RESUMEN
Numerous studies have now demonstrated that lncRNAs can influence gene expression programs leading to cell and organismal phenotypes. Typically, lncRNA perturbations and concomitant changes in gene expression are measured on the timescale of many hours to days. Thus, we currently lack a temporally grounded understanding of the primary, secondary, and tertiary relationships of lncRNA-mediated transcriptional and epigenetic regulation-a prerequisite to elucidating lncRNA mechanisms. To begin to address when and where a lncRNA regulates gene expression, we genetically engineer cell lines to temporally induce the lncRNA Firre. Using this approach, we are able to monitor lncRNA transcriptional regulatory events from 15 min to four days. We observe that upon induction, Firre RNA regulates epigenetic and transcriptional states in trans within 30 min. These early regulatory events result in much larger transcriptional changes after 12 h, well before current studies monitor lncRNA regulation. Moreover, Firre-mediated gene expression changes are epigenetically remembered for days. Overall, this study suggests that lncRNAs can rapidly regulate gene expression by establishing persistent epigenetic and transcriptional states.
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Epigénesis Genética , ARN Largo no Codificante , Transcripción Genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Humanos , Regulación de la Expresión Génica , Línea Celular , Factores de TiempoRESUMEN
Firre encodes a lncRNA involved in nuclear organization. Here, we show that Firre RNA expressed from the active X chromosome maintains histone H3K27me3 enrichment on the inactive X chromosome (Xi) in somatic cells. This trans-acting effect involves SUZ12, reflecting interactions between Firre RNA and components of the Polycomb repressive complexes. Without Firre RNA, H3K27me3 decreases on the Xi and the Xi-perinucleolar location is disrupted, possibly due to decreased CTCF binding on the Xi. We also observe widespread gene dysregulation, but not on the Xi. These effects are measurably rescued by ectopic expression of mouse or human Firre/FIRRE transgenes, supporting conserved trans-acting roles. We also find that the compact 3D structure of the Xi partly depends on the Firre locus and its RNA. In common lymphoid progenitors and T-cells Firre exerts a cis-acting effect on maintenance of H3K27me3 in a 26 Mb region around the locus, demonstrating cell type-specific trans- and cis-acting roles of this lncRNA.
Asunto(s)
Epigénesis Genética , ARN Largo no Codificante/genética , Inactivación del Cromosoma X/genética , Alelos , Animales , Secuencia de Bases , Línea Celular , Núcleo Celular/genética , Cromatina/metabolismo , ADN Complementario/genética , Femenino , Eliminación de Gen , Ontología de Genes , Sitios Genéticos , Genoma , Histonas/metabolismo , Lisina/metabolismo , Masculino , Metilación , Ratones Endogámicos C57BL , Complejo Represivo Polycomb 2/metabolismo , ARN Largo no Codificante/metabolismo , Transgenes , Regulación hacia Arriba/genética , Cromosoma X/genéticaRESUMEN
Increased consumption of fats and added sugars has been associated with an increase in metabolic syndromes. Here we show that mice chronically fed an energy-rich diet (ERD) with high fat and moderate sucrose have enhanced the absorption of a gastrointestinal fructose load, and this required expression of the arrestin domain protein Txnip in the intestinal epithelial cells. ERD feeding induced gene and protein expression of Glut5, and this required the expression of Txnip. Furthermore, Txnip interacted with Rab11a, a small GTPase that facilitates the apical localization of Glut5. We also demonstrate that ERD promoted Txnip/Glut5 complexes in the apical intestinal epithelial cell. Our findings demonstrate that ERD facilitates fructose absorption through a Txnip-dependent mechanism in the intestinal epithelial cell, suggesting that increased fructose absorption could potentially provide a mechanism for worsening of metabolic syndromes in the setting of a chronic ERD.
RESUMEN
BACKGROUND: Several long noncoding RNAs (lncRNAs) have been shown to function as components of molecular machines that play fundamental roles in biology. While the number of annotated lncRNAs in mammalian genomes has greatly expanded, studying lncRNA function has been a challenge due to their diverse biological roles and because lncRNA loci can contain multiple molecular modes that may exert function. RESULTS: We previously generated and characterized a cohort of 20 lncRNA loci knockout mice. Here, we extend this initial study and provide a more detailed analysis of the highly conserved lncRNA locus, taurine-upregulated gene 1 (Tug1). We report that Tug1-knockout male mice are sterile with underlying defects including a low number of sperm and abnormal sperm morphology. Because lncRNA loci can contain multiple modes of action, we wanted to determine which, if any, potential elements contained in the Tug1 genomic region have any activity. Using engineered mouse models and cell-based assays, we provide evidence that the Tug1 locus harbors two distinct noncoding regulatory activities, as a cis-DNA repressor that regulates neighboring genes and as a lncRNA that can regulate genes by a trans-based function. We also show that Tug1 contains an evolutionary conserved open reading frame that when overexpressed produces a stable protein which impacts mitochondrial membrane potential, suggesting a potential third coding function. CONCLUSIONS: Our results reveal an essential role for the Tug1 locus in male fertility and uncover evidence for distinct molecular modes in the Tug1 locus, thus highlighting the complexity present at lncRNA loci.
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Fertilidad/genética , ARN Largo no Codificante/genética , Animales , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Noqueados , Sistemas de Lectura Abierta , Espermatogénesis/genéticaRESUMEN
Recent advances in CRISPR/Cas gene editing technology have significantly expanded the possibilities and accelerated the pace of creating genetically engineered animal models. However, CRISPR/Cas-based strategies designed to precisely edit the genome can often yield unintended outcomes. Here, we report the use of zygotic CRISPR/Cas9 injections to generate a knock-in GFP reporter mouse at the Gdf11 locus. Phenotypic and genomic characterization of founder animals from these injections revealed a subset that contained the correct targeting event and exhibited GFP expression that, within the hematopoietic system, was restricted predominantly to lymphoid cells. Yet, in another subset of founder mice, we detected aberrant integration events at the target site that dramatically and inaccurately shifted hematopoietic GFP expression from the lymphoid to the myeloid lineage. Additionally, we recovered multiple Gdf11 deletion alleles that modified the C-terminus of the GDF11 protein. When bred to homozygosity, most of these alleles recapitulated skeletal phenotypes reported previously for Gdf11 knockout mice, suggesting that these represent null alleles. However, we also recovered one Gdf11 deletion allele that encodes a novel GDF11 variant protein ("GDF11-WE") predicted to contain two additional amino acids (tryptophan (W) and glutamic acid (E)) at the C-terminus of the mature ligand. Unlike the other Gdf11 deletion alleles recovered in this study, homozygosity for the Gdf11WE allele did not phenocopy Gdf11 knockout skeletal phenotypes. Further investigation using in vivo and in vitro approaches demonstrated that GDF11-WE retains substantial physiological function, indicating that GDF11 can tolerate at least some modifications of its C-terminus and providing unexpected insights into its biochemical activities. Altogether, our study confirms that one-step zygotic injections of CRISPR/Cas gene editing complexes provide a quick and powerful tool to generate gene-modified mouse models. Moreover, our findings underscore the critical importance of thorough characterization and validation of any modified alleles generated by CRISPR, as unintended on-target effects that fail to be detected by simple PCR screening can produce substantially altered phenotypic readouts.
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Alelos , Proteínas Morfogenéticas Óseas/genética , Sistemas CRISPR-Cas , Eliminación de Gen , Edición Génica , Factores de Diferenciación de Crecimiento/genética , Animales , Femenino , Genes Reporteros , Ingeniería Genética , Genoma , Ácido Glutámico/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células Madre Hematopoyéticas/metabolismo , Homocigoto , Ligandos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Células Mieloides/metabolismo , Fenotipo , Dominios Proteicos , Triptófano/metabolismoRESUMEN
Recent evidence has determined that the conserved X chromosome mega-structures controlled by the Firre and Dxz4 loci are not required for X chromosome inactivation (XCI) in cell lines. Here, we examined the in vivo contribution of these loci by generating mice carrying a single or double deletion of Firre and Dxz4. We found that these mutants are viable, fertile and show no defect in random or imprinted XCI. However, the lack of these elements results in many dysregulated genes on autosomes in an organ-specific manner. By comparing the dysregulated genes between the single and double deletion, we identified superloop, megadomain, and Firre locus-dependent gene sets. The largest transcriptional effect was observed in all strains lacking the Firre locus, indicating that this locus is the main driver for these autosomal expression signatures. Collectively, these findings suggest that these X-linked loci are involved in autosomal gene regulation rather than XCI biology.
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ADN Complementario/genética , Eliminación de Gen , Regulación de la Expresión Génica , ARN Largo no Codificante/genética , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos , Ratones Noqueados , Transcriptoma , Inactivación del Cromosoma XRESUMEN
RNA has been classically known to play central roles in biology, including maintaining telomeres, protein synthesis, and in sex chromosome compensation. While thousands of long noncoding RNAs (lncRNAs) have been identified, attributing RNA-based roles to lncRNA loci requires assessing whether phenotype(s) could be due to DNA regulatory elements, transcription, or the lncRNA. Here, we use the conserved X chromosome lncRNA locus Firre, as a model to discriminate between DNA- and RNA-mediated effects in vivo. We demonstrate that (i) Firre mutant mice have cell-specific hematopoietic phenotypes, and (ii) upon exposure to lipopolysaccharide, mice overexpressing Firre exhibit increased levels of pro-inflammatory cytokines and impaired survival. (iii) Deletion of Firre does not result in changes in local gene expression, but rather in changes on autosomes that can be rescued by expression of transgenic Firre RNA. Together, our results provide genetic evidence that the Firre locus produces a trans-acting lncRNA that has physiological roles in hematopoiesis.
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Sitios Genéticos , Hematopoyesis/genética , ARN Largo no Codificante/genética , Animales , Fertilidad/genética , Regulación del Desarrollo de la Expresión Génica , Inmunidad Innata/efectos de los fármacos , Lipopolisacáridos/farmacología , Ratones Noqueados , Especificidad de Órganos/genética , Fenotipo , ARN Largo no Codificante/metabolismoRESUMEN
BACKGROUND: Recently, it has become clear that some promoters function as long-range regulators of gene expression. However, direct and quantitative assessment of enhancer activity at long intergenic noncoding RNA (lincRNA) or mRNA gene bodies has not been performed. To unbiasedly assess the enhancer capacity across lincRNA and mRNA loci, we performed a massively parallel reporter assay (MPRA) on six lincRNA loci and their closest protein-coding neighbors. RESULTS: For both gene classes, we find significantly more MPRA activity in promoter regions than in gene bodies. However, three lincRNA loci, Lincp21, LincEnc1, and Peril, and one mRNA locus, Morc2a, display significant enhancer activity within their gene bodies. We hypothesize that such peaks may mark long-range enhancers, and test this in vivo using RNA sequencing from a knockout mouse model and high-throughput chromosome conformation capture (Hi-C). We find that ablation of a high-activity MPRA peak in the Peril gene body leads to consistent dysregulation of Mccc1 and Exosc9 in the neighboring topologically associated domain (TAD). This occurs irrespective of Peril lincRNA expression, demonstrating this regulation is DNA-dependent. Hi-C confirms long-range contacts with the neighboring TAD, and these interactions are altered upon Peril knockout. Surprisingly, we do not observe consistent regulation of genes within the local TAD. Together, these data suggest a long-range enhancer-like function for the Peril gene body. CONCLUSIONS: A multi-faceted approach combining high-throughput enhancer discovery with genetic models can connect enhancers to their gene targets and provides evidence of inter-TAD gene regulation.
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Regulación de la Expresión Génica , ARN Largo no Codificante , Elementos Reguladores de la Transcripción , Animales , Ratones NoqueadosRESUMEN
The binding of the transcriptional regulator CTCF to the genome has been implicated in the formation of topologically associated domains (TADs). However, the general mechanisms of folding the genome into TADs are not fully understood. Here we test the effects of deleting a CTCF-rich locus on TAD boundary formation. Using genome-wide chromosome conformation capture (Hi-C), we focus on one TAD boundary on chromosome X harboring ~ 15 CTCF binding sites and located at the long non-coding RNA (lncRNA) locus Firre. Specifically, this TAD boundary is invariant across evolution, tissues, and temporal dynamics of X-chromosome inactivation. We demonstrate that neither the deletion of this locus nor the ectopic insertion of Firre cDNA or its ectopic expression are sufficient to alter TADs in a sex-specific or allele-specific manner. In contrast, Firre's deletion disrupts the chromatin super-loop formation of the inactive X-chromosome. Collectively, our findings suggest that apart from CTCF binding, additional mechanisms may play roles in establishing TAD boundary formation.
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Factor de Unión a CCCTC/química , Cromosomas Humanos X , Eliminación de Gen , ARN Largo no Codificante/genética , Inactivación del Cromosoma X , Animales , Sitios de Unión , Factor de Unión a CCCTC/genética , Cromatina/química , ADN Complementario/metabolismo , Femenino , Biblioteca de Genes , Genoma Humano , Humanos , Células K562 , Masculino , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas , Unión Proteica , Dominios Proteicos , ARN Largo no Codificante/metabolismo , Proteínas Represoras/metabolismo , Eliminación de Secuencia , Transcripción Genética , Cromosoma XRESUMEN
GLI proteins convert Sonic hedgehog (Shh) signaling into a transcriptional output in a tissue-specific fashion. The Shh pathway has been extensively studied in the limb bud, where it helps regulate growth through a SHH-FGF feedback loop. However, the transcriptional response is still poorly understood. We addressed this by determining the gene expression patterns of approximately 200 candidate GLI-target genes and identified three discrete SHH-responsive expression domains. GLI-target genes expressed in the three domains are predominately regulated by derepression of GLI3 but have different temporal requirements for SHH. The GLI binding regions associated with these genes harbor both distinct and common DNA motifs. Given the potential for interaction between the SHH and FGF pathways, we also measured the response of GLI-target genes to inhibition of FGF signaling and found the majority were either unaffected or upregulated. These results provide the first characterization of the spatiotemporal response of a large group of GLI-target genes and lay the foundation for a systems-level understanding of the gene regulatory networks underlying SHH-mediated limb patterning.
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Tipificación del Cuerpo/fisiología , Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Hedgehog/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Esbozos de los Miembros/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Sitios de Unión/genética , Tipificación del Cuerpo/genética , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Esbozos de los Miembros/citología , Ratones , Ratones Transgénicos , Unión Proteica/genética , Estructura Terciaria de Proteína , Transducción de Señal/fisiología , Activación Transcripcional , Proteína Gli3 con Dedos de ZincRESUMEN
Mutations in the Bone Morphogenetic Protein (BMP) pathway are associated with a range of defects in skeletal formation. Genetic analysis of BMP signaling requirements is complicated by the presence of three partially redundant BMPs that are required for multiple stages of limb development. We generated an inducible allele of a BMP inhibitor, Gremlin, which reduces BMP signaling. We show that BMPs act in a dose and time dependent manner in which early reduction of BMPs result in digit loss, while inhibiting overall BMP signaling between E10.5 and E11.5 allows polydactylous digit formation. During this period, inhibiting BMPs extends the duration of FGF signaling. Sox9 is initially expressed in normal digit ray domains but at reduced levels that correlate with the reduction in BMP signaling. The persistence of elevated FGF signaling likely promotes cell proliferation and survival, inhibiting the activation of Sox9 and secondarily, inhibiting the differentiation of Sox9-expressing chondrocytes. Our results provide new insights into the timing and clarify the mechanisms underlying BMP signaling during digit morphogenesis.
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Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 4/genética , Proteína Morfogenética Ósea 7/genética , Esbozos de los Miembros/embriología , Polidactilia/genética , Animales , Apoptosis , Proteína Morfogenética Ósea 2/antagonistas & inhibidores , Proteína Morfogenética Ósea 4/antagonistas & inhibidores , Proteína Morfogenética Ósea 7/antagonistas & inhibidores , Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Proteínas Morfogenéticas Óseas/genética , Diferenciación Celular/genética , Proliferación Celular , Condrogénesis/genética , Citocinas , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Miembro Posterior/embriología , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/genética , Mesodermo/embriología , Ratones , Ratones Transgénicos , Mutación , Polidactilia/embriología , Factor de Transcripción SOX9/biosíntesis , Transducción de Señal/genéticaRESUMEN
The transcriptional response to the Hedgehog (Hh) pathway is mediated by Gli proteins, which function as context-dependent transcriptional activators or repressors. However, the mechanism by which Gli proteins regulate their target genes is poorly understood. Here, we have performed the first genetic characterization of a Gli-dependent cis-regulatory module (CRM), focusing on its regulation of Grem1 in the mouse limb bud. The CRM, termed GRE1 (Gli responsive element 1), can act as both an enhancer and a silencer. The enhancer activity requires sustained Hh signaling. As a Gli-dependent silencer, GRE1 prevents ectopic transcription of Grem1 driven through additional CRMs. In doing so, GRE1 works with additional GREs to robustly regulate Grem1. We suggest that multiple Gli CRMs may be a general mechanism for mediating a robust transcriptional response to the Hh pathway.
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Péptidos y Proteínas de Señalización Intercelular/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Esbozos de los Miembros/embriología , Esbozos de los Miembros/metabolismo , Proteínas Represoras/metabolismo , Vertebrados/embriología , Vertebrados/genética , Animales , Citocinas , Elementos de Facilitación Genéticos/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Modelos Biológicos , Transducción de Señal/genética , Factores de Tiempo , Proteína con Dedos de Zinc GLI1RESUMEN
BACKGROUND: The vertebrate limb bud is a well-established system for studying the mechanisms driving growth and patterning of an embryonic tissue. However, approaches for manipulating gene expression are currently limited to time-consuming methods. Culturing primary limb bud cells could potentially be used as a quicker assay. However, limb cells in culture quickly differentiate into cartilage under normal conditions, and approaches delivering DNA and siRNA into primary limb cells in culture are limited. These technical limitations have restricted the utility of limb buds for investigating problems that require higher-throughput approaches. RESULTS: In this report, we describe adaptations to a method for culturing primary limb bud cells in a pre-chondrogenic state, and generate a population of mouse primary limb cells that are responsive to Hedgehog (Hh) signaling. Hh-stimulated cells upregulate Hh target genes as well as an exogenous Hh-responsive reporter. We then describe a method for highly efficient delivery of plasmids and siRNAs into cultured primary limb bud cells in a 96-well format. CONCLUSIONS: Cultures of primary limb bud cells are amenable to gene manipulation under conditions that maintain the limb cells in an Hh-responsive, undifferentiated state. This approach provides a medium-throughput system to manipulate gene expression, and test DNA regulatory elements.
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Esbozos de los Miembros/metabolismo , Animales , Células Cultivadas , Electroporación , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Esbozos de los Miembros/citología , Ratones , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Maternal supplementation with folic acid is known to reduce the incidence of neural tube defects (NTDs) by as much as 70%. Despite the strong clinical link between folate and NTDs, the biochemical mechanisms through which folic acid acts during neural tube development remain undefined. The Mthfd1l gene encodes a mitochondrial monofunctional 10-formyl-tetrahydrofolate synthetase, termed MTHFD1L. This gene is expressed in adults and at all stages of mammalian embryogenesis with localized regions of higher expression along the neural tube, developing brain, craniofacial structures, limb buds, and tail bud. In both embryos and adults, MTHFD1L catalyzes the last step in the flow of one-carbon units from mitochondria to cytoplasm, producing formate from 10-formyl-THF. To investigate the role of mitochondrial formate production during embryonic development, we have analyzed Mthfd1l knockout mice. All embryos lacking Mthfd1l exhibit aberrant neural tube closure including craniorachischisis and exencephaly and/or a wavy neural tube. This fully penetrant folate-pathway mouse model does not require feeding a folate-deficient diet to cause this phenotype. Maternal supplementation with sodium formate decreases the incidence of NTDs and partially rescues the growth defect in embryos lacking Mthfd1l. These results reveal the critical role of mitochondrially derived formate in mammalian development, providing a mechanistic link between folic acid and NTDs. In light of previous studies linking a common splice variant in the human MTHFD1L gene with increased risk for NTDs, this mouse model provides a powerful system to help elucidate the specific metabolic mechanisms that underlie folate-associated birth defects, including NTDs.
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Anomalías Múltiples/genética , Aminohidrolasas/genética , Anomalías Craneofaciales/genética , Desarrollo Embrionario/genética , Formiato-Tetrahidrofolato Ligasa/genética , Metilenotetrahidrofolato Deshidrogenasa (NADP)/genética , Complejos Multienzimáticos/genética , Defectos del Tubo Neural/genética , Aminohidrolasas/deficiencia , Animales , Cartilla de ADN/genética , Desarrollo Embrionario/efectos de los fármacos , Formiato-Tetrahidrofolato Ligasa/deficiencia , Formiatos/administración & dosificación , Formiatos/farmacología , Eliminación de Gen , Genotipo , Immunoblotting , Redes y Vías Metabólicas/fisiología , Metilenotetrahidrofolato Deshidrogenasa (NADP)/deficiencia , Ratones , Ratones Noqueados , Complejos Multienzimáticos/deficiencia , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
During Drosophila melanogaster oogenesis, a germline stem cell divides forming a cyst of 16 interconnected cells. One cell enters the oogenic pathway, and the remaining 15 differentiate as nurse cells. Although directed transport and localization of oocyte differentiation factors within the single cell are indispensible for selection, maintenance, and differentiation of the oocyte, the mechanisms regulating these events are poorly understood. Mago Nashi and Tsunagi/Y14, core components of the exon junction complex (a multiprotein complex assembled on spliced RNAs), are essential for restricting oocyte fate to a single cell and for localization of oskar mRNA. Here we provide evidence that Mago Nashi and Tsunagi/Y14 form an oogenic complex with Ranshi, a protein with a zinc finger-associated domain and zinc finger domains. Genetic analyses of ranshi reveal that (1) 16-cell cysts are formed, (2) two cells retain synaptonemal complexes, (3) all cells have endoreplicated DNA (as observed in nurse cells), and (4) oocyte-specific cytoplasmic markers accumulate and persist within a single cell but are not localized within the posterior pole of the presumptive oocyte. Our results indicate that Ranshi interacts with the exon junction complex to localize components essential for oocyte differentiation within the posterior pole of the presumptive oocyte.