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1.
EMBO Mol Med ; 15(12): e18459, 2023 Dec 07.
Artículo en Inglés | MEDLINE | ID: mdl-37937685

RESUMEN

Triple-negative breast cancer (TNBC) often develops resistance to single-agent treatment, which can be circumvented using targeted combinatorial approaches. Here, we demonstrate that the simultaneous inhibition of LOXL2 and BRD4 synergistically limits TNBC proliferation in vitro and in vivo. Mechanistically, LOXL2 interacts in the nucleus with the short isoform of BRD4 (BRD4S), MED1, and the cell cycle transcriptional regulator B-MyB. These interactions sustain the formation of BRD4 and MED1 nuclear transcriptional foci and control cell cycle progression at the gene expression level. The pharmacological co-inhibition of LOXL2 and BRD4 reduces BRD4 nuclear foci, BRD4-MED1 colocalization, and the transcription of cell cycle genes, thus suppressing TNBC cell proliferation. Targeting the interaction between BRD4S and LOXL2 could be a starting point for the development of new anticancer strategies for the treatment of TNBC.


Asunto(s)
Factores de Transcripción , Neoplasias de la Mama Triple Negativas , Humanos , Aminoácido Oxidorreductasas/genética , Aminoácido Oxidorreductasas/metabolismo , Proteínas que Contienen Bromodominio , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Subunidad 1 del Complejo Mediador/genética , Subunidad 1 del Complejo Mediador/metabolismo , Proteínas Nucleares/genética , Factores de Transcripción/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Animales
2.
Mol Syst Biol ; 19(7): e11267, 2023 07 11.
Artículo en Inglés | MEDLINE | ID: mdl-37259925

RESUMEN

While cellular metabolism impacts the DNA damage response, a systematic understanding of the metabolic requirements that are crucial for DNA damage repair has yet to be achieved. Here, we investigate the metabolic enzymes and processes that are essential for the resolution of DNA damage. By integrating functional genomics with chromatin proteomics and metabolomics, we provide a detailed description of the interplay between cellular metabolism and the DNA damage response. Further analysis identified that Peroxiredoxin 1, PRDX1, contributes to the DNA damage repair. During the DNA damage response, PRDX1 translocates to the nucleus where it reduces DNA damage-induced nuclear reactive oxygen species. Moreover, PRDX1 loss lowers aspartate availability, which is required for the DNA damage-induced upregulation of de novo nucleotide synthesis. In the absence of PRDX1, cells accumulate replication stress and DNA damage, leading to proliferation defects that are exacerbated in the presence of etoposide, thus revealing a role for PRDX1 as a DNA damage surveillance factor.


Asunto(s)
Ácido Aspártico , Peroxirredoxinas , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Daño del ADN , Estrés Oxidativo/genética , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Humanos
3.
Mol Genet Genomics ; 297(2): 463-484, 2022 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-35187582

RESUMEN

BRG1 and BRM are ATPase core subunits of the human SWI/SNF chromatin remodelling complexes mainly associated with transcriptional initiation. They also have a role in alternative splicing, which has been shown for BRM-containing SWI/SNF complexes at a few genes. Here, we have identified a subset of genes which harbour alternative exons that are affected by SWI/SNF ATPases by expressing the ATPases BRG1 and BRM in C33A cells, a BRG1- and BRM-deficient cell line, and analysed the effect on splicing by RNA sequencing. BRG1- and BRM-affected sub-sets of genes favouring both exon inclusion and exon skipping, with only a minor overlap between the ATPase. Some of the changes in alternative splicing induced by BRG1 and BRM expression did not require the ATPase activity. The BRG1-ATPase independent included exons displayed an exon signature of a high GC content. By investigating three genes with exons affected by the BRG-ATPase-deficient variant, we show that these exons accumulated phosphorylated RNA pol II CTD, both serine 2 and serine 5 phosphorylation, without an enrichment of the RNA polymerase II. The ATPases were recruited to the alternative exons, together with both core and signature subunits of SWI/SNF complexes, and promoted the binding of RNA binding factors to chromatin and RNA at the alternative exons. The interaction with the nascent RNP, however, did not reflect the association to chromatin. The hnRNPL, hnRNPU and SAM68 proteins associated with chromatin in cells expressing BRG1 and BRM wild type, but the binding of hnRNPU to the nascent RNP was excluded. This suggests that SWI/SNF can regulate alternative splicing by interacting with splicing-RNA binding factor and influence their binding to the nascent pre-mRNA particle.


Asunto(s)
ADN Helicasas , Proteínas Nucleares , ARN , Factores de Transcripción , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Empalme Alternativo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , ARN/genética , ARN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
4.
FASEB J ; 34(8): 10818-10834, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32598531

RESUMEN

Regulation of ribosomal transcription is under tight control from environmental stimuli, and this control involves changes in the chromatin structure. The underlying mechanism of how chromatin changes in response to nutrient and energy supply in the cell is still unclear. The chromatin-remodeling complex B-WICH is involved in activating the ribosomal transcription, and we show here that knock down of the B-WICH component WSTF results in cells that do not respond to glucose. The promoter is less accessible, and RNA pol I and its transcription factors SL1/TIF-1B and RRN3/TIF-1A, as well as the proto-oncogene c-MYC and the activating deacetylase SIRT7 do not bind upon glucose stimulation. In contrast, the repressive chromatin state that forms after glucose deprivation is reversible, and RNA pol I factors are recruited. WSTF knock down results in an accumulation of the ATPase CHD4, a component of the NuRD chromatin remodeling complex, which is responsible for establishing a repressive poised state at the promoter. The TTF-1, which binds and affect the binding of the chromatin complexes, is important to control the association of activating chromatin component UBF. We suggest that B-WICH is required to allow for a shift to an active chromatin state upon environmental stimulation, by counteracting the repressive state induced by the NuRD complex.


Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Cromatina/genética , Glucosa/genética , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Ribosomas/genética , Transcripción Genética/genética , Adenosina Trifosfatasas/genética , Línea Celular , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Células HEK293 , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas del Complejo de Iniciación de Transcripción Pol1/genética , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-myc/genética , ARN Polimerasa I/genética , Sirtuinas/genética , Factores de Transcripción/genética
5.
F1000Res ; 9: 1336, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-34745570

RESUMEN

The COVID-19 pandemic has posed and is continuously posing enormous societal and health challenges worldwide. The research community has mobilized to develop novel projects to find a cure or a vaccine, as well as to contribute to mass testing, which has been a critical measure to contain the infection in several countries. Through this article, we share our experiences and learnings as a group of volunteers at the Centre for Genomic Regulation (CRG) in Barcelona, Spain. As members of the ORFEU project, an initiative by the Government of Catalonia to achieve mass testing of people at risk and contain the epidemic in Spain, we share our motivations, challenges and the key lessons learnt, which we feel will help better prepare the global society to address similar situations in the future.


Asunto(s)
COVID-19 , Prueba de COVID-19 , Genómica , Humanos , Pandemias , SARS-CoV-2 , Voluntarios
6.
Nucleic Acids Res ; 46(16): 8557-8573, 2018 09 19.
Artículo en Inglés | MEDLINE | ID: mdl-29860334

RESUMEN

SWI/SNF complexes associate with genes and regulate transcription by altering the chromatin at the promoter. It has recently been shown that these complexes play a role in pre-mRNA processing by associating at alternative splice sites. Here, we show that SWI/SNF complexes are involved also in pre-mRNA 3' end maturation by facilitating 3' end cleavage of specific pre-mRNAs. Comparative proteomics show that SWI/SNF ATPases interact physically with subunits of the cleavage and polyadenylation complexes in fly and human cells. In Drosophila melanogaster, the SWI/SNF ATPase Brahma (dBRM) interacts with the CPSF6 subunit of cleavage factor I. We have investigated the function of dBRM in 3' end formation in S2 cells by RNA interference, single-gene analysis and RNA sequencing. Our data show that dBRM facilitates pre-mRNA cleavage in two different ways: by promoting the association of CPSF6 to the cleavage region and by stabilizing positioned nucleosomes downstream of the cleavage site. These findings show that SWI/SNF complexes play a role also in the cleavage of specific pre-mRNAs in animal cells.


Asunto(s)
Regiones no Traducidas 3'/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Procesamiento de Término de ARN 3' , Ribonucleoproteína Nuclear Pequeña U1/genética , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Tumoral , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Células HeLa , Humanos , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Transactivadores/genética , Transactivadores/metabolismo
7.
PLoS Genet ; 11(9): e1005523, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26389589

RESUMEN

RNA surveillance factors are involved in heterochromatin regulation in yeast and plants, but less is known about the possible roles of ribonucleases in the heterochromatin of animal cells. Here we show that RRP6, one of the catalytic subunits of the exosome, is necessary for silencing heterochromatic repeats in the genome of Drosophila melanogaster. We show that a fraction of RRP6 is associated with heterochromatin, and the analysis of the RRP6 interaction network revealed physical links between RRP6 and the heterochromatin factors HP1a, SU(VAR)3-9 and RPD3. Moreover, genome-wide studies of RRP6 occupancy in cells depleted of SU(VAR)3-9 demonstrated that SU(VAR)3-9 contributes to the tethering of RRP6 to a subset of heterochromatic loci. Depletion of the exosome ribonucleases RRP6 and DIS3 stabilizes heterochromatic transcripts derived from transposons and repetitive sequences, and renders the heterochromatin less compact, as shown by micrococcal nuclease and proximity-ligation assays. Such depletion also increases the amount of HP1a bound to heterochromatic transcripts. Taken together, our results suggest that SU(VAR)3-9 targets RRP6 to a subset of heterochromatic loci where RRP6 degrades chromatin-associated non-coding RNAs in a process that is necessary to maintain the packaging of the heterochromatin.


Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Heterocromatina/metabolismo , Proteínas Represoras/metabolismo , Animales , Elementos Transponibles de ADN , Drosophila melanogaster/genética , Silenciador del Gen , Genoma , Heterocromatina/genética , Unión Proteica , ARN Mensajero/genética
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