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1.
STAR Protoc ; 2(1): 100378, 2021 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-33778777

RESUMO

Micronuclei are aberrant nuclear compartments that form when chromosomes or chromosome fragments fail to incorporate into a primary nucleus during mitotic exit. Ruptures at the micronuclear envelope are associated with DNA damage and activation of immune sensing pathways. To gain insights into these processes, we have developed a method to purify ruptured micronuclei. This method paves the way toward understanding the consequences of micronuclear envelope rupture. For complete details on the use and execution of this protocol, please refer to Mohr et al. (2021).

2.
Mol Cell ; 81(4): 724-738.e9, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33476576

RESUMO

Micronuclei are aberrant nuclear compartments that can form as a result of chromosome mis-segregation. Frequent loss of micronuclear envelope integrity exposes DNA to the cytoplasm, leading to chromosome fragmentation and immune activation. Here, we use micronuclei purification to show that the endoplasmic reticulum (ER)-associated nuclease TREX1 inhibits cGAS activation at micronuclei by degrading micronuclear DNA upon micronuclear envelope rupture. We demonstrate that the ER accesses ruptured micronuclei and plays a critical role in enabling TREX1 nucleolytic attack. TREX1 mutations, previously implicated in immune disease, untether TREX1 from the ER, disrupt TREX1 localization to micronuclei, diminish micronuclear DNA damage, and enhance cGAS activation. These results establish ER-directed resection of micronuclear DNA by TREX1 as a critical regulator of cytosolic DNA sensing in chromosomally unstable cells and provide a mechanistic basis for the importance of TREX1 ER tethering in preventing autoimmunity.


Assuntos
Dano ao DNA , Retículo Endoplasmático/metabolismo , Exodesoxirribonucleases/metabolismo , Micronúcleos com Defeito Cromossômico , Mutação , Nucleotidiltransferases/metabolismo , Fosfoproteínas/metabolismo , Retículo Endoplasmático/genética , Ativação Enzimática/genética , Exodesoxirribonucleases/genética , Células HEK293 , Humanos , Nucleotidiltransferases/genética , Fosfoproteínas/genética , Transporte Proteico/genética
3.
Nat Genet ; 52(9): 884-890, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32719516

RESUMO

Chromothripsis and kataegis are frequently observed in cancer and may arise from telomere crisis, a period of genome instability during tumorigenesis when depletion of the telomere reserve generates unstable dicentric chromosomes1-5. Here we examine the mechanism underlying chromothripsis and kataegis by using an in vitro telomere crisis model. We show that the cytoplasmic exonuclease TREX1, which promotes the resolution of dicentric chromosomes4, plays a prominent role in chromothriptic fragmentation. In the absence of TREX1, the genome alterations induced by telomere crisis primarily involve breakage-fusion-bridge cycles and simple genome rearrangements rather than chromothripsis. Furthermore, we show that the kataegis observed at chromothriptic breakpoints is the consequence of cytosine deamination by APOBEC3B. These data reveal that chromothripsis and kataegis arise from a combination of nucleolytic processing by TREX1 and cytosine editing by APOBEC3B.


Assuntos
Citidina Desaminase/genética , Exodesoxirribonucleases/genética , Fosfoproteínas/genética , Telômero/genética , Linhagem Celular Tumoral , Cromotripsia , Citosina Desaminase/genética , Instabilidade Genômica/genética , Humanos , Mutação/genética , Neoplasias/genética , Células U937
4.
Sci Adv ; 6(15): eaay3511, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32300648

RESUMO

Dyskeratosis congenita is a cancer-prone inherited bone marrow failure syndrome caused by telomere dysfunction. A mouse model recently suggested that p53 regulates telomere metabolism, but the clinical relevance of this finding remained uncertain. Here, a germline missense mutation of MDM4, a negative regulator of p53, was found in a family with features suggestive of dyskeratosis congenita, e.g., bone marrow hypocellularity, short telomeres, tongue squamous cell carcinoma, and acute myeloid leukemia. Using a mouse model, we show that this mutation (p.T454M) leads to increased p53 activity, decreased telomere length, and bone marrow failure. Variations in p53 activity markedly altered the phenotype of Mdm4 mutant mice, suggesting an explanation for the variable expressivity of disease symptoms in the family. Our data indicate that a germline activation of the p53 pathway may cause telomere dysfunction and point to polymorphisms affecting this pathway as potential genetic modifiers of telomere biology and bone marrow function.


Assuntos
Proteínas de Ciclo Celular/genética , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Proteínas Proto-Oncogênicas/genética , Homeostase do Telômero/genética , Telômero/genética , Telômero/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Alelos , Substituição de Aminoácidos , Animais , Medula Óssea/patologia , Proteínas de Ciclo Celular/metabolismo , Modelos Animais de Doenças , Família , Feminino , Estudos de Associação Genética , Humanos , Masculino , Camundongos , Camundongos Knockout , Linhagem , Fenótipo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Síndrome , Encurtamento do Telômero
5.
Cancers (Basel) ; 10(5)2018 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-29734785

RESUMO

The p53 protein has been extensively studied for its capacity to prevent proliferation of cells with a damaged genome. Surprisingly, however, our recent analysis of mice expressing a hyperactive mutant p53 that lacks the C-terminal domain revealed that increased p53 activity may alter genome maintenance. We showed that p53 downregulates genes essential for telomere metabolism, DNA repair, and centromere structure and that a sustained p53 activity leads to phenotypic traits associated with dyskeratosis congenita and Fanconi anemia. This downregulation is largely conserved in human cells, which suggests that our findings could be relevant to better understand processes involved in bone marrow failure as well as aging and tumor suppression.

6.
Genes Dev ; 31(5): 463-480, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28356341

RESUMO

In mammals, centromere definition involves the histone variant CENP-A (centromere protein A), deposited by its chaperone, HJURP (Holliday junction recognition protein). Alterations in this process impair chromosome segregation and genome stability, which are also compromised by p53 inactivation in cancer. Here we found that CENP-A and HJURP are transcriptionally up-regulated in p53-null human tumors. Using an established mouse embryonic fibroblast (MEF) model combining p53 inactivation with E1A or HRas-V12 oncogene expression, we reproduced a similar up-regulation of HJURP and CENP-A. We delineate functional CDE/CHR motifs within the Hjurp and Cenpa promoters and demonstrate their roles in p53-mediated repression. To assess the importance of HJURP up-regulation in transformed murine and human cells, we used a CRISPR/Cas9 approach. Remarkably, depletion of HJURP leads to distinct outcomes depending on their p53 status. Functional p53 elicits a cell cycle arrest response, whereas, in p53-null transformed cells, the absence of arrest enables the loss of HJURP to induce severe aneuploidy and, ultimately, apoptotic cell death. We thus tested the impact of HJURP depletion in pre-established allograft tumors in mice and revealed a major block of tumor progression in vivo. We discuss a model in which an "epigenetic addiction" to the HJURP chaperone represents an Achilles' heel in p53-deficient transformed cells.


Assuntos
Autoantígenos/metabolismo , Transformação Celular Neoplásica/genética , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes p53/genética , Oncogenes/genética , Motivos de Aminoácidos/genética , Animais , Autoantígenos/genética , Linhagem Celular , Células Cultivadas , Proteína Centromérica A , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos/genética , Proteínas de Ligação a DNA/genética , Feminino , Deleção de Genes , Instabilidade Genômica/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Animais
8.
Nat Commun ; 7: 11091, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-27033104

RESUMO

Germline mutations affecting telomere maintenance or DNA repair may, respectively, cause dyskeratosis congenita or Fanconi anaemia, two clinically related bone marrow failure syndromes. Mice expressing p53(Δ31), a mutant p53 lacking the C terminus, model dyskeratosis congenita. Accordingly, the increased p53 activity in p53(Δ31/Δ31) fibroblasts correlated with a decreased expression of 4 genes implicated in telomere syndromes. Here we show that these cells exhibit decreased mRNA levels for additional genes contributing to telomere metabolism, but also, surprisingly, for 12 genes mutated in Fanconi anaemia. Furthermore, p53(Δ31/Δ31) fibroblasts exhibit a reduced capacity to repair DNA interstrand crosslinks, a typical feature of Fanconi anaemia cells. Importantly, the p53-dependent downregulation of Fanc genes is largely conserved in human cells. Defective DNA repair is known to activate p53, but our results indicate that, conversely, an increased p53 activity may attenuate the Fanconi anaemia DNA repair pathway, defining a positive regulatory feedback loop.


Assuntos
Reparo do DNA , Regulação para Baixo , Anemia de Fanconi/genética , Proteína Supressora de Tumor p53/fisiologia , Animais , Células Cultivadas , Fator de Transcrição E2F4/genética , Fator de Transcrição E2F4/metabolismo , Fator de Transcrição E2F4/fisiologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/fisiologia , Humanos , Camundongos , Células NIH 3T3 , Transcriptoma
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