RESUMEN
Transcription by RNA polymerase II (Pol II) is coupled to pre-mRNA splicing, but the underlying mechanisms remain poorly understood. Co-transcriptional splicing requires assembly of a functional spliceosome on nascent pre-mRNA, but whether and how this influences Pol II transcription remains unclear. Here we show that inhibition of pre-mRNA branch site recognition by the spliceosome component U2 snRNP leads to a widespread and strong decrease in new RNA synthesis from human genes. Multiomics analysis reveals that inhibition of U2 snRNP function increases the duration of Pol II pausing in the promoter-proximal region, impairs recruitment of the pause release factor P-TEFb, and reduces Pol II elongation velocity at the beginning of genes. Our results indicate that efficient release of paused Pol II into active transcription elongation requires the formation of functional spliceosomes and that eukaryotic mRNA biogenesis relies on positive feedback from the splicing machinery to the transcription machinery.
Asunto(s)
ARN Polimerasa II/metabolismo , ARN Mensajero/biosíntesis , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Empalmosomas/enzimología , Elongación de la Transcripción Genética , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Retroalimentación Fisiológica , Regulación de la Expresión Génica , Células HeLa , Humanos , Células K562 , Factor B de Elongación Transcripcional Positiva/genética , Factor B de Elongación Transcripcional Positiva/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa II/genética , Precursores del ARN/genética , Precursores del ARN/metabolismo , Empalme del ARN , ARN Mensajero/genética , Ribonucleoproteína Nuclear Pequeña U2/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Empalmosomas/genética , Factores de TiempoRESUMEN
Cyclin-dependent kinase 12 (CDK12) phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (pol II) but its roles in transcription beyond the expression of DNA damage response genes remain unclear. Here, we have used TT-seq and mNET-seq to monitor the direct effects of rapid CDK12 inhibition on transcription activity and CTD phosphorylation in human cells. CDK12 inhibition causes a genome-wide defect in transcription elongation and a global reduction of CTD Ser2 and Ser5 phosphorylation. The elongation defect is explained by the loss of the elongation factors LEO1 and CDC73, part of PAF1 complex, and SPT6 from the newly-elongating pol II. Our results indicate that CDK12 is a general activator of pol II transcription elongation and indicate that it targets both Ser2 and Ser5 residues of the pol II CTD.
Asunto(s)
Quinasas Ciclina-Dependientes/fisiología , ARN Polimerasa II/metabolismo , Elongación de la Transcripción Genética , Cromatina/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Células HEK293 , Humanos , Mutación , Fosforilación , ARN/biosíntesis , ARN Polimerasa II/química , Análisis de Secuencia de ARN , Serina/metabolismo , Factores de Elongación Transcripcional/metabolismoRESUMEN
Cellular senescence is an intrinsic defense mechanism to various cellular stresses: while still metabolically active, senescent cells stop dividing and enter a proliferation arrest. Here, we identify DPY30, a member of all mammalian histone H3K4 histone methyltransferases (HMTases), as a key regulator of the proliferation potential of human primary cells. Following depletion of DPY30, cells show a severe proliferation defect and display a senescent phenotype, including a flattened and enlarged morphology, elevated level of reactive oxygen species (ROS), increased SA-ß-galactosidase activity, and formation of senescence-associated heterochromatin foci (SAHFs). While DPY30 depletion leads to a reduced level of H3K4me3-marked active chromatin, we observed a concomitant activation of CDK inhibitors, including p16INK4a, independent of H3K4me3. ChIP experiments show that key regulators of cell-cycle progression, including ID proteins, are under direct control of DPY30. Because ID proteins are negative regulators of the transcription factors ETS1/2, depletion of DPY30 leads to the transcriptional activation of p16INK4a by ETS1/2 and thus to a senescent-like phenotype. Ectoptic re-introduction of ID protein expression can partially rescue the senescence-like phenotype induced by DPY30 depletion. Thus, our data indicate that DPY30 controls proliferation by regulating ID proteins expression, which in turn lead to senescence bypass.
Asunto(s)
Senescencia Celular/fisiología , Regulación de la Expresión Génica/fisiología , Proteína 1 Inhibidora de la Diferenciación/metabolismo , Proteínas Nucleares/metabolismo , Transducción de Señal/fisiología , Western Blotting , Inmunoprecipitación de Cromatina , Ensayo de Unidades Formadoras de Colonias , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Humanos , Análisis por Micromatrices , Proteínas Nucleares/genética , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción , beta-GalactosidasaRESUMEN
Estrogen receptor-α (ERα) has central role in hormone-dependent breast cancer and its ligand-induced functions have been extensively characterized. However, evidence exists that ERα has functions that are independent of ligands. In the present work, we investigated the binding of ERα to chromatin in the absence of ligands and its functions on gene regulation. We demonstrated that in MCF7 breast cancer cells unliganded ERα binds to more than 4,000 chromatin sites. Unexpectedly, although almost entirely comprised in the larger group of estrogen-induced binding sites, we found that unliganded-ERα binding is specifically linked to genes with developmental functions, compared with estrogen-induced binding. Moreover, we found that siRNA-mediated down-regulation of ERα in absence of estrogen is accompanied by changes in the expression levels of hundreds of coding and noncoding RNAs. Down-regulated mRNAs showed enrichment in genes related to epithelial cell growth and development. Stable ERα down-regulation using shRNA, which caused cell growth arrest, was accompanied by increased H3K27me3 at ERα binding sites. Finally, we found that FOXA1 and AP2γ binding to several sites is decreased upon ERα silencing, suggesting that unliganded ERα participates, together with other factors, in the maintenance of the luminal-specific cistrome in breast cancer cells.
Asunto(s)
Neoplasias de la Mama/genética , Receptor alfa de Estrógeno/metabolismo , Genoma Humano/genética , Sitios de Unión , Neoplasias de la Mama/patología , Proliferación Celular , Inmunoprecipitación de Cromatina , Femenino , Ontología de Genes , Humanos , Ligandos , Células MCF-7 , Reacción en Cadena de la Polimerasa , ARN Interferente Pequeño/metabolismoRESUMEN
INTRODUCTION: Endocrine therapies targeting cell proliferation and survival mediated by estrogen receptor α (ERα) are among the most effective systemic treatments for ERα-positive breast cancer. However, most tumors initially responsive to these therapies acquire resistance through mechanisms that involve ERα transcriptional regulatory plasticity. Herein we identify VAV3 as a critical component in this process. METHODS: A cell-based chemical compound screen was carried out to identify therapeutic strategies against resistance to endocrine therapy. Binding to ERα was evaluated by molecular docking analyses, an agonist fluoligand assay and short hairpin (sh)RNA-mediated protein depletion. Microarray analyses were performed to identify altered gene expression. Western blot analysis of signaling and proliferation markers, and shRNA-mediated protein depletion in viability and clonogenic assays, were performed to delineate the role of VAV3. Genetic variation in VAV3 was assessed for association with the response to tamoxifen. Immunohistochemical analyses of VAV3 were carried out to determine its association with therapeutic response and different tumor markers. An analysis of gene expression association with drug sensitivity was carried out to identify a potential therapeutic approach based on differential VAV3 expression. RESULTS: The compound YC-1 was found to comparatively reduce the viability of cell models of acquired resistance. This effect was probably not due to activation of its canonical target (soluble guanylyl cyclase), but instead was likely a result of binding to ERα. VAV3 was selectively reduced upon exposure to YC-1 or ERα depletion, and, accordingly, VAV3 depletion comparatively reduced the viability of cell models of acquired resistance. In the clinical scenario, germline variation in VAV3 was associated with the response to tamoxifen in Japanese breast cancer patients (rs10494071 combined P value = 8.4 × 10-4). The allele association combined with gene expression analyses indicated that low VAV3 expression predicts better clinical outcome. Conversely, high nuclear VAV3 expression in tumor cells was associated with poorer endocrine therapy response. Based on VAV3 expression levels and the response to erlotinib in cancer cell lines, targeting EGFR signaling may be a promising therapeutic strategy. CONCLUSIONS: This study proposes VAV3 as a biomarker and a rationale for its use as a signaling target to prevent and/or overcome resistance to endocrine therapy in breast cancer.
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Neoplasias de la Mama/tratamiento farmacológico , Resistencia a Antineoplásicos/genética , Receptor alfa de Estrógeno/metabolismo , Indazoles/farmacología , Proteínas Proto-Oncogénicas c-vav/genética , Androstadienos/uso terapéutico , Antineoplásicos Hormonales/farmacología , Inhibidores de la Aromatasa/uso terapéutico , Biomarcadores de Tumor/genética , Mama/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Activadores de Enzimas/farmacología , Receptores ErbB/antagonistas & inhibidores , Clorhidrato de Erlotinib , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor alfa de Estrógeno/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Estudios de Asociación Genética , Variación Genética , Humanos , Letrozol , Células MCF-7 , Nitrilos/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Interferencia de ARN , ARN Interferente Pequeño , Tamoxifeno/farmacología , Tamoxifeno/uso terapéutico , Toremifeno/farmacología , Toremifeno/uso terapéutico , Triazoles/uso terapéuticoRESUMEN
Patients with breast cancer show altered expression of genes within the pectoralis major skeletal muscle cells of the breast. Through analyses of The Cancer Genome Atlas (TCGA)-breast cancer (BRCA), we identified three previously uncharacterized putative novel tumor suppressor genes expressed in normal muscle cells, whose expression was downregulated in breast tumors. We found that NEDD4 binding protein 2-like 1 (N4BP2L1), pleckstrin homology domain-containing family A member 4 (PLEKHA4), and brain-enriched guanylate kinase-associated protein (BEGAIN) that are normally highly expressed in breast myoepithelial cells and smooth muscle cells were significantly downregulated in breast tumor tissues of a cohort of 50 patients with this cancer. Our data revealed that the low expression of PLEKHA4 in patients with menopause below 50 years correlated with a higher risk of breast cancer. Moreover, we identified N4BP2L1 and BEGAIN as potential biomarkers of HER2-positive breast cancer. Furthermore, low BEGAIN expression in breast cancer patients with blood fat, heart problems, and diabetes correlated with a higher risk of this cancer. In addition, protein and RNA expression analysis of TCGA-BRCA revealed N4BP2L1 as a promising diagnostic protein biomarker in breast cancer. In addition, the in silico data of scRNA-seq showed high expression of these genes in several cell types of normal breast tissue, including breast myoepithelial cells and smooth muscle cells. Thus, our results suggest their possible tumor-suppressive function in breast cancer and muscle development.
RESUMEN
BACKGROUND: In the last few years several studies have shown that Transposable Elements (TEs) in the human genome are significantly associated with Transcription Factor Binding Sites (TFBSs) and that in several cases their expansion within the genome led to a substantial rewiring of the regulatory network. Another important feature of the regulatory network which has been thoroughly studied is the combinatorial organization of transcriptional regulation. In this paper we combine these two observations and suggest that TEs, besides rewiring the network, also played a central role in the evolution of particular patterns of combinatorial gene regulation. RESULTS: To address this issue we searched for TEs overlapping Estrogen Receptor α (ERα) binding peaks in two publicly available ChIP-seq datasets from the MCF7 cell line corresponding to different modalities of exposure to estrogen. We found a remarkable enrichment of a few specific classes of Transposons. Among these a prominent role was played by MIR (Mammalian Interspersed Repeats) transposons. These TEs underwent a dramatic expansion at the beginning of the mammalian radiation and then stabilized. We conjecture that the special affinity of ERα for the MIR class of TEs could be at the origin of the important role assumed by ERα in Mammalians. We then searched for TFBSs within the TEs overlapping ChIP-seq peaks. We found a strong enrichment of a few precise combinations of TFBS. In several cases the corresponding Transcription Factors (TFs) were known cofactors of ERα, thus supporting the idea of a co-regulatory role of TFBS within the same TE. Moreover, most of these correlations turned out to be strictly associated to specific classes of TEs thus suggesting the presence of a well-defined "transposon code" within the regulatory network. CONCLUSIONS: In this work we tried to shed light into the role of Transposable Elements (TEs) in shaping the regulatory network of higher eukaryotes. To test this idea we focused on a particular transcription factor: the Estrogen Receptor α (ERα) and we found that ERα preferentially targets a well defined set of TEs and that these TEs host combinations of transcriptional regulators involving several of known co-regulators of ERα. Moreover, a significant number of these TEs turned out to be conserved between human and mouse and located in the vicinity (and thus candidate to be regulators) of important estrogen-related genes.
Asunto(s)
Elementos Transponibles de ADN , Receptor alfa de Estrógeno/genética , Regulación de la Expresión Génica , Factores de Transcripción/genética , Secuencia de Bases , Sitios de Unión , Genoma Humano , Humanos , Células MCF-7 , Anotación de Secuencia Molecular , Datos de Secuencia MolecularRESUMEN
Dysregulation of alternative splicing in prostate cancer is linked to transcriptional programs activated by AR, ERG, FOXA1, and MYC. Here, we show that FOXA1 functions as the primary orchestrator of alternative splicing dysregulation across 500 primary and metastatic prostate cancer transcriptomes. We demonstrate that FOXA1 binds to the regulatory regions of splicing-related genes, including HNRNPK and SRSF1. By controlling trans-acting factor expression, FOXA1 exploits an "exon definition" mechanism calibrating alternative splicing toward dominant isoform production. This regulation especially impacts splicing factors themselves and leads to a reduction of nonsense-mediated decay (NMD)-targeted isoforms. Inclusion of the NMD-determinant FLNA exon 30 by FOXA1-controlled oncogene SRSF1 promotes cell growth in vitro and predicts disease recurrence. Overall, we report a role for FOXA1 in rewiring the alternative splicing landscape in prostate cancer through a cascade of events from chromatin access, to splicing factor regulation, and, finally, to alternative splicing of exons influencing patient survival.
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Empalme Alternativo , Neoplasias de la Próstata , Empalme Alternativo/genética , Cromatina , Factor Nuclear 3-alfa del Hepatocito/genética , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Humanos , Masculino , Recurrencia Local de Neoplasia , Neoplasias de la Próstata/genética , Factores de Empalme de ARN/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , Transactivadores/metabolismoRESUMEN
The transcription factor Oct4 is essential for the maintenance and induction of stem cell pluripotency, but its functional roles are not fully understood. Here, we investigate the functions of Oct4 by depleting and subsequently recovering it in mouse embryonic stem cells (ESCs) and conducting a time-resolved multiomics analysis. Oct4 depletion leads to an immediate loss of its binding to enhancers, accompanied by a decrease in mRNA synthesis from its target genes that are part of the transcriptional network that maintains pluripotency. Gradual decrease of Oct4 binding to enhancers does not immediately change the chromatin accessibility but reduces transcription of enhancers. Conversely, partial recovery of Oct4 expression results in a rapid increase in chromatin accessibility, whereas enhancer transcription does not fully recover. These results indicate different concentration-dependent activities of Oct4. Whereas normal ESC levels of Oct4 are required for transcription of pluripotency enhancers, low levels of Oct4 are sufficient to retain chromatin accessibility, likely together with other factors such as Sox2.
Asunto(s)
Cromatina , Células Madre Pluripotentes , Animales , Cromatina/metabolismo , Redes Reguladoras de Genes , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Pluripotentes/metabolismo , Transcripción GenéticaRESUMEN
RNA splicing is an essential part of eukaryotic gene expression. Although the mechanism of splicing has been extensively studied in vitro, in vivo kinetics for the two-step splicing reaction remain poorly understood. Here, we combine transient transcriptome sequencing (TT-seq) and mathematical modeling to quantify RNA metabolic rates at donor and acceptor splice sites across the human genome. Splicing occurs in the range of minutes and is limited by the speed of RNA polymerase elongation. Splicing kinetics strongly depends on the position and nature of nucleotides flanking splice sites, and on structural interactions between unspliced RNA and small nuclear RNAs in spliceosomal intermediates. Finally, we introduce the 'yield' of splicing as the efficiency of converting unspliced to spliced RNA and show that it is highest for mRNAs and independent of splicing kinetics. These results lead to quantitative models describing how splicing rates and yield are encoded in the human genome.
Asunto(s)
Genoma Humano , Empalme del ARN , Perfilación de la Expresión Génica , Humanos , Células K562 , Cinética , Modelos Teóricos , Análisis de Secuencia de ARN , Empalmosomas/metabolismoRESUMEN
Estrogen Receptor alpha (ERα) activation by estrogenic hormones induces luminal breast cancer cell proliferation. However, ERα plays also important hormone-independent functions to maintain breast tumor cells epithelial phenotype. We reported previously by RNA-Seq that in MCF-7 cells in absence of hormones ERα down-regulation changes the expression of several genes linked to cellular development, representing a specific subset of estrogen-induced genes. Here, we report regulation of long non-coding RNAs from the same experimental settings. A list of 133 Apo-ERα-Regulated lncRNAs (AER-lncRNAs) was identified and extensively characterized using published data from cancer cell lines and tumor tissues, or experiments on MCF-7 cells. For several features, we ran validation using cell cultures or fresh tumor biopsies. AER-lncRNAs represent a specific subset, only marginally overlapping estrogen-induced transcripts, whose expression is largely restricted to luminal cells and which is able to perfectly classify breast tumor subtypes. The most abundant AER-lncRNA, DSCAM-AS1, is expressed in ERα+ breast carcinoma, but not in pre-neoplastic lesions, and correlates inversely with EMT markers. Down-regulation of DSCAM-AS1 recapitulated, in part, the effect of silencing ERα, i.e. growth arrest and induction of EMT markers. In conclusion, we report an ERα-dependent lncRNA set representing a novel luminal signature in breast cancer cells.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Receptor alfa de Estrógeno/genética , Regulación Neoplásica de la Expresión Génica , ARN Largo no Codificante/genética , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Proliferación Celular/genética , Supervivencia Celular/genética , Regulación hacia Abajo , Activación Enzimática , Transición Epitelial-Mesenquimal/genética , Estrógenos/metabolismo , Femenino , Células HEK293 , Humanos , Ligandos , Células MCF-7 , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
Transcription regulation in metazoans often involves promoter-proximal pausing of RNA polymerase (Pol) II, which requires the 4-subunit negative elongation factor (NELF). Here we discern the functional architecture of human NELF through X-ray crystallography, protein crosslinking, biochemical assays, and RNA crosslinking in cells. We identify a NELF core subcomplex formed by conserved regions in subunits NELF-A and NELF-C, and resolve its crystal structure. The NELF-AC subcomplex binds single-stranded nucleic acids in vitro, and NELF-C associates with RNA in vivo. A positively charged face of NELF-AC is involved in RNA binding, whereas the opposite face of the NELF-AC subcomplex binds NELF-B. NELF-B is predicted to form a HEAT repeat fold, also binds RNA in vivo, and anchors the subunit NELF-E, which is confirmed to bind RNA in vivo. These results reveal the three-dimensional architecture and three RNA-binding faces of NELF.