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1.
Chembiochem ; 25(10): e202400049, 2024 May 17.
Artículo en Inglés | MEDLINE | ID: mdl-38456652

RESUMEN

Long non-coding RNAs (lncRNAs) are important regulators of gene expression and can associate with DNA as RNA : DNA heteroduplexes or RNA ⋅ DNA : DNA triple helix structures. Here, we review in vitro biochemical and biophysical experiments including electromobility shift assays (EMSA), circular dichroism (CD) spectroscopy, thermal melting analysis, microscale thermophoresis (MST), single-molecule Förster resonance energy transfer (smFRET) and nuclear magnetic resonance (NMR) spectroscopy to investigate RNA ⋅ DNA : DNA triple helix and RNA : DNA heteroduplex formation. We present the investigations of the antiparallel triplex-forming lncRNA MEG3 targeting the gene TGFB2 and the parallel triplex-forming lncRNA Fendrr with its target gene Emp2. The thermodynamic properties of these oligonucleotides lead to concentration-dependent heterogeneous mixtures, where a DNA duplex, an RNA : DNA heteroduplex and an RNA ⋅ DNA : DNA triplex coexist and their relative populations are modulated in a temperature-dependent manner. The in vitro data provide a reliable readout of triplex structures, as RNA ⋅ DNA : DNA triplexes show distinct features compared to DNA duplexes and RNA : DNA heteroduplexes. Our experimental results can be used to validate computationally predicted triple helix formation between novel disease-relevant lncRNAs and their DNA target genes.


Asunto(s)
ADN , Conformación de Ácido Nucleico , ARN Largo no Codificante , ARN Largo no Codificante/genética , ARN Largo no Codificante/química , ARN Largo no Codificante/metabolismo , ADN/química , ADN/genética , Humanos , Ácidos Nucleicos Heterodúplex/química , ARN/química , ARN/genética , ARN/metabolismo , Termodinámica
2.
NPJ Genom Med ; 9(1): 18, 2024 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-38429302

RESUMEN

CELSR3 codes for a planar cell polarity protein. We describe twelve affected individuals from eleven independent families with bi-allelic variants in CELSR3. Affected individuals presented with an overlapping phenotypic spectrum comprising central nervous system (CNS) anomalies (7/12), combined CNS anomalies and congenital anomalies of the kidneys and urinary tract (CAKUT) (3/12) and CAKUT only (2/12). Computational simulation of the 3D protein structure suggests the position of the identified variants to be implicated in penetrance and phenotype expression. CELSR3 immunolocalization in human embryonic urinary tract and transient suppression and rescue experiments of Celsr3 in fluorescent zebrafish reporter lines further support an embryonic role of CELSR3 in CNS and urinary tract formation.

3.
Nat Commun ; 14(1): 7024, 2023 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-37919291

RESUMEN

After myocardial infarction in the adult heart the remaining, non-infarcted tissue adapts to compensate the loss of functional tissue. This adaptation requires changes in gene expression networks, which are mostly controlled by transcription regulating proteins. Long non-coding transcripts (lncRNAs) are taking part in fine-tuning such gene programs. We describe and characterize the cardiomyocyte specific lncRNA Sweetheart RNA (Swhtr), an approximately 10 kb long transcript divergently expressed from the cardiac core transcription factor coding gene Nkx2-5. We show that Swhtr is dispensable for normal heart development and function but becomes essential for the tissue adaptation process after myocardial infarction in murine males. Re-expressing Swhtr from an exogenous locus rescues the Swhtr null phenotype. Genes that depend on Swhtr after cardiac stress are significantly occupied and therefore most likely regulated by NKX2-5. The Swhtr transcript interacts with NKX2-5 and disperses upon hypoxic stress in cardiomyocytes, indicating an auxiliary role of Swhtr for NKX2-5 function in tissue adaptation after myocardial injury.


Asunto(s)
Lesiones Cardíacas , Infarto del Miocardio , ARN Largo no Codificante , Masculino , Ratones , Animales , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Miocitos Cardíacos/metabolismo , Cardiomegalia/genética , Cardiomegalia/metabolismo , Infarto del Miocardio/metabolismo
4.
Nucleic Acids Res ; 51(12): 6227-6237, 2023 07 07.
Artículo en Inglés | MEDLINE | ID: mdl-37207329

RESUMEN

Long non-coding RNAs are a very versatile class of molecules that can have important roles in regulating a cells function, including regulating other genes on the transcriptional level. One of these mechanisms is that RNA can directly interact with DNA thereby recruiting additional components such as proteins to these sites via an RNA:dsDNA triplex formation. We genetically deleted the triplex forming sequence (FendrrBox) from the lncRNA Fendrr in mice and found that this FendrrBox is partially required for Fendrr function in vivo. We found that the loss of the triplex forming site in developing lungs causes a dysregulation of gene programs associated with lung fibrosis. A set of these genes contain a triplex site directly at their promoter and are expressed in lung fibroblasts. We biophysically confirmed the formation of an RNA:dsDNA triplex with target promoters in vitro. We found that Fendrr with the Wnt signalling pathway regulates these genes, implicating that Fendrr synergizes with Wnt signalling in lung fibrosis.


Asunto(s)
Fibrosis Pulmonar , ARN Largo no Codificante , Animales , Ratones , Fibrosis , Pulmón/crecimiento & desarrollo , Pulmón/metabolismo , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/metabolismo , ARN Largo no Codificante/metabolismo
5.
Nat Commun ; 14(1): 1722, 2023 04 03.
Artículo en Inglés | MEDLINE | ID: mdl-37012244

RESUMEN

Cardiogenesis relies on the precise spatiotemporal coordination of multiple progenitor populations. Understanding the specification and differentiation of these distinct progenitor pools during human embryonic development is crucial for advancing our knowledge of congenital cardiac malformations and designing new regenerative therapies. By combining genetic labelling, single-cell transcriptomics, and ex vivo human-mouse embryonic chimeras we uncovered that modulation of retinoic acid signaling instructs human pluripotent stem cells to form heart field-specific progenitors with distinct fate potentials. In addition to the classical first and second heart fields, we observed the appearance of juxta-cardiac field progenitors giving rise to both myocardial and epicardial cells. Applying these findings to stem-cell based disease modelling we identified specific transcriptional dysregulation in first and second heart field progenitors derived from stem cells of patients with hypoplastic left heart syndrome. This highlights the suitability of our in vitro differentiation platform for studying human cardiac development and disease.


Asunto(s)
Células Madre Pluripotentes , Tretinoina , Humanos , Animales , Ratones , Tretinoina/farmacología , Corazón , Miocardio , Diferenciación Celular , Miocitos Cardíacos
6.
J Med Genet ; 60(6): 587-596, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-36379543

RESUMEN

BACKGROUND: SHROOM4 is thought to play an important role in cytoskeletal modification and development of the early nervous system. Previously, single-nucleotide variants (SNVs) or copy number variations (CNVs) in SHROOM4 have been associated with the neurodevelopmental disorder Stocco dos Santos syndrome, but not with congenital anomalies of the urinary tract and the visceral or the cardiovascular system. METHODS: Here, exome sequencing and CNV analyses besides expression studies in zebrafish and mouse and knockdown (KD) experiments using a splice blocking morpholino in zebrafish were performed to study the role of SHROOM4 during embryonic development. RESULTS: In this study, we identified putative disease-causing SNVs and CNVs in SHROOM4 in six individuals from four families with congenital anomalies of the urinary tract and the anorectal, cardiovascular and central nervous systems (CNS). Embryonic mouse and zebrafish expression studies showed Shroom4 expression in the upper and lower urinary tract, the developing cloaca, the heart and the cerebral CNS. KD studies in zebrafish larvae revealed pronephric cysts, anomalies of the cloaca and the heart, decreased eye-to-head ratio and higher mortality compared with controls. These phenotypes could be rescued by co-injection of human wild-type SHROOM4 mRNA and morpholino. CONCLUSION: The identified SNVs and CNVs in affected individuals with congenital anomalies of the urinary tract, the anorectal, the cardiovascular and the central nervous systems, and subsequent embryonic mouse and zebrafish studies suggest SHROOM4 as a developmental gene for different organ systems.


Asunto(s)
Sistema Cardiovascular , Sistema Urinario , Embarazo , Femenino , Humanos , Animales , Ratones , Pez Cebra/genética , Variaciones en el Número de Copia de ADN , Morfolinos , Sistema Urinario/anomalías , Sistema Nervioso Central
7.
Circ Res ; 130(1): 67-79, 2022 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-34789007

RESUMEN

BACKGROUND: Circular RNAs (circRNAs) are generated by back splicing of mostly mRNAs and are gaining increasing attention as a novel class of regulatory RNAs that control various cellular functions. However, their physiological roles and functional conservation in vivo are rarely addressed, given the inherent challenges of their genetic inactivation. Here, we aimed to identify locus conserved circRNAs in mice and humans, which can be genetically deleted due to retained intronic elements not contained in the mRNA host gene to eventually address functional conservation. METHODS AND RESULTS: Combining published endothelial RNA-sequencing data sets with circRNAs of the circATLAS databank, we identified locus-conserved circRNA retaining intronic elements between mice and humans. CRISPR/Cas9 mediated genetic depletion of the top expressed circRNA cZfp292 resulted in an altered endothelial morphology and aberrant flow alignment in the aorta in vivo. Consistently, depletion of cZNF292 in endothelial cells in vitro abolished laminar flow-induced alterations in cell orientation, paxillin localization and focal adhesion organization. Mechanistically, we identified the protein SDOS (syndesmos) to specifically interact with cZNF292 in endothelial cells by RNA-affinity purification and subsequent mass spectrometry analysis. Silencing of SDOS or its protein binding partner Syndecan-4, or mutation of the SDOS-cZNF292 binding site, prevented laminar flow-induced cytoskeletal reorganization thereby recapitulating cZfp292 knockout phenotypes. CONCLUSIONS: Together, our data reveal a hitherto unknown role of cZNF292/cZfp292 in endothelial flow responses, which influences endothelial shape.


Asunto(s)
Proteínas de Unión al ADN , Células Endoteliales , Endotelio Vascular , ARN Circular , Factores de Transcripción , Animales , Humanos , Ratones , Circulación Sanguínea , Proteínas de Unión al ADN/genética , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones Endogámicos C57BL , Unión Proteica , ARN Circular/genética , ARN Circular/metabolismo , Sindecano-4/metabolismo , Factores de Transcripción/genética
8.
Nature ; 592(7852): 93-98, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33568816

RESUMEN

Long non-coding RNAs (lncRNAs) can be important components in gene-regulatory networks1, but the exact nature and extent of their involvement in human Mendelian disease is largely unknown. Here we show that genetic ablation of a lncRNA locus on human chromosome 2 causes a severe congenital limb malformation. We identified homozygous 27-63-kilobase deletions located 300 kilobases upstream of the engrailed-1 gene (EN1) in patients with a complex limb malformation featuring mesomelic shortening, syndactyly and ventral nails (dorsal dimelia). Re-engineering of the human deletions in mice resulted in a complete loss of En1 expression in the limb and a double dorsal-limb phenotype that recapitulates the human disease phenotype. Genome-wide transcriptome analysis in the developing mouse limb revealed a four-exon-long non-coding transcript within the deleted region, which we named Maenli. Functional dissection of the Maenli locus showed that its transcriptional activity is required for limb-specific En1 activation in cis, thereby fine-tuning the gene-regulatory networks controlling dorso-ventral polarity in the developing limb bud. Its loss results in the En1-related dorsal ventral limb phenotype, a subset of the full En1-associated phenotype. Our findings demonstrate that mutations involving lncRNA loci can result in human Mendelian disease.


Asunto(s)
Extremidades , Proteínas de Homeodominio/genética , Deformidades Congénitas de las Extremidades/genética , ARN Largo no Codificante/genética , Eliminación de Secuencia/genética , Transcripción Genética , Activación Transcripcional/genética , Animales , Línea Celular , Cromatina/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Transgénicos
9.
Elife ; 92020 10 23.
Artículo en Inglés | MEDLINE | ID: mdl-33095159

RESUMEN

While long non-coding RNA (lncRNA) genes have attracted a lot of attention in the last decade, the focus regarding their mechanisms of action has been primarily on the RNA product of these genes. Recent work on several lncRNAs genes demonstrates that not only is the produced RNA species important, but also that transcription of the lncRNA locus alone can have regulatory functions. Like the functions of lncRNA transcripts, the mechanisms that underlie these genome-based functions are varied. Here we highlight some of these examples and provide an outlook on how the functional mechanisms of a lncRNA gene can be determined.


Asunto(s)
ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo
10.
Front Cell Dev Biol ; 8: 567, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32850778

RESUMEN

Previous studies in developing Xenopus and zebrafish reported that the phosphate transporter slc20a1a is expressed in pronephric kidneys. The recent identification of SLC20A1 as a monoallelic candidate gene for cloacal exstrophy further suggests its involvement in the urinary tract and urorectal development. However, little is known of the functional role of SLC20A1 in urinary tract development. Here, we investigated this using morpholino oligonucleotide knockdown of the zebrafish ortholog slc20a1a. This caused kidney cysts and malformations of the cloaca. Moreover, in morphants we demonstrated dysfunctional voiding and hindgut opening defects mimicking imperforate anus in human cloacal exstrophy. Furthermore, we performed immunohistochemistry of an unaffected 6-week-old human embryo and detected SLC20A1 in the urinary tract and the abdominal midline, structures implicated in the pathogenesis of cloacal exstrophy. Additionally, we resequenced SLC20A1 in 690 individuals with bladder exstrophy-epispadias complex (BEEC) including 84 individuals with cloacal exstrophy. We identified two additional monoallelic de novo variants. One was identified in a case-parent trio with classic bladder exstrophy, and one additional novel de novo variant was detected in an affected mother who transmitted this variant to her affected son. To study the potential cellular impact of SLC20A1 variants, we expressed them in HEK293 cells. Here, phosphate transport was not compromised, suggesting that it is not a disease mechanism. However, there was a tendency for lower levels of cleaved caspase-3, perhaps implicating apoptosis pathways in the disease. Our results suggest SLC20A1 is involved in urinary tract and urorectal development and implicate SLC20A1 as a disease-gene for BEEC.

11.
PLoS One ; 15(6): e0234246, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32502225

RESUMEN

INTRODUCTION: Esophageal atresia with or without tracheoesophageal fistula (EA/TEF) occurs approximately 1 in 3.500 live births representing the most common malformation of the upper digestive tract. Only half a century ago, EA/TEF was fatal among affected newborns suggesting that the steady birth prevalence might in parts be due to mutational de novo events in genes involved in foregut development. METHODS: To identify mutational de novo events in EA/TEF patients, we surveyed the exome of 30 case-parent trios. Identified and confirmed de novo variants were prioritized using in silico prediction tools. To investigate the embryonic role of genes harboring prioritized de novo variants we performed targeted analysis of mouse transcriptome data of esophageal tissue obtained at the embryonic day (E) E8.5, E12.5, and postnatal. RESULTS: In total we prioritized 14 novel de novo variants in 14 different genes (APOL2, EEF1D, CHD7, FANCB, GGT6, KIAA0556, NFX1, NPR2, PIGC, SLC5A2, TANC2, TRPS1, UBA3, and ZFHX3) and eight rare de novo variants in eight additional genes (CELSR1, CLP1, GPR133, HPS3, MTA3, PLEC, STAB1, and PPIP5K2). Through personal communication during the project, we identified an additional EA/TEF case-parent trio with a rare de novo variant in ZFHX3. In silico prediction analysis of the identified variants and comparative analysis of mouse transcriptome data of esophageal tissue obtained at E8.5, E12.5, and postnatal prioritized CHD7, TRPS1, and ZFHX3 as EA/TEF candidate genes. Re-sequencing of ZFHX3 in additional 192 EA/TEF patients did not identify further putative EA/TEF-associated variants. CONCLUSION: Our study suggests that rare mutational de novo events in genes involved in foregut development contribute to the development of EA/TEF.


Asunto(s)
ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Embrión de Mamíferos/metabolismo , Atresia Esofágica/genética , Exoma/genética , Perfilación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas Represoras/genética , Fístula Traqueoesofágica/genética , Animales , Humanos , Ratones , Secuenciación del Exoma
12.
Dev Cell ; 50(5): 644-657.e8, 2019 09 09.
Artículo en Inglés | MEDLINE | ID: mdl-31422919

RESUMEN

Precisely controlled gene regulatory networks are required during embryonic development to give rise to various structures, including those of the cardiovascular system. Long non-coding RNA (lncRNA) loci are known to be important regulators of these genetic programs. We have identified a novel and essential lncRNA locus Handsdown (Hdn), active in early heart cells, and show by genetic inactivation that it is essential for murine development. Hdn displays haploinsufficiency for cardiac development as Hdn-heterozygous adult mice exhibit hyperplasia in the right ventricular wall. Transcriptional activity of the Hdn locus, independent of its RNA, suppresses its neighboring gene Hand2. We reveal a switch in a topologically associated domain in differentiation of the cardiac lineage, allowing the Hdn locus to directly interact with regulatory elements of the Hand2 locus.


Asunto(s)
Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Corazón/embriología , Miocitos Cardíacos/metabolismo , ARN Largo no Codificante/metabolismo , Animales , Células Cultivadas , Haploinsuficiencia , Ratones , Ratones Endogámicos C57BL , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Miocitos Cardíacos/citología , ARN Largo no Codificante/genética
13.
Sci Signal ; 12(581)2019 05 14.
Artículo en Inglés | MEDLINE | ID: mdl-31088978

RESUMEN

Macrophages play key roles in the immune systems of humans and other mammals. Here, we performed single-cell analyses of the mRNAs and proteins of human macrophages to compare their responses to the signaling molecules lipopolysaccharide (LPS), a component of Gram-negative bacteria, and palmitate (PAL), a free fatty acid. We found that, although both molecules signal through the cell surface protein Toll-like receptor 4 (TLR4), they stimulated the expression of different genes, resulting in specific pro- and anti-inflammatory cellular states for each signal. The effects of the glucocorticoid receptor, which antagonizes LPS signaling, and cyclic AMP-dependent transcription factor 3, which inhibits PAL-induced inflammation, on inflammatory response seemed largely determined by digital on-off events. Furthermore, the quantification of transcriptional variance and signaling entropy enabled the identification of cell state-specific deregulated molecular pathways. These data suggest that the preservation of signaling in distinct cells might confer diversity on macrophage populations essential to maintaining major cellular functions.


Asunto(s)
Variación Genética/genética , Homeostasis/genética , Macrófagos/metabolismo , Transducción de Señal/genética , Transcripción Genética/genética , Factor de Transcripción Activador 3/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-1beta/genética , Interleucina-8/genética , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Palmitatos/farmacología , Transducción de Señal/efectos de los fármacos , Análisis de la Célula Individual/métodos , Células THP-1 , Receptor Toll-Like 4/genética , Transcripción Genética/efectos de los fármacos
14.
Am J Hum Genet ; 104(5): 994-1006, 2019 05 02.
Artículo en Inglés | MEDLINE | ID: mdl-31051115

RESUMEN

Congenital lower urinary-tract obstruction (LUTO) is caused by anatomical blockage of the bladder outflow tract or by functional impairment of urinary voiding. About three out of 10,000 pregnancies are affected. Although several monogenic causes of functional obstruction have been defined, it is unknown whether congenital LUTO caused by anatomical blockage has a monogenic cause. Exome sequencing in a family with four affected individuals with anatomical blockage of the urethra identified a rare nonsense variant (c.2557C>T [p.Arg853∗]) in BNC2, encoding basonuclin 2, tracking with LUTO over three generations. Re-sequencing BNC2 in 697 individuals with LUTO revealed three further independent missense variants in three unrelated families. In human and mouse embryogenesis, basonuclin 2 was detected in lower urinary-tract rudiments. In zebrafish embryos, bnc2 was expressed in the pronephric duct and cloaca, analogs of the mammalian lower urinary tract. Experimental knockdown of Bnc2 in zebrafish caused pronephric-outlet obstruction and cloacal dilatation, phenocopying human congenital LUTO. Collectively, these results support the conclusion that variants in BNC2 are strongly implicated in LUTO etiology as a result of anatomical blockage.


Asunto(s)
Aberraciones Cromosómicas , Proteínas de Unión al ADN/genética , Enfermedades Fetales/genética , Mutación , Obstrucción del Cuello de la Vejiga Urinaria/congénito , Obstrucción del Cuello de la Vejiga Urinaria/genética , Adulto , Animales , Niño , Femenino , Enfermedades Fetales/patología , Genes Dominantes , Edad Gestacional , Humanos , Masculino , Ratones , Persona de Mediana Edad , Linaje , Embarazo , Obstrucción del Cuello de la Vejiga Urinaria/patología , Pez Cebra
15.
Circulation ; 139(24): 2778-2792, 2019 06 11.
Artículo en Inglés | MEDLINE | ID: mdl-30922078

RESUMEN

BACKGROUND: Enhancers are genomic regulatory elements conferring spatiotemporal and signal-dependent control of gene expression. Recent evidence suggests that enhancers can generate noncoding enhancer RNAs, but their (patho)biological functions remain largely elusive. METHODS: We performed chromatin immunoprecipitation-coupled sequencing of histone marks combined with RNA sequencing of left ventricular biopsies from experimental and genetic mouse models of human cardiac hypertrophy to identify transcripts revealing enhancer localization, conservation with the human genome, and hypoxia-inducible factor 1α dependence. The most promising candidate, hypoxia-inducible enhancer RNA ( HERNA)1, was further examined by investigating its capacity to modulate neighboring coding gene expression by binding to their gene promoters by using chromatin isolation by RNA purification and λN-BoxB tethering-based reporter assays. The role of HERNA1 and its neighboring genes for pathological stress-induced growth and contractile dysfunction, and the therapeutic potential of HERNA1 inhibition was studied in gapmer-mediated loss-of-function studies in vitro using human induced pluripotent stem cell-derived cardiomyocytes and various in vivo models of human pathological cardiac hypertrophy. RESULTS: HERNA1 is robustly induced on pathological stress. Production of HERNA1 is initiated by direct hypoxia-inducible factor 1α binding to a hypoxia-response element in the histoneH3-lysine27acetylation marks-enriched promoter of the enhancer and confers hypoxia responsiveness to nearby genes including synaptotagmin XVII, a member of the family of membrane-trafficking and Ca2+-sensing proteins and SMG1, encoding a phosphatidylinositol 3-kinase-related kinase. Consequently, a substrate of SMG1, ATP-dependent RNA helicase upframeshift 1, is hyperphoshorylated in a HERNA1- and SMG1-dependent manner. In vitro and in vivo inactivation of SMG1 and SYT17 revealed overlapping and distinct roles in modulating cardiac hypertrophy. Finally, in vivo administration of antisense oligonucleotides targeting HERNA1 protected mice from stress-induced pathological hypertrophy. The inhibition of HERNA1 postdisease development reversed left ventricular growth and dysfunction, resulting in increased overall survival. CONCLUSIONS: HERNA1 is a novel heart-specific noncoding RNA with key regulatory functions in modulating the growth, metabolic, and contractile gene program in disease, and reveals a molecular target amenable to therapeutic exploitation.


Asunto(s)
Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/prevención & control , Cardiomiopatía Hipertrófica/prevención & control , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Miocitos Cardíacos/metabolismo , Oligonucleótidos Antisentido/administración & dosificación , ARN no Traducido/metabolismo , Animales , Sitios de Unión , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/patología , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/metabolismo , Cardiomiopatía Hipertrófica/patología , Estudios de Casos y Controles , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/deficiencia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos Cardíacos/patología , Regiones Promotoras Genéticas , ARN no Traducido/genética , Transducción de Señal , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo
17.
Nat Commun ; 9(1): 237, 2018 01 16.
Artículo en Inglés | MEDLINE | ID: mdl-29339785

RESUMEN

Impaired or excessive growth of endothelial cells contributes to several diseases. However, the functional involvement of regulatory long non-coding RNAs in these processes is not well defined. Here, we show that the long non-coding antisense transcript of GATA6 (GATA6-AS) interacts with the epigenetic regulator LOXL2 to regulate endothelial gene expression via changes in histone methylation. Using RNA deep sequencing, we find that GATA6-AS is upregulated in endothelial cells during hypoxia. Silencing of GATA6-AS diminishes TGF-ß2-induced endothelial-mesenchymal transition in vitro and promotes formation of blood vessels in mice. We identify LOXL2, known to remove activating H3K4me3 chromatin marks, as a GATA6-AS-associated protein, and reveal a set of angiogenesis-related genes that are inversely regulated by LOXL2 and GATA6-AS silencing. As GATA6-AS silencing reduces H3K4me3 methylation of two of these genes, periostin and cyclooxygenase-2, we conclude that GATA6-AS acts as negative regulator of nuclear LOXL2 function.


Asunto(s)
Aminoácido Oxidorreductasas/metabolismo , Células Endoteliales/metabolismo , Factor de Transcripción GATA6/genética , Regulación de la Expresión Génica/genética , Hipoxia/genética , Neovascularización Fisiológica/genética , ARN sin Sentido/genética , ARN Largo no Codificante/genética , Animales , Moléculas de Adhesión Celular/genética , Ciclooxigenasa 2/genética , Epigénesis Genética , Transición Epitelial-Mesenquimal , Silenciador del Gen , Código de Histonas/genética , Histonas/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Técnicas In Vitro , Metilación , Ratones
18.
EMBO Rep ; 19(1): 118-134, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29141987

RESUMEN

T-box transcription factors play essential roles in multiple aspects of vertebrate development. Here, we show that cooperative function of BRACHYURY (T) with histone-modifying enzymes is essential for mouse embryogenesis. A single point mutation (TY88A) results in decreased histone 3 lysine 27 acetylation (H3K27ac) at T target sites, including the T locus, suggesting that T autoregulates the maintenance of its expression and functions by recruiting permissive chromatin modifications to putative enhancers during mesoderm specification. Our data indicate that T mediates H3K27ac recruitment through a physical interaction with p300. In addition, we determine that T plays a prominent role in the specification of hematopoietic and endothelial cell types. Hematopoietic and endothelial gene expression programs are disrupted in TY88A mutant embryos, leading to a defect in the differentiation of hematopoietic progenitors. We show that this role of T is mediated, at least in part, through activation of a distal Lmo2 enhancer.


Asunto(s)
Desarrollo Embrionario/genética , Proteínas Fetales/genética , Histonas/metabolismo , Mesodermo/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Proteínas de Dominio T Box/genética , Factores de Transcripción p300-CBP/genética , Acetilación , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Secuencia de Bases , Diferenciación Celular , Linaje de la Célula/genética , Cromatina/química , Cromatina/metabolismo , Embrión de Mamíferos , Células Endoteliales/citología , Células Endoteliales/metabolismo , Proteínas Fetales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Sitios Genéticos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Histonas/genética , Proteínas con Dominio LIM/genética , Proteínas con Dominio LIM/metabolismo , Mesodermo/citología , Mesodermo/crecimiento & desarrollo , Ratones , Células Madre Embrionarias de Ratones/citología , Mutación Puntual , Unión Proteica , Transducción de Señal , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción p300-CBP/metabolismo
19.
Dev Cell ; 42(5): 514-526.e7, 2017 09 11.
Artículo en Inglés | MEDLINE | ID: mdl-28826820

RESUMEN

The spinal cord and mesodermal tissues of the trunk such as the vertebral column and skeletal musculature derive from neuro-mesodermal progenitors (NMPs). Sox2, Brachyury (T), and Tbx6 have been correlated with NMP potency and lineage choice; however, their exact role and interaction in these processes have not yet been revealed. Here we present a global analysis of NMPs and their descending lineages performed on purified cells from embryonic day 8.5 wild-type and mutant embryos. We show that T, cooperatively with WNT signaling, controls the progenitor state and the switch toward the mesodermal fate. Sox2 acts antagonistically and promotes neural development. T is also involved in remodeling the chromatin for mesodermal development. Tbx6 reinforces the mesodermal fate choice, represses the progenitor state, and confers paraxial fate commitment. Our findings refine previous models and establish molecular principles underlying mammalian trunk development, comprising NMP maintenance, lineage choice, and mesoderm formation.


Asunto(s)
Linaje de la Célula/genética , Proteínas Fetales/metabolismo , Mesodermo/citología , Neuronas/citología , Factores de Transcripción SOXB1/metabolismo , Células Madre/citología , Proteínas de Dominio T Box/metabolismo , Animales , Secuencia de Bases , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina/genética , Proteínas Fetales/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Ratones , Modelos Biológicos , Neuronas/metabolismo , Factores de Transcripción SOXB1/genética , Análisis de la Célula Individual , Células Madre/metabolismo , Proteínas de Dominio T Box/genética , Factores de Transcripción/metabolismo , Vía de Señalización Wnt/genética
20.
Circ Res ; 121(4): 368-375, 2017 Aug 04.
Artículo en Inglés | MEDLINE | ID: mdl-28611075

RESUMEN

RATIONALE: Pericytes are essential for vessel maturation and endothelial barrier function. Long noncoding RNAs regulate many cellular functions, but their role in pericyte biology remains unexplored. OBJECTIVE: Here, we investigate the effect of hypoxia-induced endoplasmic reticulum stress regulating long noncoding RNAs (HypERlnc, also known as ENSG00000262454) on pericyte function in vitro and its regulation in human heart failure and idiopathic pulmonary arterial hypertension. METHODS AND RESULTS: RNA sequencing in human primary pericytes identified hypoxia-regulated long noncoding RNAs, including HypERlnc. Silencing of HypERlnc decreased cell viability and proliferation and resulted in pericyte dedifferentiation, which went along with increased endothelial permeability in cocultures consisting of human primary pericyte and human coronary microvascular endothelial cells. Consistently, Cas9-based transcriptional activation of HypERlnc was associated with increased expression of pericyte marker genes. Moreover, HypERlnc knockdown reduced endothelial-pericyte recruitment in Matrigel assays (P<0.05). Mechanistically, transcription factor reporter arrays demonstrated that endoplasmic reticulum stress-related transcription factors were prominently activated by HypERlnc knockdown, which was confirmed via immunoblotting for the endoplasmic reticulum stress markers IRE1α (P<0.001), ATF6 (P<0.01), and soluble BiP (P<0.001). Kyoto encyclopedia of genes and gene ontology pathway analyses of RNA sequencing experiments after HypERlnc knockdown indicate a role in cardiovascular disease states. Indeed, HypERlnc expression was significantly reduced in human cardiac tissue from patients with heart failure (P<0.05; n=19) compared with controls. In addition, HypERlnc expression significantly correlated with pericyte markers in human lungs derived from patients diagnosed with idiopathic pulmonary arterial hypertension and from donor lungs (n=14). CONCLUSIONS: Here, we show that HypERlnc regulates human pericyte function and the endoplasmic reticulum stress response. In addition, RNA sequencing analyses in conjunction with reduced expression of HypERlnc in heart failure and correlation with pericyte markers in idiopathic pulmonary arterial hypertension indicate a role of HypERlnc in human cardiopulmonary disease.


Asunto(s)
Estrés del Retículo Endoplásmico/fisiología , Células Endoteliales/metabolismo , Pericitos/metabolismo , ARN Largo no Codificante/biosíntesis , Animales , Secuencia de Bases , Hipoxia de la Célula/fisiología , Técnicas de Cocultivo , Células Endoteliales/patología , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/patología , Ratones , Ratones Endogámicos C57BL , Pericitos/patología , ARN Largo no Codificante/genética , Distribución Aleatoria
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