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1.
Cell ; 176(6): 1241-1243, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30849368

RESUMO

Error-prone polymerases are alleged to induce mutations while replicating damaged DNA and to increase the risk of cancer. Using in vitro studies and mice models, Yoon et al. (2019) provide evidence that the error-prone Pol θ polymerase protects against ultraviolet light-induced skin cancer despite its mutagenic potential.


Assuntos
Neoplasias Cutâneas , Raios Ultravioleta , Animais , DNA Polimerase Dirigida por DNA/genética , Camundongos , Mutagênese , DNA Polimerase teta
2.
Cell ; 156(3): 392-3, 2014 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-24485446

RESUMO

The Golgi apparatus consists of disc-like cisternae, stretching around the nucleus through forces exerted by F-actin and the Golgi membrane protein GOLPH3. Farber-Katz et al. now report that DNA damage triggers Golgi dispersal and inhibits vesicular transport through DNA-PK-mediated GOLPH3 phosphorylation, thereby linking the DNA damage response to Golgi regulation.


Assuntos
Dano ao DNA , Proteína Quinase Ativada por DNA/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Miosinas/metabolismo , Animais , Humanos
3.
Cell ; 158(3): 633-46, 2014 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-25083873

RESUMO

ATR controls chromosome integrity and chromatin dynamics. We have previously shown that yeast Mec1/ATR promotes chromatin detachment from the nuclear envelope to counteract aberrant topological transitions during DNA replication. Here, we provide evidence that ATR activity at the nuclear envelope responds to mechanical stress. Human ATR associates with the nuclear envelope during S phase and prophase, and both osmotic stress and mechanical stretching relocalize ATR to nuclear membranes throughout the cell cycle. The ATR-mediated mechanical response occurs within the range of physiological forces, is reversible, and is independent of DNA damage signaling. ATR-defective cells exhibit aberrant chromatin condensation and nuclear envelope breakdown. We propose that mechanical forces derived from chromosome dynamics and torsional stress on nuclear membranes activate ATR to modulate nuclear envelope plasticity and chromatin association to the nuclear envelope, thus enabling cells to cope with the mechanical strain imposed by these molecular processes.


Assuntos
Membrana Nuclear/metabolismo , Estresse Mecânico , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Cromatina/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Osmose , Proteínas Quinases/metabolismo
4.
Nature ; 607(7920): 790-798, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35768505

RESUMO

Ageing is intimately connected to the induction of cell senescence1,2, but why this is so remains poorly understood. A key challenge is the identification of pathways that normally suppress senescence, are lost during ageing and are functionally relevant to oppose ageing3. Here we connected the structural and functional decline of ageing tissues to attenuated function of the master effectors of cellular mechanosignalling YAP and TAZ. YAP/TAZ activity declines during physiological ageing in stromal cells, and mimicking such decline through genetic inactivation of YAP/TAZ in these cells leads to accelerated ageing. Conversely, sustaining YAP function rejuvenates old cells and opposes the emergence of ageing-related traits associated with either physiological ageing or accelerated ageing triggered by a mechano-defective extracellular matrix. Ageing traits induced by inactivation of YAP/TAZ are preceded by induction of tissue senescence. This occurs because YAP/TAZ mechanotransduction suppresses cGAS-STING signalling, to the extent that inhibition of STING prevents tissue senescence and premature ageing-related tissue degeneration after YAP/TAZ inactivation. Mechanistically, YAP/TAZ-mediated control of cGAS-STING signalling relies on the unexpected role of YAP/TAZ in preserving nuclear envelope integrity, at least in part through direct transcriptional regulation of lamin B1 and ACTR2, the latter of which is involved in building the peri-nuclear actin cap. The findings demonstrate that declining YAP/TAZ mechanotransduction drives ageing by unleashing cGAS-STING signalling, a pillar of innate immunity. Thus, sustaining YAP/TAZ mechanosignalling or inhibiting STING may represent promising approaches for limiting senescence-associated inflammation and improving healthy ageing.


Assuntos
Envelhecimento , Proteínas de Membrana , Nucleotidiltransferases , Células Estromais , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteínas de Sinalização YAP , Proteína 2 Relacionada a Actina/metabolismo , Envelhecimento/metabolismo , Senescência Celular , Matriz Extracelular , Envelhecimento Saudável , Imunidade Inata , Lamina Tipo B/metabolismo , Mecanotransdução Celular/genética , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Nucleotidiltransferases/metabolismo , Transdução de Sinais , Células Estromais/metabolismo , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/antagonistas & inibidores , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/metabolismo , Proteínas de Sinalização YAP/antagonistas & inibidores , Proteínas de Sinalização YAP/metabolismo
5.
Cell ; 149(6): 1181-3, 2012 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-22682239

RESUMO

The ATR and ATM checkpoint kinases preserve the integrity of replicating chromosomes by preventing the reversal of stalled and terminal replication forks. Hu et al. now show that the ATR pathway targets the Dna2 nuclease to process stalled forks and counteract fork reversal.

6.
Cell ; 151(4): 835-846, 2012 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-23141540

RESUMO

Transcription hinders replication fork progression and stability. The ATR checkpoint and specialized DNA helicases assist DNA synthesis across transcription units to protect genome integrity. Combining genomic and genetic approaches together with the analysis of replication intermediates, we searched for factors coordinating replication with transcription. We show that the Sen1/Senataxin DNA/RNA helicase associates with forks, promoting their progression across RNA polymerase II (RNAPII)-transcribed genes. sen1 mutants accumulate aberrant DNA structures and DNA-RNA hybrids while forks clash head-on with RNAPII transcription units. These replication defects correlate with hyperrecombination and checkpoint activation in sen1 mutants. The Sen1 function at the forks is separable from its role in RNA processing. Our data, besides unmasking a key role for Senataxin in coordinating replication with transcription, provide a framework for understanding the pathological mechanisms caused by Senataxin deficiencies and leading to the severe neurodegenerative diseases ataxia with oculomotor apraxia type 2 and amyotrophic lateral sclerosis 4.


Assuntos
DNA Helicases/metabolismo , Replicação do DNA , RNA Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Humanos , Doenças Neurodegenerativas/metabolismo , RNA Polimerase II/metabolismo
7.
Proc Natl Acad Sci U S A ; 121(29): e2404551121, 2024 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-38990945

RESUMO

Confined cell migration hampers genome integrity and activates the ATR and ATM mechano-transduction pathways. We investigated whether the mechanical stress generated by metastatic interstitial migration contributes to the enhanced chromosomal instability observed in metastatic tumor cells. We employed live cell imaging, micro-fluidic approaches, and scRNA-seq to follow the fate of tumor cells experiencing confined migration. We found that, despite functional ATR, ATM, and spindle assembly checkpoint (SAC) pathways, tumor cells dividing across constriction frequently exhibited altered spindle pole organization, chromosome mis-segregations, micronuclei formation, chromosome fragility, high gene copy number variation, and transcriptional de-regulation and up-regulation of c-MYC oncogenic transcriptional signature via c-MYC locus amplifications. In vivo tumor settings showed that malignant cells populating metastatic foci or infiltrating the interstitial stroma gave rise to cells expressing high levels of c-MYC. Altogether, our data suggest that mechanical stress during metastatic migration contributes to override the checkpoint controls and boosts genotoxic and oncogenic events. Our findings may explain why cancer aneuploidy often does not correlate with mutations in SAC genes and why c-MYC amplification is strongly linked to metastatic tumors.


Assuntos
Movimento Celular , Amplificação de Genes , Proteínas Proto-Oncogênicas c-myc , Estresse Mecânico , Humanos , Movimento Celular/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Linhagem Celular Tumoral , Camundongos , Mitose/genética , Instabilidade Cromossômica , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Neoplasias/patologia , Neoplasias/metabolismo
8.
Cell ; 146(2): 233-46, 2011 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-21784245

RESUMO

Transcription hinders replication fork progression and stability, and the Mec1/ATR checkpoint protects fork integrity. Examining checkpoint-dependent mechanisms controlling fork stability, we find that fork reversal and dormant origin firing due to checkpoint defects are rescued in checkpoint mutants lacking THO, TREX-2, or inner-basket nucleoporins. Gene gating tethers transcribed genes to the nuclear periphery and is counteracted by checkpoint kinases through phosphorylation of nucleoporins such as Mlp1. Checkpoint mutants fail to detach transcribed genes from nuclear pores, thus generating topological impediments for incoming forks. Releasing this topological complexity by introducing a double-strand break between a fork and a transcribed unit prevents fork collapse. Mlp1 mutants mimicking constitutive checkpoint-dependent phosphorylation also alleviate checkpoint defects. We propose that the checkpoint assists fork progression and stability at transcribed genes by phosphorylating key nucleoporins and counteracting gene gating, thus neutralizing the topological tension generated at nuclear pore gated genes.


Assuntos
Replicação do DNA , Poro Nuclear/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Quinase do Ponto de Checagem 2 , Quebras de DNA de Cadeia Dupla , Hidroxiureia/farmacologia , Mutação , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo
9.
Nature ; 577(7792): 701-705, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31969709

RESUMO

Transcription challenges the integrity of replicating chromosomes by generating topological stress and conflicts with forks1,2. The DNA topoisomerases Top1 and Top2 and the HMGB family protein Hmo1 assist DNA replication and transcription3-6. Here we describe the topological architecture of genes in Saccharomyces cerevisiae during the G1 and S phases of the cell cycle. We found under-wound DNA at gene boundaries and over-wound DNA within coding regions. This arrangement does not depend on Pol II or S phase. Top2 and Hmo1 preserve negative supercoil at gene boundaries, while Top1 acts at coding regions. Transcription generates RNA-DNA hybrids within coding regions, independently of fork orientation. During S phase, Hmo1 protects under-wound DNA from Top2, while Top2 confines Pol II and Top1 at coding units, counteracting transcription leakage and aberrant hybrids at gene boundaries. Negative supercoil at gene boundaries prevents supercoil diffusion and nucleosome repositioning at coding regions. DNA looping occurs at Top2 clusters. We propose that Hmo1 locks gene boundaries in a cruciform conformation and, with Top2, modulates the architecture of genes that retain the memory of the topological arrangements even when transcription is repressed.


Assuntos
DNA Fúngico/química , DNA Super-Helicoidal/química , Genes Fúngicos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Montagem e Desmontagem da Cromatina , Replicação do DNA , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , DNA Cruciforme/química , DNA Cruciforme/genética , DNA Cruciforme/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA Super-Helicoidal/genética , DNA Super-Helicoidal/metabolismo , Fase G1 , Regulação Fúngica da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/metabolismo , Mutação , Hibridização de Ácido Nucleico , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Fases de Leitura Aberta/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Fúngico/química , RNA Fúngico/genética , RNA Fúngico/metabolismo , Fase S , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Gênica
10.
Mol Cell ; 70(4): 628-638.e5, 2018 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-29775579

RESUMO

Cell survival to replication stress depends on the activation of the Mec1ATR-Rad53 checkpoint response that protects the integrity of stalled forks and controls the origin firing program. Here we found that Mad2, a member of the spindle assembly checkpoint (SAC), contributes to efficient origin firing and to cell survival in response to replication stress. We show that Rad53 and Mad2 promote S-phase cyclin expression through different mechanisms: while Rad53 influences Clb5,6 degradation, Mad2 promotes their protein synthesis. We found that Mad2 co-sediments with polysomes and modulates the association of the translation inhibitor Caf204E-BP with the translation machinery and the initiation factor eIF4E. This Mad2-dependent translational regulatory process does not depend on other SAC proteins. Altogether our observations indicate that Mad2 has an additional function outside of mitosis to control DNA synthesis and collaborates with the Mec1-Rad53 regulatory axis to allow cell survival in response to replication stress.


Assuntos
Ciclinas/genética , Replicação do DNA , Proteínas Mad2/metabolismo , Mitose , Biossíntese de Proteínas , Fase S , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Ciclina B/genética , Ciclina B/metabolismo , Ciclinas/metabolismo , Proteínas Mad2/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Origem de Replicação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
11.
Mol Cell ; 67(2): 266-281.e4, 2017 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-28648781

RESUMO

Mec1ATR mediates the DNA damage response (DDR), integrating chromosomal signals and mechanical stimuli. We show that the PP2A phosphatases, ceramide-activated enzymes, couple cell metabolism with the DDR. Using genomic screens, metabolic analysis, and genetic and pharmacological studies, we found that PP2A attenuates the DDR and that three metabolic circuits influence the DDR by modulating PP2A activity. Irc21, a putative cytochrome b5 reductase that promotes the condensation reaction generating dihydroceramides (DHCs), and Ppm1, a PP2A methyltransferase, counteract the DDR by activating PP2A; conversely, the nutrient-sensing TORC1-Tap42 axis sustains DDR activation by inhibiting PP2A. Loss-of-function mutations in IRC21, PPM1, and PP2A and hyperactive tap42 alleles rescue mec1 mutants. Ceramides synergize with rapamycin, a TORC1 inhibitor, in counteracting the DDR. Hence, PP2A integrates nutrient-sensing and metabolic pathways to attenuate the Mec1ATR response. Our observations imply that metabolic changes affect genome integrity and may help with exploiting therapeutic options and repositioning known drugs.


Assuntos
Dano ao DNA , Reparo do DNA , DNA Fúngico/metabolismo , Metabolismo Energético , Genoma Fúngico , Instabilidade Genômica , Proteína Fosfatase 2/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ceramidas/metabolismo , Ceramidas/farmacologia , Citocromo-B(5) Redutase/genética , Citocromo-B(5) Redutase/metabolismo , Reparo do DNA/efeitos dos fármacos , DNA Fúngico/genética , Ativação Enzimática , Regulação Fúngica da Expressão Gênica , Genoma Fúngico/efeitos dos fármacos , Instabilidade Genômica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metabolômica , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Metiltransferases/genética , Proteínas Metiltransferases/metabolismo , Proteína Fosfatase 2/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Sirolimo/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
12.
Nat Mater ; 22(5): 644-655, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36581770

RESUMO

The process in which locally confined epithelial malignancies progressively evolve into invasive cancers is often promoted by unjamming, a phase transition from a solid-like to a liquid-like state, which occurs in various tissues. Whether this tissue-level mechanical transition impacts phenotypes during carcinoma progression remains unclear. Here we report that the large fluctuations in cell density that accompany unjamming result in repeated mechanical deformations of cells and nuclei. This triggers a cellular mechano-protective mechanism involving an increase in nuclear size and rigidity, heterochromatin redistribution and remodelling of the perinuclear actin architecture into actin rings. The chronic strains and stresses associated with unjamming together with the reduction of Lamin B1 levels eventually result in DNA damage and nuclear envelope ruptures, with the release of cytosolic DNA that activates a cGAS-STING (cyclic GMP-AMP synthase-signalling adaptor stimulator of interferon genes)-dependent cytosolic DNA response gene program. This mechanically driven transcriptional rewiring ultimately alters the cell state, with the emergence of malignant traits, including epithelial-to-mesenchymal plasticity phenotypes and chemoresistance in invasive breast carcinoma.


Assuntos
Actinas , Neoplasias , DNA , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Citosol/metabolismo , Transdução de Sinais
13.
Cell ; 137(2): 247-58, 2009 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-19361851

RESUMO

In response to replication stress, the Mec1/ATR and SUMO pathways control stalled- and damaged-fork stability. We investigated the S phase response at forks encountering a broken template (termed the terminal fork). We show that double-strand break (DSB) formation can locally trigger dormant origin firing. Irreversible fork resolution at the break does not impede progression of the other fork in the same replicon (termed the sister fork). The Mre11-Tel1/ATM response acts at terminal forks, preventing accumulation of cruciform DNA intermediates that tether sister chromatids and can undergo nucleolytic processing. We conclude that sister forks can be uncoupled during replication and that, after DSB-induced fork termination, replication is rescued by dormant origin firing or adjacent replicons. We have uncovered a Tel1/ATM- and Mre11-dependent response controlling terminal fork integrity. Our findings have implications for those genome instability syndromes that accumulate DNA breaks during S phase and for forks encountering eroding telomeres.


Assuntos
Replicação do DNA , Replicon , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Reparo do DNA , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Endonucleases , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Origem de Replicação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
14.
Cell ; 138(5): 870-84, 2009 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-19737516

RESUMO

Specialized topoisomerases solve the topological constraints arising when replication forks encounter transcription. We have investigated the contribution of Top2 in S phase transcription. Specifically in S phase, Top2 binds intergenic regions close to transcribed genes. The Top2-bound loci exhibit low nucleosome density and accumulate gammaH2A when Top2 is defective. These intergenic loci associate with the HMG protein Hmo1 throughout the cell cycle and are refractory to the histone variant Htz1. In top2 mutants, Hmo1 is deleterious and accumulates at pericentromeric regions in G2/M. Our data indicate that Top2 is dispensable for transcription and that Hmo1 and Top2 bind in the proximity of genes transcribed in S phase suppressing chromosome fragility at the M-G1 transition. We propose that an Hmo1-dependent epigenetic signature together with Top2 mediate an S phase architectural pathway to preserve genome integrity.


Assuntos
Replicação do DNA , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Fase S , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Fragilidade Cromossômica , Epigênese Genética , Genoma Fúngico , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia
15.
Nucleic Acids Res ; 49(21): e121, 2021 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-34500456

RESUMO

We report a rapid experimental procedure based on high-density in vivo psoralen inter-strand DNA cross-linking coupled to spreading of naked purified DNA, positive staining, low-angle rotary shadowing, and transmission electron microscopy (TEM) that allows quick visualization of the dynamic of heavy strand (HS) and light strand (LS) human mitochondrial DNA replication. Replication maps built on linearized mitochondrial genomes and optimized rotary shadowing conditions enable clear visualization of the progression of the mitochondrial DNA synthesis and visualization of replication intermediates carrying long single-strand DNA stretches. One variant of this technique, called denaturing spreading, allowed the inspection of the fine chromatin structure of the mitochondrial genome and was applied to visualize the in vivo three-strand DNA structure of the human mitochondrial D-loop intermediate with unprecedented clarity.


Assuntos
Replicação do DNA , DNA Mitocondrial/ultraestrutura , DNA de Cadeia Simples/ultraestrutura , Microscopia Eletrônica de Transmissão/métodos , Mitocôndrias , Humanos , Mitocôndrias/genética , Mitocôndrias/ultraestrutura
16.
Circulation ; 144(20): 1629-1645, 2021 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-34636652

RESUMO

BACKGROUND: PALMD (palmdelphin) belongs to the family of paralemmin proteins implicated in cytoskeletal regulation. Single nucleotide polymorphisms in the PALMD locus that result in reduced expression are strong risk factors for development of calcific aortic valve stenosis and predict severity of the disease. METHODS: Immunodetection and public database screening showed dominant expression of PALMD in endothelial cells (ECs) in brain and cardiovascular tissues including aortic valves. Mass spectrometry, coimmunoprecipitation, and immunofluorescent staining allowed identification of PALMD partners. The consequence of loss of PALMD expression was assessed in small interferring RNA-treated EC cultures, knockout mice, and human valve samples. RNA sequencing of ECs and transcript arrays on valve samples from an aortic valve study cohort including patients with the single nucleotide polymorphism rs7543130 informed about gene regulatory changes. RESULTS: ECs express the cytosolic PALMD-KKVI splice variant, which associated with RANGAP1 (RAN GTP hydrolyase activating protein 1). RANGAP1 regulates the activity of the GTPase RAN and thereby nucleocytoplasmic shuttling via XPO1 (Exportin1). Reduced PALMD expression resulted in subcellular relocalization of RANGAP1 and XPO1, and nuclear arrest of the XPO1 cargoes p53 and p21. This indicates an important role for PALMD in nucleocytoplasmic transport and consequently in gene regulation because of the effect on localization of transcriptional regulators. Changes in EC responsiveness on loss of PALMD expression included failure to form a perinuclear actin cap when exposed to flow, indicating lack of protection against mechanical stress. Loss of the actin cap correlated with misalignment of the nuclear long axis relative to the cell body, observed in PALMD-deficient ECs, Palmd-/- mouse aorta, and human aortic valve samples derived from patients with calcific aortic valve stenosis. In agreement with these changes in EC behavior, gene ontology analysis showed enrichment of nuclear- and cytoskeleton-related terms in PALMD-silenced ECs. CONCLUSIONS: We identify RANGAP1 as a PALMD partner in ECs. Disrupting the PALMD/RANGAP1 complex alters the subcellular localization of RANGAP1 and XPO1, and leads to nuclear arrest of the XPO1 cargoes p53 and p21, accompanied by gene regulatory changes and loss of actin-dependent nuclear resilience. Combined, these consequences of reduced PALMD expression provide a mechanistic underpinning for PALMD's contribution to calcific aortic valve stenosis pathology.


Assuntos
Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Endoteliais/metabolismo , Endotélio/metabolismo , Proteínas de Membrana/genética , Estresse Mecânico , Idoso , Animais , Comunicação Celular/genética , Linhagem Celular , Movimento Celular/genética , Células Cultivadas , Biologia Computacional/métodos , Bases de Dados Genéticas , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Ontologia Genética , Humanos , Imuno-Histoquímica , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Transporte Proteico
17.
Nat Rev Mol Cell Biol ; 11(3): 208-19, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20177396

RESUMO

Aberrant DNA replication is a major source of the mutations and chromosome rearrangements that are associated with pathological disorders. When replication is compromised, DNA becomes more prone to breakage. Secondary structures, highly transcribed DNA sequences and damaged DNA stall replication forks, which then require checkpoint factors and specialized enzymatic activities for their stabilization and subsequent advance. These mechanisms ensure that the local DNA damage response, which enables replication fork progression and DNA repair in S phase, is coupled with cell cycle transitions. The mechanisms that operate in eukaryotic cells to promote replication fork integrity and coordinate replication with other aspects of chromosome maintenance are becoming clear.


Assuntos
Replicação do DNA/genética , Instabilidade Genômica , Origem de Replicação/genética , Animais , DNA/química , DNA/genética , Dano ao DNA , Reparo do DNA , Humanos , Modelos Biológicos , Conformação de Ácido Nucleico
18.
Acta Neuropathol ; 142(4): 609-627, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34274995

RESUMO

Heterogeneous nuclear ribonucleoproteins (HnRNPs) are a group of ubiquitously expressed RNA-binding proteins implicated in the regulation of all aspects of nucleic acid metabolism. HnRNP K is a member of this highly versatile hnRNP family. Pathological redistribution of hnRNP K to the cytoplasm has been linked to the pathogenesis of several malignancies but, until now, has been underexplored in the context of neurodegenerative disease. Here we show hnRNP K mislocalisation in pyramidal neurons of the frontal cortex to be a novel neuropathological feature that is associated with both frontotemporal lobar degeneration and ageing. HnRNP K mislocalisation is mutually exclusive to TDP-43 and tau pathological inclusions in neurons and was not observed to colocalise with mitochondrial, autophagosomal or stress granule markers. De-repression of cryptic exons in RNA targets following TDP-43 nuclear depletion is an emerging mechanism of potential neurotoxicity in frontotemporal lobar degeneration and the mechanistically overlapping disorder amyotrophic lateral sclerosis. We silenced hnRNP K in neuronal cells to identify the transcriptomic consequences of hnRNP K nuclear depletion. Intriguingly, by performing RNA-seq analysis we find that depletion of hnRNP K induces 101 novel cryptic exon events. We validated cryptic exon inclusion in an SH-SY5Y hnRNP K knockdown and in FTLD brain exhibiting hnRNP K nuclear depletion. We, therefore, present evidence for hnRNP K mislocalisation to be associated with FTLD and for this to induce widespread changes in splicing.


Assuntos
Envelhecimento/metabolismo , Envelhecimento/patologia , Degeneração Lobar Frontotemporal/metabolismo , Degeneração Lobar Frontotemporal/patologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Splicing de RNA/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/genética , Estudos de Casos e Controles , Feminino , Degeneração Lobar Frontotemporal/genética , Humanos , Masculino , Pessoa de Meia-Idade
19.
Nat Rev Mol Cell Biol ; 9(4): 297-308, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18285803

RESUMO

The repair of DNA lesions that occur endogenously or in response to diverse genotoxic stresses is indispensable for genome integrity. DNA lesions activate checkpoint pathways that regulate specific DNA-repair mechanisms in the different phases of the cell cycle. Checkpoint-arrested cells resume cell-cycle progression once damage has been repaired, whereas cells with unrepairable DNA lesions undergo permanent cell-cycle arrest or apoptosis. Recent studies have provided insights into the mechanisms that contribute to DNA repair in specific cell-cycle phases and have highlighted the mechanisms that ensure cell-cycle progression or arrest in normal and cancerous cells.


Assuntos
Ciclo Celular , Reparo do DNA , Animais , Dano ao DNA , Humanos , Proteínas Quinases/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitinação
20.
Mol Cell ; 45(6): 710-8, 2012 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-22464441

RESUMO

DNA and RNA polymerases clash along the genome as they compete for the same DNA template. Cells have evolved specialized strategies to prevent and resolve replication and transcription interference. Here, we review the topology and architecture at sites of replication fork clashes with transcription bubbles as well as the regulatory circuits that control replication fork passage across transcribed genes. In the case of RNA polymerase II-transcribed genes, cotranscriptional processes such as mRNA maturation, splicing, and export influence the integrity of replication forks and transcribed loci. Fork passage likely contributes to reset the epigenetic landscape, influencing gene expression and transcriptional memory. When any of these processes are not properly coordinated, aberrant outcomes such as fork reversal and R-loop formation arise and trigger unscheduled recombinogenic events and genome rearrangements. The evolutionary implications of such conflicts on genome dynamics and their potential impact on oncogenic stress are discussed.


Assuntos
Replicação do DNA , Instabilidade Genômica , Transcrição Gênica , Cromatina/química , Cromatina/genética , DNA/química , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Splicing de RNA
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