congenital abnormalities and very early onset of various cancers. This includes acute leukemias, Wilms tumor, medulloblastoma and neuroblastomas. Also, heterozygous germ-line mutations of PALB2, like mutations in several other essential HR genes listed above, yield an increased susceptibility to breast and pancreatic cancer.

13.
J Med Genet ; 54(11): 721-731, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28866612

RESUMO

Mutation of BRCA1 and BRCA2 is the most common cause of inherited breast and ovarian cancer. Genetic screens to detect carriers of variants can aid in cancer prevention by identifying individuals with a greater cancer risk and can potentially be used to predict the responsiveness of tumours to therapy. Frequently, classification cannot be performed based on traditional approaches such as segregation analyses, including for many missense variants, which are therefore referred to as variants of uncertain significance (VUS). Functional assays provide an important alternative for classification of BRCA1 and BRCA2 VUS. As reviewed here, both of these tumour suppressors promote the maintenance of genome stability via homologous recombination. Thus, related assays may be particularly relevant to cancer risk. Progress in implementing functional assays to assess missense variants of BRCA1 and BRCA2 is considered here, along with current limitations and the path to more impactful assay systems. While functional assays have been developed to independently evaluate BRCA1 and BRCA2 VUS, high-throughput assays with sufficient sensitivity to characterise the large number of identified variants are lacking. Additionally, because of relatively low conservation of certain domains of BRCA1, and of BRCA2, between humans and rodents, heterologous expression in rodent cells may have limited reliability or capacity to assess variants present throughout either protein. Moving forward, it will be important to perform assays in human cell lines with relevance to particular tumour types, and to strengthen risk predictions based on multifactorial statistical analyses that also include available data on cosegregation and tumour pathology.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , Triagem de Portadores Genéticos/métodos , Neoplasias Ovarianas/genética , Linhagem Celular , Reparo do DNA , Feminino , Mutação em Linhagem Germinativa , Humanos , Mutação de Sentido Incorreto , Determinação de Necessidades de Cuidados de Saúde
14.
Genes Dev ; 31(16): 1693-1703, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28924034

RESUMO

During spermatogenesis, a large number of germline genes essential for male fertility are coordinately activated. However, it remains unknown how timely activation of this group of germline genes is accomplished. Here we show that Polycomb-repressive complex 1 (PRC1) directs timely activation of germline genes during spermatogenesis. Inactivation of PRC1 in male germ cells results in the gradual loss of a stem cell population and severe differentiation defects, leading to male infertility. In the stem cell population, RNF2, the dominant catalytic subunit of PRC1, activates transcription of Sall4, which codes for a transcription factor essential for subsequent spermatogenic differentiation. Furthermore, RNF2 and SALL4 together occupy transcription start sites of germline genes in the stem cell population. Once differentiation commences, these germline genes are activated to enable the progression of spermatogenesis. Our study identifies a novel mechanism by which Polycomb directs the developmental process by activating a group of lineage-specific genes.


Assuntos
Complexo Repressor Polycomb 1/fisiologia , Espermatogênese/genética , Ativação Transcricional , Animais , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Masculino , Camundongos , Camundongos Transgênicos , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Espermatogônias/citologia , Espermatogônias/metabolismo , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
15.
Proc Natl Acad Sci U S A ; 114(24): E4782-E4791, 2017 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-28533414

RESUMO

Antigen-activated lymphocytes undergo extraordinarily rapid cell division in the course of immune responses. We hypothesized that this unique aspect of lymphocyte biology leads to unusual genomic stress in recently antigen-activated lymphocytes and that targeted manipulation of DNA damage-response (DDR) signaling pathways would allow for selective therapeutic targeting of pathological T cells in disease contexts. Consistent with these hypotheses, we found that activated mouse and human T cells display a pronounced DDR in vitro and in vivo. Upon screening a variety of small-molecule compounds, we found that potentiation of p53 (via inhibition of MDM2) or impairment of cell cycle checkpoints (via inhibition of CHK1/2 or WEE1) led to the selective elimination of activated, pathological T cells in vivo. The combination of these strategies [which we termed "p53 potentiation with checkpoint abrogation" (PPCA)] displayed therapeutic benefits in preclinical disease models of hemophagocytic lymphohistiocytosis and multiple sclerosis, which are driven by foreign antigens or self-antigens, respectively. PPCA therapy targeted pathological T cells but did not compromise naive, regulatory, or quiescent memory T-cell pools, and had a modest nonimmune toxicity profile. Thus, PPCA is a therapeutic modality for selective, antigen-specific immune modulation with significant translational potential for diverse immune-mediated diseases.


Assuntos
Dano ao DNA/imunologia , Doenças do Sistema Imunitário/terapia , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/imunologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/terapia , Etoposídeo/administração & dosagem , Humanos , Doenças do Sistema Imunitário/imunologia , Ativação Linfocitária , Linfo-Histiocitose Hemofagocítica/imunologia , Linfo-Histiocitose Hemofagocítica/terapia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Esclerose Múltipla/imunologia , Esclerose Múltipla/terapia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/imunologia , Transdução de Sinais/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/patologia , Proteína Supressora de Tumor p53/imunologia
16.
Sci Rep ; 7: 45626, 2017 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-28378742

RESUMO

Fancd2 is a component of the Fanconi anemia (FA) DNA repair pathway, which is frequently found defective in human cancers. The full repertoire of Fancd2 functions in normal development and tumorigenesis remains to be determined. Here we developed a Flag- and hemagglutinin-tagged Fancd2 knock-in mouse strain that allowed a high throughput mass spectrometry approach to search for Fancd2-binding proteins in different mouse organs. In addition to DNA repair partners, we observed that many Fancd2-interacting proteins are mitochondrion-specific. Fancd2 localizes in the mitochondrion and associates with the nucleoid complex components Atad3 and Tufm. The Atad3-Tufm complex is disrupted in Fancd2-/- mice and those deficient for the FA core component Fanca. Fancd2 mitochondrial localization requires Atad3. Collectively, these findings provide evidence for Fancd2 as a crucial regulator of mitochondrion biosynthesis, and of a molecular link between FA and mitochondrial homeostasis.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Fator Tu de Elongação de Peptídeos/metabolismo , Mapas de Interação de Proteínas , Animais , Técnicas de Introdução de Genes , Espectrometria de Massas , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica
17.
Sci Rep ; 7: 44662, 2017 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-28317934

RESUMO

DEK is a highly conserved chromatin-bound protein whose upregulation across cancer types correlates with genotoxic therapy resistance. Loss of DEK induces genome instability and sensitizes cells to DNA double strand breaks (DSBs), suggesting defects in DNA repair. While these DEK-deficiency phenotypes were thought to arise from a moderate attenuation of non-homologous end joining (NHEJ) repair, the role of DEK in DNA repair remains incompletely understood. We present new evidence demonstrating the observed decrease in NHEJ is insufficient to impact immunoglobulin class switching in DEK knockout mice. Furthermore, DEK knockout cells were sensitive to apoptosis with NHEJ inhibition. Thus, we hypothesized DEK plays additional roles in homologous recombination (HR). Using episomal and integrated reporters, we demonstrate that HR repair of conventional DSBs is severely compromised in DEK-deficient cells. To define responsible mechanisms, we tested the role of DEK in the HR repair cascade. DEK-deficient cells were impaired for γH2AX phosphorylation and attenuated for RAD51 filament formation. Additionally, DEK formed a complex with RAD51, but not BRCA1, suggesting a potential role regarding RAD51 filament formation, stability, or function. These findings define DEK as an important and multifunctional mediator of HR, and establish a synthetic lethal relationship between DEK loss and NHEJ inhibition.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Recombinação Homóloga , Proteínas Oncogênicas/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Feminino , Células HeLa , Histonas/metabolismo , Recombinação Homóloga/efeitos dos fármacos , Recombinação Homóloga/efeitos da radiação , Humanos , Masculino , Camundongos Knockout , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/efeitos da radiação , Inibidores de Proteínas Quinases/farmacologia , Rad51 Recombinase/metabolismo , Radiação Ionizante , Proteína de Replicação A/metabolismo
18.
Chromosoma ; 126(3): 417-430, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-27277787

RESUMO

Fanconi anemia (FA) is a chromosome instability syndrome and the 20 identified FA proteins are organized into two main arms which are thought to function at distinct steps in the repair of DNA interstrand crosslinks (ICLs). These two arms include the upstream FA pathway, which culminates in the monoubiquitination of FANCD2 and FANCI, and downstream breast cancer (BRCA)-associated proteins that interact in protein complexes. How, and whether, these two groups of FA proteins are integrated is unclear. Here, we show that FANCD2 and PALB2, as indicators of the upstream and downstream arms, respectively, colocalize independently of each other in response to DNA damage induced by mitomycin C (MMC). We also show that ubiquitin chains are induced by MMC and colocalize with both FANCD2 and PALB2. Our finding that the RNF8 E3 ligase has a role in recruiting FANCD2 and PALB2 also provides support for the hypothesis that the two branches of the FA-BRCA pathway are coordinated by ubiquitin signaling. Interestingly, we find that the RNF8 partner, MDC1, as well as the ubiquitin-binding protein, RAP80, specifically recruit PALB2, while a different ubiquitin-binding protein, FAAP20, functions only in the recruitment of FANCD2. Thus, FANCD2 and PALB2 are not recruited in a single linear pathway, rather we define how their localization is coordinated and integrated by a network of ubiquitin-related proteins. We propose that such regulation may enable upstream and downstream FA proteins to act at distinct steps in the repair of ICLs.


Assuntos
Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação N da Anemia de Fanconi/metabolismo , Anemia de Fanconi/metabolismo , Ubiquitina/metabolismo , Dano ao DNA , Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Humanos , Ligação Proteica , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
19.
Cell Rep ; 17(4): 1141-1157, 2016 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-27760317

RESUMO

Precise epigenetic regulation of the sex chromosomes is vital for the male germline. Here, we analyze meiosis in eight mouse models deficient for various DNA damage response (DDR) factors, including Fanconi anemia (FA) proteins. We reveal a network of FA and DDR proteins in which FA core factors FANCA, FANCB, and FANCC are essential for FANCD2 foci formation, whereas BRCA1 (FANCS), MDC1, and RNF8 are required for BRCA2 (FANCD1) and SLX4 (FANCP) accumulation on the sex chromosomes during meiosis. In addition, FA proteins modulate distinct histone marks on the sex chromosomes: FA core proteins and FANCD2 regulate H3K9 methylation, while FANCD2 and RNF8 function together to regulate H3K4 methylation independently of FA core proteins. Our data suggest that RNF8 integrates the FA-BRCA pathway. Taken together, our study reveals distinct functions for FA proteins and illuminates the male sex chromosomes as a model to dissect the function of the FA-BRCA pathway.


Assuntos
Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Histonas/metabolismo , Meiose , Processamento de Proteína Pós-Traducional , Animais , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Quebras de DNA de Cadeia Dupla , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisina/metabolismo , Masculino , Metilação , Camundongos , Rad51 Recombinase/metabolismo , Recombinação Genética/genética , Cromossomos Sexuais/metabolismo , Fatores de Tempo
20.
J Med Genet ; 53(10): 672-680, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27208205

RESUMO

BACKGROUND: Fanconi anaemia (FA) is a heterogeneous inherited disorder clinically characterised by progressive bone marrow failure, congenital anomalies and a predisposition to malignancies. OBJECTIVE: Determine, based on correction of cellular phenotypes, whether XRCC2 is a FA gene. METHODS: Cells (900677A) from a previously identified patient with biallelic mutation of XRCC2, among other mutations, were genetically complemented with wild-type XRCC2. RESULTS: Wild-type XRCC2 corrects each of three phenotypes characteristic of FA cells, all related to the repair of DNA interstrand crosslinks, including increased sensitivity to mitomycin C (MMC), chromosome breakage and G2-M accumulation in the cell cycle. Further, the p.R215X mutant of XRCC2, which is harboured by the patient, is unstable. This provides an explanation for the pathogenesis of this mutant, as does the fact that 900677A cells have reduced levels of other proteins in the XRCC2-RAD51B-C-D complex. Also, FANCD2 monoubiquitination and foci formation, but not assembly of RAD51 foci, are normal in 900677A cells. Thus, XRCC2 acts late in the FA-BRCA pathway as also suggested by hypersensitivity of 900677A cells to ionising radiation. These cells also share milder sensitivities towards olaparib and formaldehyde with certain other FA cells. CONCLUSIONS: XRCC2/FANCU is a FA gene, as is another RAD51 paralog gene, RAD51C/FANCO. Notably, similar to a subset of FA genes that act downstream of FANCD2, biallelic mutation of XRCC2/FANCU has not been associated with bone marrow failure. Taken together, our results yield important insights into phenotypes related to FA and its genetic origins.


Assuntos
Adutos de DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/genética , Anemia de Fanconi/metabolismo , Mutação , Criança , Dano ao DNA , Anemia de Fanconi/genética , Humanos , Masculino
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