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1.
J Cell Sci ; 133(4)2020 Feb 24.
Artículo en Inglés | MEDLINE | ID: mdl-32005696

RESUMEN

USP16 (also known as UBP-M) has emerged as a histone H2AK119 deubiquitylase (DUB) implicated in the regulation of chromatin-associated processes and cell cycle progression. Despite this, available evidence suggests that this DUB is also present in the cytoplasm. How the nucleo-cytoplasmic transport of USP16, and hence its function, is regulated has remained elusive. Here, we show that USP16 is predominantly cytoplasmic in all cell cycle phases. We identified the nuclear export signal (NES) responsible for maintaining USP16 in the cytoplasm. We found that USP16 is only transiently retained in the nucleus following mitosis and then rapidly exported from this compartment. We also defined a non-canonical nuclear localization signal (NLS) sequence that plays a minimal role in directing USP16 into the nucleus. We further established that this DUB does not accumulate in the nucleus following DNA damage. Instead, only enforced nuclear localization of USP16 abolishes DNA double-strand break (DSB) repair, possibly due to unrestrained DUB activity. Thus, in contrast to the prevailing view, our data indicate that USP16 is actively excluded from the nucleus and that this DUB might indirectly regulate DSB repair.This article has an associated First Person interview with the first author of the paper.

2.
PLoS Genet ; 15(11): e1008463, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31675375

RESUMEN

Nucleophosmin (NPM1) is frequently mutated or subjected to chromosomal translocation in acute myeloid leukemia (AML). NPM protein is primarily located in the nucleus, but the recurrent NPMc+ mutation, which creates a nuclear export signal, is characterized by cytoplasmic localization and leukemogenic properties. Similarly, the NPM-MLF1 translocation product favors the partial cytoplasmic retention of NPM. Regardless of their common cellular distribution, NPM-MLF1 malignancies engender different effects on hematopoiesis compared to NPMc+ counterparts, highlighting possible aberrant nuclear function(s) of NPM in NPMc+ and NPM-MLF1 AML. We performed a proteomic analysis and found that NPM and NPM-MLF1 interact with various nuclear proteins including subunits of the chromatin remodeling complexes ISWI, NuRD and P/BAF. Accordingly, NPM and NPM-MLF1 are recruited to transcriptionally active or repressed genes along with NuRD subunits. Although the overall gene expression program in NPM knockdown cells is similar to that resulting from NPMc+, NPM-MLF1 expression differentially altered gene transcription regulated by NPM. The abnormal gene regulation imposed by NPM-MLF1 can be characterized by the enhanced recruitment of NuRD to gene regulatory regions. Thus, different mechanisms would orchestrate the dysregulation of NPM function in NPMc+- versus NPM1-MLF1-associated leukemia.

3.
J Vis Exp ; (149)2019 Jul 25.
Artículo en Inglés | MEDLINE | ID: mdl-31403616

RESUMEN

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.  Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

4.
Elife ; 82019 04 10.
Artículo en Inglés | MEDLINE | ID: mdl-30969169

RESUMEN

The ability to isolate rare live cells within a heterogeneous population based solely on visual criteria remains technically challenging, due largely to limitations imposed by existing sorting technologies. Here, we present a new method that permits labeling cells of interest by attaching streptavidin-coated magnetic beads to their membranes using the lasers of a confocal microscope. A simple magnet allows highly specific isolation of the labeled cells, which then remain viable and proliferate normally. As proof of principle, we tagged, isolated, and expanded individual cells based on three biologically relevant visual characteristics: i) presence of multiple nuclei, ii) accumulation of lipid vesicles, and iii) ability to resolve ionizing radiation-induced DNA damage foci. Our method constitutes a rapid, efficient, and cost-effective approach for isolation and subsequent characterization of rare cells based on observable traits such as movement, shape, or location, which in turn can generate novel mechanistic insights into important biological processes.

5.
Lab Chip ; 19(3): 464-474, 2019 01 29.
Artículo en Inglés | MEDLINE | ID: mdl-30570636

RESUMEN

On-chip high-throughput phenotyping of single cells has gained a lot of interest recently due to the discrimination capability of label-free biomarkers such as whole-cell deformability and refractive index. Here we present on-chip refractive index cytometry (RIC) for whole-cell deformability at a high measurement rate. We have further exploited a previously published on-chip optical characterization method which enhances cellular discrimination through the refractive index measurement of single cells. The proposed on-chip RIC can simultaneously probe the cellular refractive index, effective volume and whole-cell deformability while reaching a measurement rate up to 5000 cells per second. Additionally, the relative position of the nucleus inside the cell is reflected by the asymmetry of the measured curve. This particular finding is confirmed by our numerical simulation model and emphasized by a modified cytoskeleton HL-60 cells model. Furthermore, the proposed device discriminated HL-60 derived myeloid cells such as neutrophils, basophils and promyelocytes, which are indistinguishable using flow cytometry. To our knowledge, this is the first integrated device to simultaneously characterize the cellular refractive index and whole-cell deformability, yielding enhanced discrimination of large myeloid cell populations.


Asunto(s)
Citometría de Flujo/instrumentación , Dispositivos Laboratorio en un Chip , Fenómenos Mecánicos , Refractometría/instrumentación , Análisis de la Célula Individual/instrumentación , Fenómenos Biomecánicos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Citocalasina B/farmacología , Células HL-60 , Humanos , Fenómenos Mecánicos/efectos de los fármacos
7.
Cell Death Differ ; 25(11): 1885-1904, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30323273

RESUMEN

The relative contribution of intrinsic genetic factors and extrinsic environmental ones to cancer aetiology and natural history is a lengthy and debated issue. Gene-environment interactions (G x E) arise when the combined presence of both a germline genetic variant and a known environmental factor modulates the risk of disease more than either one alone. A panel of experts discussed our current understanding of cancer aetiology, known examples of G × E interactions in cancer, and the expanded concept of G × E interactions to include somatic cancer mutations and iatrogenic environmental factors such as anti-cancer treatment. Specific genetic polymorphisms and genetic mutations increase susceptibility to certain carcinogens and may be targeted in the near future for prevention and treatment of cancer patients with novel molecularly based therapies. There was general consensus that a better understanding of the complexity and numerosity of G × E interactions, supported by adequate technological, epidemiological, modelling and statistical resources, will further promote our understanding of cancer and lead to novel preventive and therapeutic approaches.


Asunto(s)
Interacción Gen-Ambiente , Neoplasias/genética , Medicina de Precisión , Carcinogénesis , Consenso , Daño del ADN , Estudio de Asociación del Genoma Completo , Humanos , Neoplasias/epidemiología , Neoplasias/patología , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo
8.
Nat Commun ; 9(1): 4385, 2018 10 22.
Artículo en Inglés | MEDLINE | ID: mdl-30349006

RESUMEN

The tumor suppressor and deubiquitinase (DUB) BAP1 and its Drosophila ortholog Calypso assemble DUB complexes with the transcription regulators Additional sex combs-like (ASXL1, ASXL2, ASXL3) and Asx respectively. ASXLs and Asx use their DEUBiquitinase ADaptor (DEUBAD) domain to stimulate BAP1/Calypso DUB activity. Here we report that monoubiquitination of the DEUBAD is a general feature of ASXLs and Asx. BAP1 promotes DEUBAD monoubiquitination resulting in an increased stability of ASXL2, which in turn stimulates BAP1 DUB activity. ASXL2 monoubiquitination is directly catalyzed by UBE2E family of Ubiquitin-conjugating enzymes and regulates mammalian cell proliferation. Remarkably, Calypso also regulates Asx monoubiquitination and transgenic flies expressing monoubiquitination-defective Asx mutant exhibit developmental defects. Finally, the protein levels of ASXL2, BAP1 and UBE2E enzymes are highly correlated in mesothelioma tumors suggesting the importance of this signaling axis for tumor suppression. We propose that monoubiquitination orchestrates a molecular symbiosis relationship between ASXLs and BAP1.


Asunto(s)
Proteínas de Drosophila/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Animales , Sistemas CRISPR-Cas/genética , Ciclo Celular/genética , Ciclo Celular/fisiología , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Drosophila , Proteínas de Drosophila/genética , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Inmunoprecipitación , ARN Interferente Pequeño/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteínas Supresoras de Tumor/genética , Ubiquitina Tiolesterasa/genética , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación/genética , Ubiquitinación/fisiología
9.
J Clin Invest ; 128(10): 4525-4542, 2018 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-30222135

RESUMEN

The E3 ubiquitin ligase RNF8 plays critical roles in maintaining genomic stability by promoting the repair of DNA double-strand breaks (DSBs) through ubiquitin signaling. Abnormal activation of Notch signaling and defective repair of DSBs promote breast cancer risk. Here, we found that low expression of the full-length RNF8 correlated with poor prognosis for breast cancer patients. Our data revealed that in addition to its role in the repair of DSBs, RNF8 regulated Notch1 signaling and cell-fate determination of mammary luminal progenitors. Mechanistically, RNF8 acted as a negative regulator of Notch signaling by ubiquitylating the active NOTCH1 protein (N1ICD), leading to its degradation. Consistent with abnormal activation of Notch signaling and impaired repair of DSBs in Rnf8-mutant mammary epithelial cells, we observed increased risk of mammary tumorigenesis in mouse models for RNF8 deficiency. Notably, deficiency of RNF8 sensitized breast cancer cells to combination of pharmacological inhibitors of Notch signaling and poly(ADP-ribose) polymerase (PARP), suggesting implications for treatment of breast cancer associated with impaired RNF8 expression or function.


Asunto(s)
Carcinogénesis/metabolismo , Glándulas Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/metabolismo , Proteínas de Neoplasias/metabolismo , Receptor Notch1/metabolismo , Transducción de Señal , Ubiquitina-Proteína Ligasas/biosíntesis , Animales , Carcinogénesis/genética , Carcinogénesis/patología , Roturas del ADN de Doble Cadena , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/patología , Ratones , Ratones Noqueados , Proteínas de Neoplasias/genética , Receptor Notch1/genética , Ubiquitina-Proteína Ligasas/genética
10.
Nature ; 546(7659): 549-553, 2017 06 22.
Artículo en Inglés | MEDLINE | ID: mdl-28614305

RESUMEN

BRCA1-associated protein 1 (BAP1) is a potent tumour suppressor gene that modulates environmental carcinogenesis. All carriers of inherited heterozygous germline BAP1-inactivating mutations (BAP1+/-) developed one and often several BAP1-/- malignancies in their lifetime, mostly malignant mesothelioma, uveal melanoma, and so on. Moreover, BAP1-acquired biallelic mutations are frequent in human cancers. BAP1 tumour suppressor activity has been attributed to its nuclear localization, where it helps to maintain genome integrity. The possible activity of BAP1 in the cytoplasm is unknown. Cells with reduced levels of BAP1 exhibit chromosomal abnormalities and decreased DNA repair by homologous recombination, indicating that BAP1 dosage is critical. Cells with extensive DNA damage should die and not grow into malignancies. Here we discover that BAP1 localizes at the endoplasmic reticulum. Here, it binds, deubiquitylates, and stabilizes type 3 inositol-1,4,5-trisphosphate receptor (IP3R3), modulating calcium (Ca2+) release from the endoplasmic reticulum into the cytosol and mitochondria, promoting apoptosis. Reduced levels of BAP1 in BAP1+/- carriers cause reduction both of IP3R3 levels and of Ca2+ flux, preventing BAP1+/- cells that accumulate DNA damage from executing apoptosis. A higher fraction of cells exposed to either ionizing or ultraviolet radiation, or to asbestos, survive genotoxic stress, resulting in a higher rate of cellular transformation. We propose that the high incidence of cancers in BAP1+/- carriers results from the combined reduced nuclear and cytoplasmic activities of BAP1. Our data provide a mechanistic rationale for the powerful ability of BAP1 to regulate gene-environment interaction in human carcinogenesis.


Asunto(s)
Calcio/metabolismo , Transformación Celular Neoplásica , Citoplasma/metabolismo , Retículo Endoplásmico/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Mitocondrias/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Apoptosis/genética , Asbestos/toxicidad , Señalización del Calcio , Núcleo Celular/metabolismo , Supervivencia Celular , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/efectos de la radiación , Células Cultivadas , Daño del ADN , Epitelio , Fibroblastos , Interacción Gen-Ambiente , Humanos , Unión Proteica , Estabilidad Proteica , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética , Ubiquitina/metabolismo , Ubiquitina Tiolesterasa/deficiencia , Ubiquitina Tiolesterasa/genética
11.
Sci Rep ; 6: 29389, 2016 07 08.
Artículo en Inglés | MEDLINE | ID: mdl-27388124

RESUMEN

APE1 is an essential DNA repair protein that also possesses the ability to regulate transcription. It has a unique cysteine residue C65, which maintains the reduce state of several transcriptional activators such as NF-κB. How APE1 is being recruited to execute the various biological functions remains unknown. Herein, we show that APE1 interacts with a novel partner PRDX1, a peroxidase that can also prevent oxidative damage to proteins by serving as a chaperone. PRDX1 knockdown did not interfere with APE1 expression level or its DNA repair activities. However, PRDX1 knockdown greatly facilitates APE1 detection within the nucleus by indirect immunofluorescence analysis, even though APE1 level was unchanged. The loss of APE1 interaction with PRDX1 promotes APE1 redox function to activate binding of the transcription factor NF-κB onto the promoter of a target gene, the proinflammatory chemokine IL-8 involved in cancer invasion and metastasis, resulting in its upregulation. Depletion of APE1 blocked the upregulation of IL-8 in the PRDX1 knockdown cells. Our findings suggest that the interaction of PRDX1 with APE1 represents a novel anti-inflammatory function of PRDX1, whereby the association safeguards APE1 from reducing transcription factors and activating superfluous gene expression, which otherwise could trigger cancer invasion and metastasis.


Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Interleucina-8/genética , FN-kappa B/metabolismo , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Neoplasias Gástricas/genética , Núcleo Celular/metabolismo , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Peróxido de Hidrógeno/farmacología , Invasividad Neoplásica , Metástasis de la Neoplasia , Estrés Oxidativo , Regiones Promotoras Genéticas , Neoplasias Gástricas/metabolismo , Activación Transcripcional
12.
Nat Commun ; 7: 11636, 2016 05 20.
Artículo en Inglés | MEDLINE | ID: mdl-27198043

RESUMEN

The ability to conduct image-based, non-invasive cell tagging, independent of genetic engineering, is key to cell biology applications. Here we introduce cell labelling via photobleaching (CLaP), a method that enables instant, specific tagging of individual cells based on a wide array of criteria such as shape, behaviour or positional information. CLaP uses laser illumination to crosslink biotin onto the plasma membrane, coupled with streptavidin conjugates to label individual cells for genomic, cell-tracking, flow cytometry or ultra-microscopy applications. We show that the incorporated mark is stable, non-toxic, retained for several days, and transferred by cell division but not to adjacent cells in culture. To demonstrate the potential of CLaP for genomic applications, we combine CLaP with microfluidics-based single-cell capture followed by transcriptome-wide next-generation sequencing. Finally, we show that CLaP can also be exploited for inducing transient cell adhesion to substrates for microengineering cultures with spatially patterned cell types.


Asunto(s)
Fotoblanqueo , Análisis de la Célula Individual/métodos , Coloración y Etiquetado/métodos , Animales , Perros , Genómica/métodos , Humanos , Rayos Láser , Células de Riñón Canino Madin Darby
13.
Proc Natl Acad Sci U S A ; 113(5): 1393-8, 2016 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-26764384

RESUMEN

Oncogenic transcription factors are commonly activated in acute leukemias and subvert normal gene expression networks to reprogram hematopoietic progenitors into preleukemic stem cells, as exemplified by LIM-only 2 (LMO2) in T-cell acute lymphoblastic leukemia (T-ALL). Whether or not these oncoproteins interfere with other DNA-dependent processes is largely unexplored. Here, we show that LMO2 is recruited to DNA replication origins by interaction with three essential replication enzymes: DNA polymerase delta (POLD1), DNA primase (PRIM1), and minichromosome 6 (MCM6). Furthermore, tethering LMO2 to synthetic DNA sequences is sufficient to transform these sequences into origins of replication. We next addressed the importance of LMO2 in erythroid and thymocyte development, two lineages in which cell cycle and differentiation are tightly coordinated. Lowering LMO2 levels in erythroid progenitors delays G1-S progression and arrests erythropoietin-dependent cell growth while favoring terminal differentiation. Conversely, ectopic expression in thymocytes induces DNA replication and drives these cells into cell cycle, causing differentiation blockade. Our results define a novel role for LMO2 in directly promoting DNA synthesis and G1-S progression.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Replicación del ADN/genética , Células Madre Hematopoyéticas/metabolismo , Proteínas con Dominio LIM/genética , Animales , Células Madre Hematopoyéticas/citología , Ratones , Origen de Réplica , Fase S
14.
J Biol Chem ; 290(48): 28643-63, 2015 Nov 27.
Artículo en Inglés | MEDLINE | ID: mdl-26416890

RESUMEN

The deubiquitinase (DUB) and tumor suppressor BAP1 catalyzes ubiquitin removal from histone H2A Lys-119 and coordinates cell proliferation, but how BAP1 partners modulate its function remains poorly understood. Here, we report that BAP1 forms two mutually exclusive complexes with the transcriptional regulators ASXL1 and ASXL2, which are necessary for maintaining proper protein levels of this DUB. Conversely, BAP1 is essential for maintaining ASXL2, but not ASXL1, protein stability. Notably, cancer-associated loss of BAP1 expression results in ASXL2 destabilization and hence loss of its function. ASXL1 and ASXL2 use their ASXM domains to interact with the C-terminal domain (CTD) of BAP1, and these interactions are required for ubiquitin binding and H2A deubiquitination. The deubiquitination-promoting effect of ASXM requires intramolecular interactions between catalytic and non-catalytic domains of BAP1, which generate a composite ubiquitin-binding interface (CUBI). Notably, the CUBI engages multiple interactions with ubiquitin involving (i) the ubiquitin carboxyl hydrolase catalytic domain of BAP1, which interacts with the hydrophobic patch of ubiquitin, and (ii) the CTD domain, which interacts with a charged patch of ubiquitin. Significantly, we identified cancer-associated mutations of BAP1 that disrupt the CUBI and notably an in-frame deletion in the CTD that inhibits its interaction with ASXL1/2 and DUB activity and deregulates cell proliferation. Moreover, we demonstrated that BAP1 interaction with ASXL2 regulates cell senescence and that ASXL2 cancer-associated mutations disrupt BAP1 DUB activity. Thus, inactivation of the BAP1/ASXL2 axis might contribute to cancer development.


Asunto(s)
Proliferación Celular , Neoplasias/metabolismo , Proteínas Represoras/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Células HEK293 , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Neoplasias/genética , Neoplasias/patología , Proteínas Represoras/genética , Proteínas Supresoras de Tumor/genética , Ubiquitina Tiolesterasa/genética , Proteasas Ubiquitina-Específicas/genética
15.
Epigenetics ; 10(8): 677-91, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26075789

RESUMEN

O-GlcNAcylation is a posttranslational modification catalyzed by the O-Linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and reversed by O-GlcNAcase (OGA). Numerous transcriptional regulators, including chromatin modifying enzymes, transcription factors, and co-factors, are targeted by O-GlcNAcylation, indicating that this modification is central for chromatin-associated processes. Recently, OGT-mediated O-GlcNAcylation was reported to be a novel histone modification, suggesting a potential role in directly coordinating chromatin structure and function. In contrast, using multiple biochemical approaches, we report here that histone O-GlcNAcylation is undetectable in mammalian cells. Conversely, O-GlcNAcylation of the transcription regulators Host Cell Factor-1 (HCF-1) and Ten-Eleven Translocation protein 2 (TET2) could be readily observed. Our study raises questions on the occurrence and abundance of O-GlcNAcylation as a histone modification in mammalian cells and reveals technical complications regarding the detection of genuine protein O-GlcNAcylation. Therefore, the identification of the specific contexts in which histone O-GlcNAcylation might occur is still to be established.


Asunto(s)
Proteínas de Unión al ADN/genética , Epigénesis Genética , Histonas/genética , Factor C1 de la Célula Huésped/genética , Proteínas Proto-Oncogénicas/genética , beta-N-Acetilhexosaminidasas/genética , Acilación , Animales , Cromatina/genética , Glicosilación , Células HEK293 , Histonas/metabolismo , Humanos , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Procesamiento Proteico-Postraduccional/genética , beta-N-Acetilhexosaminidasas/metabolismo
16.
PLoS Genet ; 10(12): e1004827, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25474253

RESUMEN

IKAROS is a critical regulator of hematopoietic cell fate and its dynamic expression pattern is required for proper hematopoiesis. In collaboration with the Nucleosome Remodeling and Deacetylase (NuRD) complex, it promotes gene repression and activation. It remains to be clarified how IKAROS can support transcription activation while being associated with the HDAC-containing complex NuRD. IKAROS also binds to the Positive-Transcription Elongation Factor b (P-TEFb) at gene promoters. Here, we demonstrate that NuRD and P-TEFb are assembled in a complex that can be recruited to specific genes by IKAROS. The expression level of IKAROS influences the recruitment of the NuRD-P-TEFb complex to gene regulatory regions and facilitates transcription elongation by transferring the Protein Phosphatase 1α (PP1α), an IKAROS-binding protein and P-TEFb activator, to CDK9. We show that an IKAROS mutant that is unable to bind PP1α cannot sustain gene expression and impedes normal differentiation of Ik(NULL) hematopoietic progenitors. Finally, the knock-down of the NuRD subunit Mi2 reveals that the occupancy of the NuRD complex at transcribed regions of genes favors the relief of POL II promoter-proximal pausing and thereby, promotes transcription elongation.


Asunto(s)
Ensamble y Desensamble de Cromatina , Hematopoyesis , Factor de Transcripción Ikaros/metabolismo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Factor B de Elongación Transcripcional Positiva/metabolismo , Animales , Células COS , Ensamble y Desensamble de Cromatina/genética , Hematopoyesis/genética , Humanos , Factor de Transcripción Ikaros/genética , Células Jurkat , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Ratones , Ratones Noqueados , Nucleosomas/metabolismo , Unión Proteica , ARN Polimerasa II/metabolismo , Activación Transcripcional
17.
Mol Cell ; 54(3): 392-406, 2014 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-24703950

RESUMEN

The tumor suppressor BAP1 interacts with chromatin-associated proteins and regulates cell proliferation, but its mechanism of action and regulation remain poorly defined. We show that the ubiquitin-conjugating enzyme UBE2O multi-monoubiquitinates the nuclear localization signal of BAP1, thereby inducing its cytoplasmic sequestration. This activity is counteracted by BAP1 autodeubiquitination through intramolecular interactions. Significantly, we identified cancer-derived BAP1 mutations that abrogate autodeubiquitination and promote its cytoplasmic retention, indicating that BAP1 autodeubiquitination ensures tumor suppression. The antagonistic relationship between UBE2O and BAP1 is also observed during adipogenesis, whereby UBE2O promotes differentiation and cytoplasmic localization of BAP1. Finally, we established a putative targeting consensus sequence of UBE2O and identified numerous chromatin remodeling factors as potential targets, several of which tested positive for UBE2O-mediated ubiquitination. Thus, UBE2O defines an atypical ubiquitin-signaling pathway that coordinates the function of BAP1 and establishes a paradigm for regulation of nuclear trafficking of chromatin-associated proteins.


Asunto(s)
Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación , Células 3T3-L1 , Adipocitos/fisiología , Secuencia de Aminoácidos , Animales , Diferenciación Celular , Secuencia de Consenso , Citoplasma/metabolismo , Células HEK293 , Humanos , Ratones , Datos de Secuencia Molecular , Mutación Missense , Neoplasias/genética , Señales de Localización Nuclear , Transporte de Proteínas , Transducción de Señal , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Ubiquitina Tiolesterasa/química , Ubiquitina Tiolesterasa/genética
18.
Proc Natl Acad Sci U S A ; 111(1): 285-90, 2014 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-24347639

RESUMEN

The cellular response to highly genotoxic DNA double-strand breaks (DSBs) involves the exquisite coordination of multiple signaling and repair factors. Here, we conducted a functional RNAi screen and identified BAP1 as a deubiquitinase required for efficient assembly of the homologous recombination (HR) factors BRCA1 and RAD51 at ionizing radiation (IR) -induced foci. BAP1 is a chromatin-associated protein frequently inactivated in cancers of various tissues. To further investigate the role of BAP1 in DSB repair, we used a gene targeting approach to knockout (KO) this deubiquitinase in chicken DT40 cells. We show that BAP1-deficient cells are (i) sensitive to IR and other agents that induce DSBs, (ii) defective in HR-mediated immunoglobulin gene conversion, and (iii) exhibit an increased frequency of chromosomal breaks after IR treatment. We also show that BAP1 is recruited to chromatin in the proximity of a single site-specific I-SceI-induced DSB. Finally, we identified six IR-induced phosphorylation sites in BAP1 and showed that mutation of these residues inhibits BAP1 recruitment to DSB sites. We also found that both BAP1 catalytic activity and its phosphorylation are critical for promoting DNA repair and cellular recovery from DNA damage. Our data reveal an important role for BAP1 in DSB repair by HR, thereby providing a possible molecular basis for its tumor suppressor function.


Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Regulación Neoplásica de la Expresión Génica , Recombinación Homóloga , Neoplasias/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Animales , Proteína BRCA1/metabolismo , Línea Celular , Línea Celular Tumoral , Pollos , Daño del ADN , Células HEK293 , Células HeLa , Humanos , Inmunoglobulinas/genética , Células MCF-7 , Microscopía Fluorescente , Mutación , Neoplasias/genética , Fenotipo , Fosforilación , Recombinasa Rad51 , Radiación Ionizante
19.
Mol Cell Biol ; 33(16): 3064-76, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23732910

RESUMEN

Ikaros (Ik) is a critical regulator of hematopoietic gene expression. Here, we established that the Ik interactions with GATA transcription factors and cyclin-dependent kinase 9 (Cdk9), a component of the positive transcription elongation factor b (P-TEFb), are required for transcriptional activation of Ik target genes. A detailed dissection of Ik-GATA and Ik-Cdk9 protein interactions indicated that the C-terminal zinc finger domain of Ik interacts directly with the C-terminal zinc fingers of GATA1, GATA2, and GATA3, whereas the N-terminal zinc finger domain of Ik is required for interaction with the kinase and T-loop domains of Cdk9. The relevance of these interactions was demonstrated in vivo in COS-7 and primary hematopoietic cells, in which Ik facilitated Cdk9 and GATA protein recruitment to gene promoters and transcriptional activation. Moreover, the oncogenic isoform Ik6 did not efficiently interact with Cdk9 or GATA proteins in vivo and perturbed Cdk9/P-TEFb recruitment to Ik target genes, thereby affecting transcription elongation. Finally, characterization of a novel nuclear Ik isoform revealed that Ik exon 6 is dispensable for interactions with Mi2 and GATA proteins but is essential for the Cdk9 interaction. Thus, Ik is central to the Ik-GATA-Cdk9 regulatory network, which is broadly utilized for gene regulation in hematopoietic cells.


Asunto(s)
Quinasa 9 Dependiente de la Ciclina/metabolismo , Factor de Transcripción GATA1/metabolismo , Factor de Transcripción GATA2/metabolismo , Factor de Transcripción GATA3/metabolismo , Hematopoyesis , Factor de Transcripción Ikaros/metabolismo , Activación Transcripcional , Animales , Línea Celular , Células Cultivadas , Quinasa 9 Dependiente de la Ciclina/química , Factor de Transcripción GATA1/química , Factor de Transcripción GATA2/química , Factor de Transcripción GATA3/química , Factor de Transcripción Ikaros/química , Ratones , Dominios y Motivos de Interacción de Proteínas , Mapas de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo
20.
Proteomics ; 13(6): 982-91, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23335398

RESUMEN

The development of electron-based, unimolecular dissociation MS, i.e. electron capture and electron transfer dissociation (ECD and ETD, respectively), has greatly increased the speed and reliability of labile PTM site assignment. The field of intracellular O-GlcNAc (O-linked N-acetylglucosamine) signaling has especially advanced with the advent of ETD MS. Only within the last five years have proteomic-scale experiments utilizing ETD allowed the assignment of hundreds of O-GlcNAc sites within cells and subcellular structures. Our ability to identify and unambiguously assign the site of O-GlcNAc modifications using ETD is rapidly increasing our understanding of this regulatory glycosylation and its potential interaction with other PTMs. Here, we discuss the advantages of using ETD, complimented with collisional-activation MS, in a study of the extensively O-GlcNAcylated protein Host Cell Factor C1 (HCF-1). HCF-1 is a transcriptional coregulator that forms a stable complex with O-GlcNAc transferase and controls cell cycle progression. ETD, along with higher energy collisional dissociation (HCD) MS, was employed to assign the PTMs of the HCF-1 protein isolated from HEK293T cells. These include 19 sites of O-GlcNAcylation, two sites of phosphorylation, and two sites bearing dimethylarginine, and showcase the residue-specific, PTM complexity of this regulator of cell proliferation.


Asunto(s)
Factor C1 de la Célula Huésped/química , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Glicopéptidos/química , Glicopéptidos/aislamiento & purificación , Glicosilación , Células HEK293 , Factor C1 de la Célula Huésped/aislamiento & purificación , Factor C1 de la Célula Huésped/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos
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