RESUMEN
The architectural chromatin protein HMGA1 and the transcription factor Fra-1 are both overexpressed in aggressive triple-negative breast cancers (TNBC), where they both favor epithelial-to-mesenchymal transition, invasion, and metastasis. We therefore explored the possibility that Fra-1 might be involved in enhanced transcription of the HMGA1 gene in TNBCs by exploiting cancer transcriptome datasets and resorting to functional studies combining RNA interference, mRNA and transcriptional run-on assays, chromatin immunoprecipitation, and chromosome conformation capture approaches in TNBC model cell lines. Our bioinformatic analysis indicated that Fra-1 and HMGA1 expressions positively correlate in primary samples of patients with TNBC. Our functional studies showed that Fra-1 regulates HMGA1 mRNA expression at the transcriptional level via binding to enhancer elements located in the last two introns of the gene. Although Fra-1 binding is required for p300/CBP recruitment at the enhancer domain, this recruitment did not appear essential for Fra-1-stimulated HMGA1 gene expression. Strikingly, Fra-1 binding is required for efficient recruitment of RNA Polymerase II at the HMGA1 promoter. This is permitted owing to chromatin interactions bringing about the intragenic Fra-1-binding enhancers and the gene promoter region. Fra-1 is, however, not instrumental for chromatin loop formation at the HMGA1 locus but rather exerts its transcriptional activity by exploiting chromatin interactions preexisting to its binding. IMPLICATIONS: We demonstrate that Fra-1 bound to an intragenic enhancer region is required for RNA Pol II recruitement at the HMGA1 promoter. Thereby, we provide novel insights into the mechanisms whereby Fra-1 exerts its prooncogenic transcriptional actions in the TNBC pathologic context.
Asunto(s)
Proteína HMGA1a/genética , Oncogenes/genética , Factor de Transcripción AP-1/genética , Transcripción Genética/genética , Neoplasias de la Mama Triple Negativas/genética , Línea Celular Tumoral , Femenino , HumanosRESUMEN
Plau codes for the urokinase-type plasminogen activator (uPA), critical in cancer metastasis. While the mechanisms driving its overexpression in tumorigenic processes are unknown, it is regulated by the AP-1 transcriptional complex in diverse situations. The AP-1 component Fra-1 being overexpressed in aggressive breast cancers, we have addressed its role in the overexpression of Plau in the highly metastatic breast cancer model cell line MDA-MB231 using ChIP, pharmacological and RNAi approaches. Plau transcription appears controlled by 2 AP-1 enhancers located -1.9 (ABR-1.9) and -4.1 kb (ABR-4.1) upstream of the transcription start site (TSS) of the uPA-coding mRNA, Plau-001, that bind Fra-1. Surprisingly, RNA Pol II is not recruited only at the Plau-001 TSS but also upstream in the ABR-1.9 and ABR-4.1 region. Most Pol II molecules transcribe short and unstable RNAs while tracking down toward the TSS, where there are converted into Plau-001 mRNA-productive species. Moreover, a minority of Pol II molecules transcribes a low abundance mRNA of unknown function called Plau-004 from the ABR-1.9 domain, whose expression is tempered by Fra-1. Thus, we unveil a heretofore-unsuspected transcriptional complexity at Plau in a reference metastatic breast cancer cell line with pleiotropic effects for Fra-1, providing novel information on AP-1 transcriptional action.
Asunto(s)
Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Proto-Oncogénicas c-fos/fisiología , Factor de Transcripción AP-1/fisiología , Transcripción Genética , Activador de Plasminógeno de Tipo Uroquinasa/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Cromatina/química , Femenino , Sitios Genéticos , Humanos , Metástasis de la Neoplasia , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-fos/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
The junb gene behaves as an immediate early gene in bacterial lipopolysaccharide (LPS)-stimulated dendritic cells (DCs), where its transient transcriptional activation is necessary for the induction of inflammatory cytokines. junb is a short gene and its transcriptional activation by LPS depends on the binding of NF-κB to an enhancer located just downstream of its 3' UTR. Here, we have addressed the mechanisms underlying the transcriptional hyper-reactivity of junb. Using transfection and pharmacological assays to complement chromatin immunoprecipitation analyses addressing the localization of histones, polymerase II, negative elongation factor (NELF)-, DRB sensitivity-inducing factor (DSIF)- and Positive Transcription Factor b complexes, we demonstrate that junb is a RNA Pol II-paused gene where Pol II is loaded in the transcription start site domain but poorly active. Moreover, High salt-Recovered Sequence, chromosome conformation capture (3C)- and gene transfer experiments show that (i) junb is organized in a nuclear chromatin loop bringing into close spatial proximity the upstream promoter region and the downstream enhancer and (ii) this configuration permits immediate Pol II release on the junb body on binding of LPS-activated NF-κB to the enhancer. Thus, our work unveils a novel topological framework underlying fast junb transcriptional response in DCs. Moreover, it also points to a novel layer of complexity in the modes of action of NF-κB.
Asunto(s)
Cromatina/química , Células Dendríticas/metabolismo , Factores de Transcripción/genética , Activación Transcripcional , Animales , Línea Celular , Células Dendríticas/química , Células Dendríticas/enzimología , Elementos de Facilitación Genéticos , Sitios Genéticos , Histonas/análisis , Humanos , Lipopolisacáridos/farmacología , Ratones , Conformación de Ácido Nucleico , ARN Polimerasa II/análisis , Factores de Transcripción/biosíntesis , Sitio de Iniciación de la TranscripciónRESUMEN
Mesenchymal stem cells (MSCs) exert immunomodulatory properties via the inhibition of T cell activation and proliferation. Because of the deleterious role of Th17 cells in the pathogenesis of inflammatory disease, we investigated whether proinflammatory cytokines could modify the expression of adhesion molecules on human MSCs, thereby contributing to increased Th17 cell adhesion to MSCs and, as a consequence, modulating the function of the latter cells. IFN-gamma and TNF-alpha synergistically enhanced the expression of CD54 by MSCs, enabling the CCR6 chemokine ligand CCL20 to induce in vitro adhesion of Th17 cells to MSCs. MSCs prevented the in vitro differentiation of naive CD4(+) T cells into Th17 cells and inhibited the production of IL-17, IL-22, IFN-gamma, and TNF-alpha by fully differentiated Th17 cells; this was mediated, in part, via PGE(2), the production of which was enhanced in cocultures with Th17 cells. Moreover, MSCs induced the production of IL-10 and trimethylation of histone H3K4me3 at the promoter of the FOXP3 gene locus, whereas it suppressed trimethylation of the corresponding region in the RORC gene in Th17 cells. These epigenetic changes were associated with the induction of fork head box p3 and the acquisition by Th17 cells of the capacity to inhibit in vitro proliferative responses of activated CD4(+) T cells, which was enhanced when MSCs were preincubated with IFN-gamma and TNF-alpha. These results showed that, under inflammatory conditions, MSCs mediate the adhesion of Th17 cells via CCR6 and exert anti-inflammatory effects through the induction of a T cell regulatory phenotype in these cells.
Asunto(s)
Diferenciación Celular/inmunología , Inhibidores de Crecimiento/fisiología , Interleucina-17/antagonistas & inhibidores , Células Madre Mesenquimatosas/inmunología , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Antígeno CD11a/metabolismo , Antígeno CD11a/fisiología , Antígenos CD18/metabolismo , Antígenos CD18/fisiología , Adhesión Celular/inmunología , Comunicación Celular/inmunología , Movimiento Celular/inmunología , Células Cultivadas , Técnicas de Cocultivo , Humanos , Inmunofenotipificación , Mediadores de Inflamación/metabolismo , Mediadores de Inflamación/fisiología , Molécula 1 de Adhesión Intercelular/biosíntesis , Interferón gamma/fisiología , Interleucina-17/biosíntesis , Interleucina-17/fisiología , Células L , Ligandos , Células Madre Mesenquimatosas/patología , Ratones , Receptores CCR6/metabolismo , Receptores CCR6/fisiología , Linfocitos T Reguladores/patología , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
The c-Fos proto-oncogenic transcription factor defines a multigene family controlling many processes both at the cell and the whole organism level. To bind to its target AP-1/12-O-tetradecanoylphorbol-13-acetate-responsive element or cAMP-responsive element DNA sequences in gene promoters and exert its transcriptional part, c-Fos must heterodimerize with other bZip proteins, its best studied partners being the Jun proteins (c-Jun, JunB, and JunD). c-Fos expression is regulated at many transcriptional and post-transcriptional levels, yet little is known on how its localization is dynamically regulated in the cell. Here we have investigated its intranuclear mobility using fluorescence recovery after photobleaching, genetic, and biochemical approaches. Whereas monomeric c-Fos is highly mobile and distributed evenly with nucleolar exclusion in the nucleus, heterodimerization with c-Jun entails intranuclear redistribution and dramatic reduction in mobility of c-Fos caused by predominant association with the nuclear matrix independently of any binding to AP-1/12-O-tetradecanoylphorbol-13-acetate-responsive element or cAMP-responsive element sequences. In contrast to c-Jun, dimerization with JunB does not detectably affect c-Fos mobility. However, dimerization with JunB affects intranuclear distribution with significant differences in the localization of c-Fos.c-Jun and c-Fos.JunB dimers. Moreover, c-Jun and JunB exert comparable effects on another Fos family member, Fra-1. Thus, we report a novel regulation, i.e. differentially regulated intranuclear mobility and distribution of Fos proteins by their Jun partners, and suggest the existence of intranuclear storage sites for latent c-Fos.c-Jun AP-1 complexes. This may affect the numerous physiopathological functions these transcription factors control.
Asunto(s)
Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factor de Transcripción AP-1/metabolismo , Animales , Línea Celular , Núcleo Celular/química , Núcleo Celular/metabolismo , Células HeLa , Humanos , Ratones , Complejos Multiproteicos/metabolismo , Multimerización de Proteína , Transporte de Proteínas , Ratas , Factores de Transcripción/metabolismoRESUMEN
c-Fos proto-oncoprotein defines a family of closely related transcription factors (Fos proteins) also comprising Fra-1, Fra-2, FosB and DeltaFosB, the latter two proteins being generated by alternative splicing. Through the regulation of many genes, most of them still unidentified, they regulate major functions from the cell level up to the whole organism. Thus they are involved in the control of proliferation, differentiation and apoptosis, as well as in the control of responses to stresses, and they play important roles in organogenesis, immune responses and control of cognitive functions, among others. Fos proteins are intrinsically unstable. We have studied how two of them, c-Fos and Fra-1, are degraded. Departing from the classical scenario where unstable key cell regulators are hydrolysed by the proteasome after polyubiquitination, we showed that the bulk of c-Fos and Fra-1 can be broken down independently of any prior ubiquitination. Certain conserved structural domains suggest that similar mechanisms may also apply to Fra-2 and FosB. Computer search indicates that certain motifs shared by the Fos proteins and putatively responsible for instability are found in no other protein, suggesting the existence of degradation mechanisms specific for this protein family. Under particular signalling conditions, others have shown that a part of cytoplasmic c-Fos requires ubiquitination for fast turnover. This poses the question of the multiplicity of degradation pathways that apply to proteins depending on their intracellular localization.