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1.
Cell ; 176(1-2): 182-197.e23, 2019 01 10.
Artículo en Inglés | MEDLINE | ID: mdl-30595450

RESUMEN

During development, the precise relationships between transcription and chromatin modifications often remain unclear. We use the X chromosome inactivation (XCI) paradigm to explore the implication of chromatin changes in gene silencing. Using female mouse embryonic stem cells, we initiate XCI by inducing Xist and then monitor the temporal changes in transcription and chromatin by allele-specific profiling. This reveals histone deacetylation and H2AK119 ubiquitination as the earliest chromatin alterations during XCI. We show that HDAC3 is pre-bound on the X chromosome and that, upon Xist coating, its activity is required for efficient gene silencing. We also reveal that first PRC1-associated H2AK119Ub and then PRC2-associated H3K27me3 accumulate initially at large intergenic domains that can then spread into genes only in the context of histone deacetylation and gene silencing. Our results reveal the hierarchy of chromatin events during the initiation of XCI and identify key roles for chromatin in the early steps of transcriptional silencing.


Asunto(s)
Cromatina/metabolismo , Inactivación del Cromosoma X/genética , Inactivación del Cromosoma X/fisiología , Acetilación , Animales , Cromatina/genética , Células Madre Embrionarias , Epigenómica/métodos , Femenino , Silenciador del Gen , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Ratones , Proteínas del Grupo Polycomb/metabolismo , Procesamiento Proteico-Postraduccional , ARN Largo no Codificante/metabolismo , Transcripción Genética , Ubiquitinación , Cromosoma X/metabolismo
2.
Mol Cell ; 83(20): 3659-3668.e10, 2023 10 19.
Artículo en Inglés | MEDLINE | ID: mdl-37832547

RESUMEN

The integrity of the nuclear envelope (NE) is essential for maintaining the structural stability of the nucleus. Rupture of the NE has been frequently observed in cancer cells, especially in the context of mechanical challenges, such as physical confinement and migration. However, spontaneous NE rupture events, without any obvious physical challenges to the cell, have also been described. The molecular mechanism(s) of these spontaneous NE rupture events remain to be explored. Here, we show that DNA damage and subsequent ATR activation leads to NE rupture. Upon DNA damage, lamin A/C is phosphorylated in an ATR-dependent manner, leading to changes in lamina assembly and, ultimately, NE rupture. In addition, we show that cancer cells with intrinsic DNA repair defects undergo frequent events of DNA-damage-induced NE rupture, which renders them extremely sensitive to further NE perturbations. Exploiting this NE vulnerability could provide a new angle to complement traditional, DNA-damage-based chemotherapy.


Asunto(s)
Lamina Tipo A , Membrana Nuclear , Membrana Nuclear/metabolismo , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Fosforilación , Daño del ADN , ADN/metabolismo , Núcleo Celular/metabolismo
3.
Mol Cell ; 82(13): 2458-2471.e9, 2022 07 07.
Artículo en Inglés | MEDLINE | ID: mdl-35550257

RESUMEN

Many cancers are characterized by gene fusions encoding oncogenic chimeric transcription factors (TFs) such as EWS::FLI1 in Ewing sarcoma (EwS). Here, we find that EWS::FLI1 induces the robust expression of a specific set of novel spliced and polyadenylated transcripts within otherwise transcriptionally silent regions of the genome. These neogenes (NGs) are virtually undetectable in large collections of normal tissues or non-EwS tumors and can be silenced by CRISPR interference at regulatory EWS::FLI1-bound microsatellites. Ribosome profiling and proteomics further show that some NGs are translated into highly EwS-specific peptides. More generally, we show that hundreds of NGs can be detected in diverse cancers characterized by chimeric TFs. Altogether, this study identifies the transcription, processing, and translation of novel, specific, highly expressed multi-exonic transcripts from otherwise silent regions of the genome as a new activity of aberrant TFs in cancer.


Asunto(s)
Carcinogénesis , Regulación Neoplásica de la Expresión Génica , Proteínas de Fusión Oncogénica , Proteína Proto-Oncogénica c-fli-1 , Factores de Transcripción , Carcinogénesis/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/genética , Silenciador del Gen , Genoma/genética , Genómica , Humanos , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Oncogenes/genética , Proteína Proto-Oncogénica c-fli-1/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patología , Factores de Transcripción/genética , Transcripción Genética/genética
4.
Nature ; 621(7978): 415-422, 2023 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-37674080

RESUMEN

DNA double-strand breaks (DSBs) are deleterious lesions that challenge genome integrity. To mitigate this threat, human cells rely on the activity of multiple DNA repair machineries that are tightly regulated throughout the cell cycle1. In interphase, DSBs are mainly repaired by non-homologous end joining and homologous recombination2. However, these pathways are completely inhibited in mitosis3-5, leaving the fate of mitotic DSBs unknown. Here we show that DNA polymerase theta6 (Polθ) repairs mitotic DSBs and thereby maintains genome integrity. In contrast to other DSB repair factors, Polθ function is activated in mitosis upon phosphorylation by Polo-like kinase 1 (PLK1). Phosphorylated Polθ is recruited by a direct interaction with the BRCA1 C-terminal domains of TOPBP1 to mitotic DSBs, where it mediates joining of broken DNA ends. Loss of Polθ leads to defective repair of mitotic DSBs, resulting in a loss of genome integrity. This is further exacerbated in cells that are deficient in homologous recombination, where loss of mitotic DSB repair by Polθ results in cell death. Our results identify mitotic DSB repair as the underlying cause of synthetic lethality between Polθ and homologous recombination. Together, our findings reveal the critical importance of mitotic DSB repair in the maintenance of genome integrity.


Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , ADN Polimerasa Dirigida por ADN , Mitosis , Proteínas Serina-Treonina Quinasas , Humanos , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular/metabolismo , Muerte Celular/genética , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Recombinación Homóloga/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Mutaciones Letales Sintéticas , ADN Polimerasa theta , Quinasa Tipo Polo 1
5.
Nature ; 617(7960): 386-394, 2023 May.
Artículo en Inglés | MEDLINE | ID: mdl-37100912

RESUMEN

Inflammation is a complex physiological process triggered in response to harmful stimuli1. It involves cells of the immune system capable of clearing sources of injury and damaged tissues. Excessive inflammation can occur as a result of infection and is a hallmark of several diseases2-4. The molecular bases underlying inflammatory responses are not fully understood. Here we show that the cell surface glycoprotein CD44, which marks the acquisition of distinct cell phenotypes in the context of development, immunity and cancer progression, mediates the uptake of metals including copper. We identify a pool of chemically reactive copper(II) in mitochondria of inflammatory macrophages that catalyses NAD(H) redox cycling by activating hydrogen peroxide. Maintenance of NAD+ enables metabolic and epigenetic programming towards the inflammatory state. Targeting mitochondrial copper(II) with supformin (LCC-12), a rationally designed dimer of metformin, induces a reduction of the NAD(H) pool, leading to metabolic and epigenetic states that oppose macrophage activation. LCC-12 interferes with cell plasticity in other settings and reduces inflammation in mouse models of bacterial and viral infections. Our work highlights the central role of copper as a regulator of cell plasticity and unveils a therapeutic strategy based on metabolic reprogramming and the control of epigenetic cell states.


Asunto(s)
Plasticidad de la Célula , Cobre , Inflamación , Transducción de Señal , Animales , Ratones , Cobre/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , NAD/metabolismo , Transducción de Señal/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Peróxido de Hidrógeno/metabolismo , Epigénesis Genética/efectos de los fármacos , Metformina/análogos & derivados , Oxidación-Reducción , Plasticidad de la Célula/efectos de los fármacos , Plasticidad de la Célula/genética , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/genética
6.
EMBO J ; 42(1): e111485, 2023 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-36385434

RESUMEN

Sleep intensity is adjusted by the length of previous awake time, and under tight homeostatic control by protein phosphorylation. Here, we establish microglia as a new cellular component of the sleep homeostasis circuit. Using quantitative phosphoproteomics of the mouse frontal cortex, we demonstrate that microglia-specific deletion of TNFα perturbs thousands of phosphorylation sites during the sleep period. Substrates of microglial TNFα comprise sleep-related kinases such as MAPKs and MARKs, and numerous synaptic proteins, including a subset whose phosphorylation status encodes sleep need and determines sleep duration. As a result, microglial TNFα loss attenuates the build-up of sleep need, as measured by electroencephalogram slow-wave activity and prevents immediate compensation for loss of sleep. Our data suggest that microglia control sleep homeostasis by releasing TNFα which acts on neuronal circuitry through dynamic control of phosphorylation.


Asunto(s)
Microglía , Factor de Necrosis Tumoral alfa , Ratones , Animales , Microglía/metabolismo , Fosforilación , Factor de Necrosis Tumoral alfa/metabolismo , Sueño/fisiología , Homeostasis/fisiología
7.
EMBO J ; 42(24): e113590, 2023 Dec 11.
Artículo en Inglés | MEDLINE | ID: mdl-38073509

RESUMEN

Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or ENPs) that may play a role in intercellular communication. Tumor-derived EVs have been proposed to induce immune priming of antigen presenting cells or to be immuno-suppressive agents. We suspect that such disparate functions are due to variable compositions in EV subtypes and ENPs. We aimed to characterize the array of secreted EVs and ENPs of murine tumor cell lines. Unexpectedly, we identified virus-like particles (VLPs) from endogenous murine leukemia virus in preparations of EVs produced by many tumor cells. We established a protocol to separate small EVs from VLPs and ENPs. We compared their protein composition and analyzed their functional interaction with target dendritic cells. ENPs were poorly captured and did not affect dendritic cells. Small EVs specifically induced dendritic cell death. A mixed large/dense EV/VLP preparation was most efficient to induce dendritic cell maturation and antigen presentation. Our results call for systematic re-evaluation of the respective proportions and functions of non-viral EVs and VLPs produced by murine tumors and their contribution to tumor progression.


Asunto(s)
Retrovirus Endógenos , Vesículas Extracelulares , Neoplasias , Animales , Ratones , Vesículas Extracelulares/metabolismo , Línea Celular Tumoral , Diferenciación Celular , Células Dendríticas , Neoplasias/metabolismo
8.
EMBO J ; 41(12): e108306, 2022 06 14.
Artículo en Inglés | MEDLINE | ID: mdl-35506364

RESUMEN

Influenza virus infection causes considerable morbidity and mortality, but current therapies have limited efficacy. We hypothesized that investigating the metabolic signaling during infection may help to design innovative antiviral approaches. Using bronchoalveolar lavages of infected mice, we here demonstrate that influenza virus induces a major reprogramming of lung metabolism. We focused on mitochondria-derived succinate that accumulated both in the respiratory fluids of virus-challenged mice and of patients with influenza pneumonia. Notably, succinate displays a potent antiviral activity in vitro as it inhibits the multiplication of influenza A/H1N1 and A/H3N2 strains and strongly decreases virus-triggered metabolic perturbations and inflammatory responses. Moreover, mice receiving succinate intranasally showed reduced viral loads in lungs and increased survival compared to control animals. The antiviral mechanism involves a succinate-dependent posttranslational modification, that is, succinylation, of the viral nucleoprotein at the highly conserved K87 residue. Succinylation of viral nucleoprotein altered its electrostatic interactions with viral RNA and further impaired the trafficking of viral ribonucleoprotein complexes. The finding that succinate efficiently disrupts the influenza replication cycle opens up new avenues for improved treatment of influenza pneumonia.


Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Gripe Humana , Infecciones por Orthomyxoviridae , Neumonía , Animales , Antivirales/farmacología , Humanos , Subtipo H3N2 del Virus de la Influenza A/metabolismo , Ratones , Proteínas de la Nucleocápside , Nucleoproteínas/metabolismo , Ácido Succínico/metabolismo , Ácido Succínico/farmacología , Ácido Succínico/uso terapéutico , Replicación Viral
9.
Nature ; 578(7795): 455-460, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-32025035

RESUMEN

Xist represents a paradigm for the function of long non-coding RNA in epigenetic regulation, although how it mediates X-chromosome inactivation (XCI) remains largely unexplained. Several proteins that bind to Xist RNA have recently been identified, including the transcriptional repressor SPEN1-3, the loss of which has been associated with deficient XCI at multiple loci2-6. Here we show in mice that SPEN is a key orchestrator of XCI in vivo and we elucidate its mechanism of action. We show that SPEN is essential for initiating gene silencing on the X chromosome in preimplantation mouse embryos and in embryonic stem cells. SPEN is dispensable for maintenance of XCI in neural progenitors, although it significantly decreases the expression of genes that escape XCI. We show that SPEN is immediately recruited to the X chromosome upon the upregulation of Xist, and is targeted to enhancers and promoters of active genes. SPEN rapidly disengages from chromatin upon gene silencing, suggesting that active transcription is required to tether SPEN to chromatin. We define the SPOC domain as a major effector of the gene-silencing function of SPEN, and show that tethering SPOC to Xist RNA is sufficient to mediate gene silencing. We identify the protein partners of SPOC, including NCoR/SMRT, the m6A RNA methylation machinery, the NuRD complex, RNA polymerase II and factors involved in the regulation of transcription initiation and elongation. We propose that SPEN acts as a molecular integrator for the initiation of XCI, bridging Xist RNA with the transcription machinery-as well as with nucleosome remodellers and histone deacetylases-at active enhancers and promoters.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Epigénesis Genética , Silenciador del Gen , Proteínas de Unión al ARN/metabolismo , Transcripción Genética , Inactivación del Cromosoma X/genética , Cromosoma X/genética , Animales , Blastocisto/citología , Blastocisto/metabolismo , Cromatina/genética , Cromatina/metabolismo , Proteínas de Unión al ADN/química , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos/genética , Femenino , Histona Desacetilasas/metabolismo , Masculino , Metilación , Ratones , Regiones Promotoras Genéticas/genética , Dominios Proteicos , ARN Largo no Codificante/genética , Proteínas de Unión al ARN/química
10.
J Biol Chem ; : 107601, 2024 Jul 24.
Artículo en Inglés | MEDLINE | ID: mdl-39059493

RESUMEN

Ubiquitination plays a crucial role in cellular homeostasis by regulating the degradation, localization, and activity of proteins, ensuring proper cell function and balance. Among E3 ubiquitin ligases, WWP1 is implicated in cell proliferation, survival and apoptosis. Notably WWP1 is frequently amplified in breast cancer and associated with poor prognosis. Here we identify the protein CYYR1 that had previously no assigned function, as a regulator of WWP1 activity and stability. We show that CYYR1 binds to the WW domains of the E3 ubiquitin ligase WWP1 through its PPxY motifs. This interaction triggers K63-linked auto-ubiquitination and subsequent degradation of WWP1. We furthermore demonstrate that CYYR1 localizes to late endosomal vesicles and directs poly-ubiquitinated WWP1 toward lysosomal degradation through binding to ANKRD13A. Moreover, we found that CYYR1 expression attenuates breast cancer cell growth in anchorage-dependent and independent colony formation assays in a PPxY-dependent manner. Finally, we highlight that CYYR1 expression is significantly decreased in breast cancer and is associated with beneficial clinical outcome. Taken together our study suggests tumor suppressor properties for CYYR1 through regulation of WWP1 auto-ubiquitination and lysosomal degradation.

11.
EMBO J ; 40(8): e105492, 2021 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-33709510

RESUMEN

Cells release diverse types of extracellular vesicles (EVs), which transfer complex signals to surrounding cells. Specific markers to distinguish different EVs (e.g. exosomes, ectosomes, enveloped viruses like HIV) are still lacking. We have developed a proteomic profiling approach for characterizing EV subtype composition and applied it to human Jurkat T cells. We generated an interactive database to define groups of proteins with similar profiles, suggesting release in similar EVs. Biochemical validation confirmed the presence of preferred partners of commonly used exosome markers in EVs: CD81/ADAM10/ITGB1, and CD63/syntenin. We then compared EVs from control and HIV-1-infected cells. HIV infection altered EV profiles of several cellular proteins, including MOV10 and SPN, which became incorporated into HIV virions, and SERINC3, which was re-routed to non-viral EVs in a Nef-dependent manner. Furthermore, we found that SERINC3 controls the surface composition of EVs. Our workflow provides an unbiased approach for identifying candidate markers and potential regulators of EV subtypes. It can be widely applied to in vitro experimental systems for investigating physiological or pathological modifications of EV release.


Asunto(s)
Vesículas Extracelulares/metabolismo , Infecciones por VIH/metabolismo , Proteoma/metabolismo , Células Cultivadas , Células HEK293 , VIH-1 , Humanos , Células Jurkat , Leucosialina/metabolismo , Glicoproteínas de Membrana/metabolismo , ARN Helicasas/metabolismo
12.
Am J Hum Genet ; 109(12): 2196-2209, 2022 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-36459980

RESUMEN

The TERT/CLPTM1L risk locus on chromosome 5p15.33 is a pleiotropic cancer risk locus in which multiple independent risk alleles have been identified, across well over ten cancer types. We previously conducted a genome-wide association study in uveal melanoma (UM), which uncovered a role for the TERT/CLPTM1L risk locus in this intraocular tumor and identified multiple highly correlated risk alleles. Aiming to unravel the biological mechanisms in UM of this locus, which contains a domain enriched in active chromatin marks and enhancer elements, we demonstrated the allele-specific enhancer activity of this risk region using reporter assays. In UM, we identified the functional variant rs452384, of which the C risk allele is associated with higher gene expression, increased CLPTM1L expression in UM tumors, and a longer telomere length in peripheral blood mononuclear cells. Electrophoretic mobility shift assays and quantitative mass spectrometry identified NKX2.4 as an rs452384-T-specific binding protein, whereas GATA4 preferentially interacted with rs452384-C. Knockdown of NKX2.4 but not GATA4 resulted in increased TERT and CLPTM1L expression. In summary, the UM risk conferred by the 5p locus is at least partly due to rs452384, for which NKX2.4 presents strong differential binding activity and regulates CLPTM1L and TERT expression. Altogether, our work unraveled some of the complex regulatory mechanisms at the 5p15.33 susceptibility region in UM, and this might also shed light on shared mechanisms with other tumor types affected by this susceptibility region.


Asunto(s)
Estudio de Asociación del Genoma Completo , Neoplasias de la Úvea , Humanos , Alelos , Leucocitos Mononucleares , Neoplasias de la Úvea/genética
14.
BMC Biol ; 21(1): 73, 2023 04 07.
Artículo en Inglés | MEDLINE | ID: mdl-37024974

RESUMEN

BACKGROUND: E3 ubiquitin ligases play critical roles in regulating cellular signaling pathways by inducing ubiquitylation of key components. RNF111/Arkadia is a RING E3 ubiquitin ligase that activates TGF-ß signaling by inducing ubiquitylation and proteasomal degradation of the transcriptional repressor SKIL/SnoN. In this study, we have sought to identify novel regulators of the E3 ubiquitin ligase activity of RNF111 by searching for proteins that specifically interacts with its RING domain. RESULTS: We found that UBXN7, a member of the UBA-UBX family, directly interacts with the RING domain of RNF111 or its related E3 RNF165/ARK2C that shares high sequence homology with RNF111. We showed that UBXN7 docks on RNF111 or RNF165 RING domain through its UAS thioredoxin-like domain. Overexpression of UBXN7 or its UAS domain increases endogenous RNF111, while an UBXN7 mutant devoid of UAS domain has no effect. Conversely, depletion of UBXN7 decreases RNF111 protein level. As a consequence, we found that UBXN7 can modulate degradation of the RNF111 substrate SKIL in response to TGF-ß signaling. We further unveiled this mechanism of regulation by showing that docking of the UAS domain of UBXN7 inhibits RNF111 ubiquitylation by preventing interaction of the RING domain with the E2 conjugating enzymes. By analyzing the interactome of the UAS domain of UBXN7, we identified that it also interacts with the RING domain of the E3 TOPORS and similarly regulates its E3 ubiquitin ligase activity by impairing E2 binding. CONCLUSIONS: Taken together, our results demonstrate that UBXN7 acts as a direct regulator for the E3 ubiquitin ligases RNF111, RNF165, and TOPORS and reveal that a thioredoxin-like domain can dock on specific RING domains to regulate their E3 ubiquitin ligase activity.


Asunto(s)
Ubiquitina-Proteína Ligasas , Ubiquitinas , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Factor de Crecimiento Transformador beta/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo
15.
Genes Dev ; 30(11): 1313-26, 2016 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-27284163

RESUMEN

The CENP-T/-W histone fold complex, as an integral part of the inner kinetochore, is essential for building a proper kinetochore at the centromere in order to direct chromosome segregation during mitosis. Notably, CENP-T/-W is not inherited at centromeres, and new deposition is absolutely required at each cell cycle for kinetochore function. However, the mechanisms underlying this new deposition of CENP-T/-W at centromeres are unclear. Here, we found that CENP-T deposition at centromeres is uncoupled from DNA synthesis. We identified Spt16 and SSRP1, subunits of the H2A-H2B histone chaperone facilitates chromatin transcription (FACT), as CENP-W binding partners through a proteomic screen. We found that the C-terminal region of Spt16 binds specifically to the histone fold region of CENP-T/-W. Furthermore, depletion of Spt16 impairs CENP-T and CENP-W deposition at endogenous centromeres, and site-directed targeting of Spt16 alone is sufficient to ensure local de novo CENP-T accumulation. We propose a model in which the FACT chaperone stabilizes the soluble CENP-T/-W complex in the cell and promotes dynamics of exchange, enabling CENP-T/-W deposition at centromeres.


Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Chaperonas de Histonas/metabolismo , Cinetocoros/metabolismo , Factores de Elongación Transcripcional/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Centrómero/metabolismo , Proteínas de Unión al ADN/genética , Células HeLa , Proteínas del Grupo de Alta Movilidad/genética , Humanos , Unión Proteica , Dominios Proteicos , Pliegue de Proteína , Proteómica , Proteínas Recombinantes/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Elongación Transcripcional/genética
16.
Nat Mater ; 21(3): 366-377, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-34663953

RESUMEN

Mechanotransduction is a process by which cells sense the mechanical properties of their surrounding environment and adapt accordingly to perform cellular functions such as adhesion, migration and differentiation. Integrin-mediated focal adhesions are major sites of mechanotransduction and their connection with the actomyosin network is crucial for mechanosensing as well as for the generation and transmission of forces onto the substrate. Despite having emerged as major regulators of cell adhesion and migration, the contribution of microtubules to mechanotransduction still remains elusive. Here, we show that talin- and actomyosin-dependent mechanosensing of substrate rigidity controls microtubule acetylation (a tubulin post-translational modification) by promoting the recruitment of α-tubulin acetyltransferase 1 (αTAT1) to focal adhesions. Microtubule acetylation tunes the mechanosensitivity of focal adhesions and Yes-associated protein (YAP) translocation. Microtubule acetylation, in turn, promotes the release of the guanine nucleotide exchange factor GEF-H1 from microtubules to activate RhoA, actomyosin contractility and traction forces. Our results reveal a fundamental crosstalk between microtubules and actin in mechanotransduction that contributes to mechanosensitive cell adhesion and migration.


Asunto(s)
Mecanotransducción Celular , Microtúbulos , Citoesqueleto de Actina/metabolismo , Adhesión Celular , Adhesiones Focales/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo
17.
Blood ; 138(1): 57-70, 2021 07 08.
Artículo en Inglés | MEDLINE | ID: mdl-33881493

RESUMEN

Follicular lymphoma (FL) originates in the lymph nodes (LNs) and infiltrates bone marrow (BM) early in the course of the disease. BM FL B cells are characterized by a lower cytological grade, decreased proliferation, and a specific phenotypic and subclonal profile. Mesenchymal stromal cells (MSCs) obtained from FL BM display a specific gene expression profile (GEP), including enrichment for a lymphoid stromal cell signature, and an increased capacity to sustain FL B-cell growth. However, the mechanisms triggering the formation of the medullar FL permissive stromal niche have not been identified. In the current work, we demonstrate that FL B cells produce extracellular vesicles (EVs) that can be internalized by BM-MSCs, making them more efficient to support FL B-cell survival and quiescence. Accordingly, EVs purified from FL BM plasma activate transforming growth factor ß-dependent and independent pathways in BM-MSCs and modify their GEP, triggering an upregulation of factors classically associated with hematopoietic stem cell niche, including CXCL12 and angiopoietin-1. Moreover, we provide the first characterization of BM FL B-cell GEP, allowing the definition of the landscape of molecular interactions they could engage with EV-primed BM-MSCs. This work identifies FL-derived EVs as putative mediators of BM stroma polarization and supports further investigation of their clinical interest for targeting the crosstalk between BM-MSCs and malignant B cells.


Asunto(s)
Linfocitos B/patología , Células de la Médula Ósea/patología , Polaridad Celular , Vesículas Extracelulares/patología , Linfoma Folicular/patología , Secuencia de Bases , Células de la Médula Ósea/metabolismo , Comunicación Celular , Diferenciación Celular , Endocitosis , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/ultraestructura , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Humanos , Linfoma Folicular/genética , Heterotrímero de Linfotoxina alfa1 y beta2/metabolismo , Células Madre Mesenquimatosas/metabolismo , Fenotipo , Transducción de Señal , Células del Estroma/metabolismo , Células del Estroma/patología , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba/genética
18.
Mol Cell ; 60(5): 784-796, 2015 Dec 03.
Artículo en Inglés | MEDLINE | ID: mdl-26656161

RESUMEN

Stalling of RNA Polymerase II (RNAPII) on chromatin during transcriptional stress results in polyubiquitination and degradation of the largest subunit of RNAPII, Rpb1, by the ubiquitin proteasome system (UPS). Here, we report that the ATP-dependent chromatin remodeling complex INO80 is required for turnover of chromatin-bound RNAPII in yeast. INO80 interacts physically and functionally with Cdc48/p97/VCP, a component of UPS required for degradation of RNAPII. Cells lacking INO80 are defective in Rpb1 degradation and accumulate tightly bound ubiquitinated Rpb1 on chromatin. INO80 forms a ternary complex with RNAPII and Cdc48 and targets Rpb1 primed for degradation. The function of INO80 in RNAPII turnover is required for cell growth and survival during genotoxic stress. Our results identify INO80 as a bona fide component of the proteolytic pathway for RNAPII degradation and suggest that INO80 nucleosome remodeling activity promotes the dissociation of ubiquitinated Rpb1 from chromatin to protect the integrity of the genome.


Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Genoma Fúngico , Saccharomyces cerevisiae/metabolismo , Ubiquitinación , Proteína que Contiene Valosina
19.
Mol Cell Proteomics ; 20: 100173, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34740826

RESUMEN

RNF111/Arkadia is an E3 ubiquitin ligase that activates the transforming growth factor-ß (TGF-ß) pathway by degrading transcriptional repressors SKIL/SnoN and SKI. Truncations of the RING C-terminal domain of RNF111 that abolish its E3 function and subsequently activate TGF-ß signaling are observed in some cancers. In the present study, we sought to perform a comprehensive analysis of RNF111 endogenous substrates upon TGF-ß signaling activation using an integrative proteomic approach. In that aim, we carried out label-free quantitative proteomics after the enrichment of ubiquitylated proteins (ubiquitylome) in parental U2OS cell line compared with U2OS CRISPR engineered clones expressing a truncated form of RNF111 devoid of its C-terminal RING domain. We compared two methods of enrichment for ubiquitylated proteins before proteomics analysis by mass spectrometry, the diGlycine (diGly) remnant peptide immunoprecipitation with a K-ε-GG antibody, and a novel approach using protein immunoprecipitation with a ubiquitin pan nanobody that recognizes all ubiquitin chains and monoubiquitylation. Although we detected SKIL ubiquitylation among 108 potential RNF111 substrates with the diGly method, we found that the ubiquitin pan nanobody method also constitutes a powerful approach because it enabled the detection of 52 potential RNF111 substrates including SKI, SKIL, and RNF111. Integrative comparison of the RNF111-dependent proteome and ubiquitylomes enabled the identification of SKI and SKIL as the only targets ubiquitylated and degraded by RNF111 E3 ligase function in the presence of TGF-ß. Our results indicate that lysine 343 localized in the SAND domain of SKIL constitutes a target for RNF111 ubiquitylation and demonstrate that RNF111 E3 ubiquitin ligase function specifically targets SKI and SKIL ubiquitylation and degradation upon TGF-ß pathway activation.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Nucleares/genética , Proteoma/metabolismo , Proteínas Proto-Oncogénicas/genética , Transducción de Señal , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación
20.
Nucleic Acids Res ; 49(8): 4522-4533, 2021 05 07.
Artículo en Inglés | MEDLINE | ID: mdl-33823531

RESUMEN

Meiotic recombination ensures proper chromosome segregation to form viable gametes and results in gene conversions events between homologs. Conversion tracts are shorter in meiosis than in mitotically dividing cells. This results at least in part from the binding of a complex, containing the Mer3 helicase and the MutLß heterodimer, to meiotic recombination intermediates. The molecular actors inhibited by this complex are elusive. The Pif1 DNA helicase is known to stimulate DNA polymerase delta (Pol δ) -mediated DNA synthesis from D-loops, allowing long synthesis required for break-induced replication. We show that Pif1 is also recruited genome wide to meiotic DNA double-strand break (DSB) sites. We further show that Pif1, through its interaction with PCNA, is required for the long gene conversions observed in the absence of MutLß recruitment to recombination sites. In vivo, Mer3 interacts with the PCNA clamp loader RFC, and in vitro, Mer3-MutLß ensemble inhibits Pif1-stimulated D-loop extension by Pol δ and RFC-PCNA. Mechanistically, our results suggest that Mer3-MutLß may compete with Pif1 for binding to RFC-PCNA. Taken together, our data show that Pif1's activity that promotes meiotic DNA repair synthesis is restrained by the Mer3-MutLß ensemble which in turn prevents long gene conversion tracts and possibly associated mutagenesis.


Asunto(s)
ADN Helicasas/metabolismo , Conversión Génica , Recombinación Homóloga , Meiosis/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuenciación de Inmunoprecipitación de Cromatina , Roturas del ADN de Doble Cadena , ADN Helicasas/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Espectrometría de Masas , Proteínas MutL/genética , Proteínas MutL/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Recombinantes , Proteína de Replicación C/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
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