RESUMEN
Loss of retinal ganglion cells (RGCs) is central to the pathogenesis of optic neuropathies such as glaucoma. Increased RGC cAMP signaling is neuroprotective. We have shown that displacement of the cAMP-specific phosphodiesterase PDE4D3 from an RGC perinuclear compartment by expression of the modified PDE4D3 N-terminal peptide 4D3(E) increases perinuclear cAMP and protein kinase A activity in cultured neurons and in vivo RGC survival after optic nerve crush (ONC) injury. To explore mechanisms by which PDE4D3 displacement promotes neuroprotection, in this study mice intravitreally injected with an adeno-associated virus to express an mCherry-tagged 4D3(E) peptide were subjected to ONC injury and analyzed by single cell RNA-sequencing (scRNA-seq). 4D3(E)-mCherry expression was associated with an attenuation of injury-induced changes in gene expression, thereby supporting the hypothesis that enhanced perinuclear PKA signaling promotes neuroprotective RGC gene expression.
Asunto(s)
Ratones Endogámicos C57BL , Traumatismos del Nervio Óptico , Células Ganglionares de la Retina , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patología , Células Ganglionares de la Retina/efectos de los fármacos , Animales , Ratones , Traumatismos del Nervio Óptico/metabolismo , Traumatismos del Nervio Óptico/tratamiento farmacológico , Traumatismos del Nervio Óptico/genética , Regulación de la Expresión Génica , Modelos Animales de Enfermedad , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Compresión Nerviosa , Supervivencia Celular , Inyecciones Intravítreas , Transducción de Señal , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Masculino , Células CultivadasRESUMEN
Retinal ganglion cell (RGC) differentiation is tightly controlled by extrinsic and intrinsic factors. Growth and differentiation factor 15 (GDF-15) promotes RGC differentiation, opposite to GDF-11 which inhibits RGC differentiation, both in the mouse retina and in human stem cells. To deepen our understanding of how these two closely related molecules confer opposing effects on retinal development, here we assess the transcriptional profiles of mouse retinal progenitors exposed to exogenous GDF-11 or -15. We find a dichotomous effect of GDF-15 on RGC differentiation, decreasing RGCs expressing residual pro-proliferative genes and increasing RGCs expressing non-proliferative genes, suggestive of greater RGC maturation. Furthermore, GDF-11 promoted the differentiation of photoreceptors and amacrine cells. These data enhance our understanding of the mechanisms underlying the differentiation of RGCs and photoreceptors from retinal progenitors and suggest new approaches to the optimization of protocols for the differentiation of these cell types.
RESUMEN
The intrinsic mechanisms that regulate neurotoxic versus neuroprotective astrocyte phenotypes and their effects on central nervous system degeneration and repair remain poorly understood. Here we show that injured white matter astrocytes differentiate into two distinct C3-positive and C3-negative reactive populations, previously simplified as neurotoxic (A1) and neuroprotective (A2)1,2, which can be further subdivided into unique subpopulations defined by proliferation and differential gene expression signatures. We find the balance of neurotoxic versus neuroprotective astrocytes is regulated by discrete pools of compartmented cyclic adenosine monophosphate derived from soluble adenylyl cyclase and show that proliferating neuroprotective astrocytes inhibit microglial activation and downstream neurotoxic astrocyte differentiation to promote retinal ganglion cell survival. Finally, we report a new, therapeutically tractable viral vector to specifically target optic nerve head astrocytes and show that raising nuclear or depleting cytoplasmic cyclic AMP in reactive astrocytes inhibits deleterious microglial or macrophage cell activation and promotes retinal ganglion cell survival after optic nerve injury. Thus, soluble adenylyl cyclase and compartmented, nuclear- and cytoplasmic-localized cyclic adenosine monophosphate in reactive astrocytes act as a molecular switch for neuroprotective astrocyte reactivity that can be targeted to inhibit microglial activation and neurotoxic astrocyte differentiation to therapeutic effect. These data expand on and define new reactive astrocyte subtypes and represent a step towards the development of gliotherapeutics for the treatment of glaucoma and other optic neuropathies.
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Astrocitos , Neuroprotección , Adenilil Ciclasas/metabolismo , Astrocitos/citología , Astrocitos/enzimología , Astrocitos/metabolismo , Diferenciación Celular , Núcleo Celular/metabolismo , Supervivencia Celular , AMP Cíclico/metabolismo , Citoplasma/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Microglía/metabolismo , Microglía/patología , Traumatismos del Nervio Óptico/metabolismo , Traumatismos del Nervio Óptico/patología , Traumatismos del Nervio Óptico/terapia , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismo , Sustancia Blanca/metabolismo , Sustancia Blanca/patología , Glaucoma/patología , Glaucoma/terapiaRESUMEN
Retinal ganglion cell (RGC) replacement therapy could restore vision in glaucoma and other optic neuropathies. We developed a rapid protocol for directly induced RGC (iRGC) differentiation from human stem cells, leveraging overexpression of NGN2. Neuronal morphology and neurite growth were observed within 1 week of induction; characteristic RGC-specific gene expression confirmed identity. Calcium imaging demonstrated γ-aminobutyric acid (GABA)-induced excitation characteristic of immature RGCs. Single-cell RNA sequencing showed more similarities between iRGCs and early-stage fetal human RGCs than retinal organoid-derived RGCs. Intravitreally transplanted iRGCs survived and migrated into host retinas independent of prior optic nerve trauma, but iRGCs protected host RGCs from neurodegeneration. These data demonstrate rapid iRGC generation in vitro into an immature cell with high similarity to human fetal RGCs and capacity for retinal integration after transplantation and neuroprotective function after optic nerve injury. The simplicity of this system may benefit translational studies on human RGCs.
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Glaucoma , Traumatismos del Nervio Óptico , Humanos , Células Ganglionares de la Retina , Traumatismos del Nervio Óptico/metabolismo , Retina , Células MadreRESUMEN
Adeno-associated viruses (AAVs) targeting specific cell types are powerful tools for studying distinct cell types in the central nervous system (CNS). Cis-regulatory modules (CRMs), e.g., enhancers, are highly cell-type-specific and can be integrated into AAVs to render cell type specificity. Chromatin accessibility has been commonly used to nominate CRMs, which have then been incorporated into AAVs and tested for cell type specificity in the CNS. However, chromatin accessibility data alone cannot accurately annotate active CRMs, as many chromatin-accessible CRMs are not active and fail to drive gene expression in vivo. Using available large-scale datasets on chromatin accessibility, such as those published by the ENCODE project, here we explored strategies to increase efficiency in identifying active CRMs for AAV-based cell-type-specific labeling and manipulation. We found that prescreening of chromatin-accessible putative CRMs based on the density of cell-type-specific transcription factor binding sites (TFBSs) can significantly increase efficiency in identifying active CRMs. In addition, generation of synthetic CRMs by stitching chromatin-accessible regions flanking cell-type-specific genes can render cell type specificity in many cases. Using these straightforward strategies, we generated AAVs that can target the extensively studied interneuron and glial cell types in the retina and brain. Both strategies utilize available genomic datasets and can be employed to generate AAVs targeting specific cell types in CNS without conducting comprehensive screening and sequencing experiments, making a step forward in cell-type-specific research.
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Encéfalo , Dependovirus , Retina , Coloración y Etiquetado , Factores de Transcripción , Animales , Sitios de Unión , Encéfalo/citología , Encéfalo/metabolismo , Cromatina/genética , Cromatina/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Ratones , Retina/citología , Retina/metabolismo , Coloración y Etiquetado/métodos , Factores de Transcripción/metabolismoRESUMEN
The size of the transcriptional program of long non-coding RNAs in the mammalian genome has engendered discussions about their biological roles1, particularly the promoter antisense (PAS) transcripts2,3. Here we report the development of an assay-referred to as chromatin isolation by RNA-Cas13a complex-to quantitatively detect the distribution of RNA in the genome. The assay revealed that PAS RNAs serve as a key gatekeeper of a broad transcriptional pause release program, based on decommissioning the 7SK small nuclear RNA-dependent inhibitory P-TEFb complex. Induction of PAS RNAs by liganded ERα led to a significant loss of H3K9me3 and the release of basally recruited HP1α and KAP1 on activated target gene promoters. This release was due to PAS RNA-dependent recruitment of H3K9me3 demethylases, which required interactions with a compact stem-loop structure in the PAS RNAs, an apparent feature of similarly regulated PAS RNAs. Activation of the ERα-bound MegaTrans enhancer, which is essential for robust pause release, required the recruitment of phosphorylated KAP1, with its transfer to the cognate promoters permitting 17ß-oestradiol-induced pause release and activation of the target gene. This study reveals a mechanism, based on RNA structure, that mediates the function of PAS RNAs in gene regulation.
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Conformación de Ácido Nucleico , Regiones Promotoras Genéticas/genética , ARN sin Sentido/química , ARN sin Sentido/genética , Activación Transcripcional/genética , Línea Celular , Homólogo de la Proteína Chromobox 5/metabolismo , Proteína Sustrato Asociada a CrK , Receptor alfa de Estrógeno/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Ligandos , Factor B de Elongación Transcripcional Positiva/metabolismo , ARN Polimerasa II/metabolismo , Estabilidad del ARN , Proteína 28 que Contiene Motivos Tripartito/metabolismoRESUMEN
The failure of adult CNS neurons to survive and regenerate their axons after injury or in neurodegenerative disease remains a major target for basic and clinical neuroscience. Recent data demonstrated in the adult mouse that exogenous expression of Sry-related high-mobility-box 11 (Sox11) promotes optic nerve regeneration after optic nerve injury but exacerbates the death of a subset of retinal ganglion cells (RGCs), α-RGCs. During development, Sox11 is required for RGC differentiation from retinal progenitor cells (RPCs), and we found that mutation of a single residue to prevent SUMOylation at lysine 91 (K91) increased Sox11 nuclear localization and RGC differentiation in vitro Here, we explored whether this Sox11 manipulation similarly has stronger effects on RGC survival and optic nerve regeneration. In vitro, we found that non-SUMOylatable Sox11K91A leads to RGC death and suppresses axon outgrowth in primary neurons. We furthermore found that Sox11K91A more strongly promotes axon regeneration but also increases RGC death after optic nerve injury in vivo in the adult mouse. RNA sequence (RNA-seq) data showed that Sox11 and Sox11K91A increase the expression of key signaling pathway genes associated with axon growth and regeneration but downregulated Spp1 and Opn4 expression in RGC cultures, consistent with negatively regulating the survival of α-RGCs and ipRGCs. Thus, Sox11 and its SUMOylation site at K91 regulate gene expression, survival and axon growth in RGCs, and may be explored further as potential regenerative therapies for optic neuropathy.
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Enfermedades Neurodegenerativas , Traumatismos del Nervio Óptico , Animales , Axones/metabolismo , Supervivencia Celular , Ratones , Regeneración Nerviosa , Enfermedades Neurodegenerativas/metabolismo , Traumatismos del Nervio Óptico/metabolismo , Procesamiento Proteico-Postraduccional , Factores de Transcripción SOXC/genética , Factores de Transcripción SOXC/metabolismoRESUMEN
Loss of retinal ganglion cells (RGCs) in optic neuropathies results in permanent partial or complete blindness. Myocyte enhancer factor 2 (MEF2) transcription factors have been shown to play a pivotal role in neuronal systems, and in particular MEF2A knockout was shown to enhance RGC survival after optic nerve crush injury. Here we expanded these prior data to study bi-allelic, tri-allelic and heterozygous allele deletion. We observed that deletion of all MEF2A, MEF2C, and MEF2D alleles had no effect on RGC survival during development. Our extended experiments suggest that the majority of the neuroprotective effect was conferred by complete deletion of MEF2A but that MEF2D knockout, although not sufficient to increase RGC survival on its own, increased the positive effect of MEF2A knockout. Conversely, MEF2A over-expression in wildtype mice worsened RGC survival after optic nerve crush. Interestingly, MEF2 transcription factors are regulated by post-translational modification, including by calcineurin-catalyzed dephosphorylation of MEF2A Ser-408 known to increase MEF2A-dependent transactivation in neurons. However, neither phospho-mimetic nor phospho-ablative mutation of MEF2A Ser-408 affected the ability of MEF2A to promote RGC death in vivo after optic nerve injury. Together these findings demonstrate that MEF2 gene expression opposes RGC survival following axon injury in a complex hierarchy, and further support the hypothesis that loss of or interference with MEF2A expression might be beneficial for RGC neuroprotection in diseases such as glaucoma and other optic neuropathies.
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Factores de Transcripción MEF2/metabolismo , Traumatismos del Nervio Óptico/metabolismo , Traumatismos del Nervio Óptico/patología , Células Ganglionares de la Retina/patología , Alelos , Animales , Recuento de Células , Humanos , Factores de Transcripción MEF2/deficiencia , Factores de Transcripción MEF2/genética , Ratones , Traumatismos del Nervio Óptico/genética , Mutación Puntual , Transducción de SeñalRESUMEN
Osteosarcoma is a highly aggressive cancer for which treatment has remained essentially unchanged for more than 30 years. Osteosarcoma is characterized by widespread and recurrent somatic copy-number alterations (SCNA) and structural rearrangements. In contrast, few recurrent point mutations in protein-coding genes have been identified, suggesting that genes within SCNAs are key oncogenic drivers in this disease. SCNAs and structural rearrangements are highly heterogeneous across osteosarcoma cases, suggesting the need for a genome-informed approach to targeted therapy. To identify patient-specific candidate drivers, we used a simple heuristic based on degree and rank order of copy-number amplification (identified by whole-genome sequencing) and changes in gene expression as identified by RNA sequencing. Using patient-derived tumor xenografts, we demonstrate that targeting of patient-specific SCNAs leads to significant decrease in tumor burden, providing a road map for genome-informed treatment of osteosarcoma. SIGNIFICANCE: Osteosarcoma is treated with a chemotherapy regimen established 30 years ago. Although osteosarcoma is genomically complex, we hypothesized that tumor-specific dependencies could be identified within SCNAs. Using patient-derived tumor xenografts, we found a high degree of response for "genome-matched" therapies, demonstrating the utility of a targeted genome-informed approach.This article is highlighted in the In This Issue feature, p. 1.
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Neoplasias Óseas/terapia , Variación Estructural del Genoma , Terapia Molecular Dirigida , Osteosarcoma/terapia , Animales , Neoplasias Óseas/genética , Variaciones en el Número de Copia de ADN , Genómica , Humanos , Ratones , Osteosarcoma/genética , Análisis de Secuencia de ARN , Secuenciación Completa del Genoma , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Nuclear receptors induce both transcriptional activation and repression programs responsible for development, homeostasis, and disease. Here, we report a previously overlooked enhancer decommissioning strategy underlying a large estrogen receptor alpha (ERα)-dependent transcriptional repression program. The unexpected signature for this E2-induced program resides in indirect recruitment of ERα to a large cohort of pioneer factor basally active FOXA1-bound enhancers that lack cognate ERα DNA-binding elements. Surprisingly, these basally active estrogen-repressed (BAER) enhancers are decommissioned by ERα-dependent recruitment of the histone demethylase KDM2A, functioning independently of its demethylase activity. Rather, KDM2A tethers the E3 ubiquitin-protein ligase NEDD4 to ubiquitylate/dismiss Pol II to abrogate eRNA transcription, with consequent target gene downregulation. Thus, our data reveal that Pol II ubiquitylation/dismissal may serve as a potentially broad strategy utilized by indirectly bound nuclear receptors to abrogate large programs of pioneer factor-mediated, eRNA-producing enhancers.
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Elementos de Facilitación Genéticos , Receptor alfa de Estrógeno/genética , Proteínas F-Box/genética , Factor Nuclear 3-alfa del Hepatocito/genética , Histona Demetilasas con Dominio de Jumonji/genética , Ubiquitina-Proteína Ligasas Nedd4/genética , ARN Polimerasa II/genética , Secuencia de Bases , Sitios de Unión , Sistemas CRISPR-Cas , Estradiol/farmacología , Receptor alfa de Estrógeno/metabolismo , Proteínas F-Box/metabolismo , Edición Génica/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Células MCF-7 , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Unión Proteica , ARN/genética , ARN/metabolismo , ARN Polimerasa II/metabolismo , Transducción de Señal , Transcripción Genética/efectos de los fármacos , Ubiquitinación/efectos de los fármacosRESUMEN
As most of the mitochondrial proteome is encoded in the nucleus, mitochondrial functions critically depend on nuclear gene expression and bidirectional mito-nuclear communication. However, mitochondria-to-nucleus communication pathways in mammals are incompletely understood. Here, we identify G-Protein Pathway Suppressor 2 (GPS2) as a mediator of mitochondrial retrograde signaling and a transcriptional activator of nuclear-encoded mitochondrial genes. GPS2-regulated translocation from mitochondria to nucleus is essential for the transcriptional activation of a nuclear stress response to mitochondrial depolarization and for supporting basal mitochondrial biogenesis in differentiating adipocytes and brown adipose tissue (BAT) from mice. In the nucleus, GPS2 recruitment to target gene promoters regulates histone H3K9 demethylation and RNA POL2 activation through inhibition of Ubc13-mediated ubiquitination. These findings, together, reveal an additional layer of regulation of mitochondrial gene transcription, uncover a direct mitochondria-nuclear communication pathway, and indicate that GPS2 retrograde signaling is a key component of the mitochondrial stress response in mammals.
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Núcleo Celular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mitocondrias/metabolismo , Biogénesis de Organelos , Transducción de Señal/fisiología , Células 3T3-L1 , Transporte Activo de Núcleo Celular/fisiología , Animales , Núcleo Celular/genética , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Metilación , Ratones , Mitocondrias/genética , Regiones Promotoras Genéticas/fisiología , Activación Transcripcional/fisiologíaRESUMEN
Non-proteolytic ubiquitin signaling mediated by Lys63 ubiquitin chains plays a critical role in multiple pathways that are key to the development and activation of immune cells. Our previous work indicates that GPS2 (G-protein Pathway Suppressor 2) is a multifunctional protein regulating TNFα signaling and lipid metabolism in the adipose tissue through modulation of Lys63 ubiquitination events. However, the full extent of GPS2-mediated regulation of ubiquitination and the underlying molecular mechanisms are unknown. Here, we report that GPS2 is required for restricting the activation of TLR and BCR signaling pathways and the AKT/FOXO1 pathway in immune cells based on direct inhibition of Ubc13 enzymatic activity. Relevance of this regulatory strategy is confirmed in vivo by B cell-targeted deletion of GPS2, resulting in developmental defects at multiple stages of B cell differentiation. Together, these findings reveal that GPS2 genomic and non-genomic functions are critical for the development and cellular homeostasis of B cells.
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Linfocitos B/citología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Enzimas Ubiquitina-Conjugadoras/antagonistas & inhibidores , Animales , Células de la Médula Ósea/citología , Diferenciación Celular , Perfilación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , UbiquitinaciónRESUMEN
Experimental evidence indicates that about 60% of miRNA-binding activity does not follow the canonical rule about the seed matching between miRNA and target mRNAs, but rather a non-canonical miRNA targeting activity outside the seed or with a seed-like motifs. Here, we propose a new unbiased method to identify canonical and non-canonical miRNA-binding sites from peaks identified by Ago2 Cross-Linked ImmunoPrecipitation associated to high-throughput sequencing (CLIP-seq). Since the quality of peaks is of pivotal importance for the final output of the proposed method, we provide a comprehensive benchmarking of four peak detection programs, namely CIMS, PIPE-CLIP, Piranha and Pyicoclip, on four publicly available Ago2-HITS-CLIP datasets and one unpublished in-house Ago2-dataset in stem cells. We measured the sensitivity, the specificity and the position accuracy toward miRNA binding sites identification, and the agreement with TargetScan. Secondly, we developed a new pipeline, called miRBShunter, to identify canonical and non-canonical miRNA-binding sites based on de novo motif identification from Ago2 peaks and prediction of miRNA::RNA heteroduplexes. miRBShunter was tested and experimentally validated on the in-house Ago2-dataset and on an Ago2-PAR-CLIP dataset in human stem cells. Overall, we provide guidelines to choose a suitable peak detection program and a new method for miRNA-target identification.
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Secuenciación de Nucleótidos de Alto Rendimiento , MicroARNs/metabolismo , Secuencias de Aminoácidos , Proteínas Argonautas/química , Proteínas Argonautas/genética , Benchmarking , Sitios de Unión , Humanos , MicroARNs/química , Conformación de Ácido Nucleico , Sensibilidad y Especificidad , Programas InformáticosRESUMEN
Enhancers instruct spatio-temporally specific gene expression in a manner tightly linked to higher-order chromatin architecture. Critical chromatin architectural regulators condensin I and condensin II play non-redundant roles controlling mitotic chromosomes. But the chromosomal locations of condensins and their functional roles in interphase are poorly understood. Here we report that both condensin complexes exhibit an unexpected, dramatic estrogen-induced recruitment to estrogen receptor α (ER-α)-bound eRNA(+) active enhancers in interphase breast cancer cells, exhibiting non-canonical interaction with ER-α via its DNA-binding domain (DBD). Condensins positively regulate ligand-dependent enhancer activation at least in part by recruiting an E3 ubiquitin ligase, HECTD1, to modulate the binding of enhancer-associated coactivators/corepressors, including p300 and RIP140, permitting full eRNA transcription, formation of enhancer:promoter looping, and the resultant coding gene activation. Collectively, our results reveal an important, unanticipated transcriptional role of interphase condensins in modulating estrogen-regulated enhancer activation and coding gene transcriptional program.
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Adenosina Trifosfatasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Receptor alfa de Estrógeno/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/genética , Secuencia de Bases , Sitios de Unión , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cromatina/genética , Cromatina/metabolismo , ADN de Neoplasias/genética , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Estradiol/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Interfase , Células MCF-7 , Modelos Biológicos , Datos de Secuencia Molecular , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/genética , Proteínas Nucleares/metabolismo , Proteína de Interacción con Receptores Nucleares 1 , Regiones Promotoras Genéticas , Unión Proteica , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
FOXO family transcription factors are downstream effectors of Insulin/IGF-1 signaling (IIS) and major determinants of aging in organisms ranging from worms to man. The molecular mechanisms that actively promote DAF16/FOXO stability and function are unknown. Here we identify the deubiquitylating enzyme MATH-33 as an essential DAF-16 regulator in IIS, which stabilizes active DAF-16 protein levels and, as a consequence, influences DAF-16 functions, such as metabolism, stress response, and longevity in C. elegans. MATH-33 associates with DAF-16 in cellulo and in vitro. MATH-33 functions as a deubiquitylase by actively removing ubiquitin moieties from DAF-16, thus counteracting the action of the RLE-1 E3-ubiquitin ligase. Our findings support a model in which MATH-33 promotes DAF-16 stability in response to decreased IIS by directly modulating its ubiquitylation state, suggesting that regulated oscillations in the stability of DAF-16 protein play an integral role in controlling processes such as metabolism and longevity.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Endopeptidasas/metabolismo , Factores de Transcripción Forkhead/metabolismo , Animales , Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/química , Factores de Transcripción Forkhead/química , Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Longevidad , Estabilidad Proteica , Transducción de Señal , UbiquitinaciónRESUMEN
Substantial evidence supports the hypothesis that enhancers are critical regulators of cell-type determination, orchestrating both positive and negative transcriptional programs; however, the basic mechanisms by which enhancers orchestrate interactions with cognate promoters during activation and repression events remain incompletely understood. Here we report the required actions of LIM domain-binding protein 1 (LDB1)/cofactor of LIM homeodomain protein 2/nuclear LIM interactor, interacting with the enhancer-binding protein achaete-scute complex homolog 1, to mediate looping to target gene promoters and target gene regulation in corticotrope cells. LDB1-mediated enhancer:promoter looping appears to be required for both activation and repression of these target genes. Although LDB1-dependent activated genes are regulated at the level of transcriptional initiation, the LDB1-dependent repressed transcription units appear to be regulated primarily at the level of promoter pausing, with LDB1 regulating recruitment of metastasis-associated 1 family, member 2, a component of the nucleosome remodeling deacetylase complex, on these negative enhancers, required for the repressive enhancer function. These results indicate that LDB1-dependent looping events can deliver repressive cargo to cognate promoters to mediate promoter pausing events in a pituitary cell type.
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Corticotrofos/fisiología , Proteínas de Unión al ADN/fisiología , Elementos de Facilitación Genéticos , Proteínas con Dominio LIM/fisiología , Regiones Promotoras Genéticas , Animales , Línea Celular , Proteínas de Unión al ADN/metabolismo , Proteínas con Dominio LIM/metabolismo , Ratones , Ratones NoqueadosRESUMEN
Bevacizumab, the recombinant antibody targeting vascular endothelial growth factor (VEGF), improves progression-free but not overall survival in metastatic breast cancer. To seek further insights in resistance mechanisms to bevacizumab at the molecular level, we developed VEGF and non-VEGF-driven ER-positive MCF7-derived xenograft models allowing comparison of tumor response at different timepoints. VEGF gene (MV165) overexpressing xenografts were initially sensitive to bevacizumab, but eventually acquired resistance. In contrast, parental MCF7 cells derived tumors were de novo insensitive to bevacizumab. Microarray analysis with qRT-PCR validation revealed that Follistatin (FST) and NOTCH were the top signaling pathways associated with resistance in VEGF-driven tumors (P<0.05). Based on the presence of VEGF, treatment with bevacizumab resulted in altered patterns of metagenes and PAM50 gene expression. In VEGF-driven model after short and long-term bevacizumab treatments, a change in the intrinsic subtype (luminal to myoepithelial/basal-like) was observed in association with increased expression of genes implicated with cancer stem cell phenotype (P<0.05). Our results show that the presence or absence of VEGF expression affects the response to bevacizumab therapy and gene pathways. In particular, long-term bevacizumab treatment shifts the cancer cells to a more aggressive myoepithelial/basal subtype in VEGF-expressing model, but not in non-VEGF model. These findings could shed light on variable results to anti-VEGF therapy in patients and emphasize the importance of patient stratification based on the VEGF expression. Our data strongly suggest consideration of patient subgroups for treatment and designing novel combinatory therapies in the clinical setting.
RESUMEN
Understanding the mechanisms by which compounds discovered using cell-based phenotypic screening strategies might exert their effects would be highly augmented by new approaches exploring their potential interactions with the genome. For example, altered androgen receptor (AR) transcriptional programs, including castration resistance and subsequent chromosomal translocations, play key roles in prostate cancer pathological progression, making the quest for identification of new therapeutic agents and an understanding of their actions a continued priority. Here we report an approach that has permitted us to uncover the sites and mechanisms of action of a drug, referred to as "SD70," initially identified by phenotypic screening for inhibitors of ligand and genotoxic stress-induced translocations in prostate cancer cells. Based on synthesis of a derivatized form of SD70 that permits its application for a ChIP-sequencing-like approach, referred to as "Chem-seq," we were next able to efficiently map the genome-wide binding locations of this small molecule, revealing that it largely colocalized with AR on regulatory enhancers. Based on these observations, we performed the appropriate global analyses to ascertain that SD70 inhibits the androgen-dependent AR program, and prostate cancer cell growth, acting, at least in part, by functionally inhibiting the Jumonji domain-containing demethylase, KDM4C. Global location of candidate drugs represents a powerful strategy for new drug development by mapping genome-wide location of small molecules, a powerful adjunct to contemporary drug development strategies.
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Antagonistas de Receptores Androgénicos/farmacología , Sistemas de Liberación de Medicamentos/métodos , Proteínas de Neoplasias/genética , Neoplasias de la Próstata/genética , Receptores Androgénicos/genética , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Animales , Antineoplásicos , Línea Celular Tumoral , Análisis Mutacional de ADN/métodos , Humanos , Masculino , Ratones , Proteínas de Neoplasias/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/metabolismo , Translocación GenéticaRESUMEN
Timely and selective recruitment of transcription factors to their appropriate DNA-binding sites represents a critical step in regulating gene activation; however, the regulatory strategies underlying each factor's effective recruitment to specific promoter and/or enhancer regions are not fully understood. Here, we identify an unexpected regulatory mechanism by which promoter-specific binding, and therefore function, of peroxisome proliferator-activator receptor γ (PPARγ) in adipocytes requires G protein suppressor 2 (GPS2) to prime the local chromatin environment via inhibition of the ubiquitin ligase RNF8 and stabilization of the H3K9 histone demethylase KDM4A/JMJD2. Integration of genome-wide profiling data indicates that the pioneering activity of GPS2/KDM4A is required for PPARγ-mediated regulation of a specific transcriptional program, including the lipolytic enzymes adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL). Hence, our findings reveal that GPS2 exerts a biologically important function in adipose tissue lipid mobilization by directly regulating ubiquitin signaling and indirectly modulating chromatin remodeling to prime selected genes for activation.
Asunto(s)
Histona Demetilasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , PPAR gamma/metabolismo , Regiones Promotoras Genéticas , Adipocitos/metabolismo , Animales , Ensamble y Desensamble de Cromatina , Lipasa/genética , Lipasa/metabolismo , Lipólisis/genética , Ratones , Esterol Esterasa/genética , Esterol Esterasa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Although recent studies have indicated roles of long non-coding RNAs (lncRNAs) in physiological aspects of cell-type determination and tissue homeostasis, their potential involvement in regulated gene transcription programs remains rather poorly understood. The androgen receptor regulates a large repertoire of genes central to the identity and behaviour of prostate cancer cells, and functions in a ligand-independent fashion in many prostate cancers when they become hormone refractory after initial androgen deprivation therapy. Here we report that two lncRNAs highly overexpressed in aggressive prostate cancer, PRNCR1 (also known as PCAT8) and PCGEM1, bind successively to the androgen receptor and strongly enhance both ligand-dependent and ligand-independent androgen-receptor-mediated gene activation programs and proliferation in prostate cancer cells. Binding of PRNCR1 to the carboxy-terminally acetylated androgen receptor on enhancers and its association with DOT1L appear to be required for recruitment of the second lncRNA, PCGEM1, to the androgen receptor amino terminus that is methylated by DOT1L. Unexpectedly, recognition of specific protein marks by PCGEM1-recruited pygopus 2 PHD domain enhances selective looping of androgen-receptor-bound enhancers to target gene promoters in these cells. In 'resistant' prostate cancer cells, these overexpressed lncRNAs can interact with, and are required for, the robust activation of both truncated and full-length androgen receptor, causing ligand-independent activation of the androgen receptor transcriptional program and cell proliferation. Conditionally expressed short hairpin RNA targeting these lncRNAs in castration-resistant prostate cancer cell lines strongly suppressed tumour xenograft growth in vivo. Together, these results indicate that these overexpressed lncRNAs can potentially serve as a required component of castration-resistance in prostatic tumours.