RESUMO
Transcriptional networks governing cardiac precursor cell (CPC) specification are incompletely understood owing, in part, to limitations in distinguishing CPCs from non-cardiac mesoderm in early gastrulation. We leveraged detection of early cardiac lineage transgenes within a granular single-cell transcriptomic time course of mouse embryos to identify emerging CPCs and describe their transcriptional profiles. Mesp1, a transiently expressed mesodermal transcription factor, is canonically described as an early regulator of cardiac specification. However, we observed perdurance of CPC transgene-expressing cells in Mesp1 mutants, albeit mislocalized, prompting us to investigate the scope of the role of Mesp1 in CPC emergence and differentiation. Mesp1 mutant CPCs failed to robustly activate markers of cardiomyocyte maturity and crucial cardiac transcription factors, yet they exhibited transcriptional profiles resembling cardiac mesoderm progressing towards cardiomyocyte fates. Single-cell chromatin accessibility analysis defined a Mesp1-dependent developmental breakpoint in cardiac lineage progression at a shift from mesendoderm transcriptional networks to those necessary for cardiac patterning and morphogenesis. These results reveal Mesp1-independent aspects of early CPC specification and underscore a Mesp1-dependent regulatory landscape required for progression through cardiogenesis.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Epigenômica , Miócitos Cardíacos , Animais , Camundongos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/metabolismo , Miócitos Cardíacos/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Localization of RNAs at specific subcellular locations regulating various local cellular events has gained much attention recently. Like most other classes of RNAs, the function of newly discovered circular RNAs (circRNAs) is predominantly determined by their association with different cellular factors in the cell. CircRNAs function as transcriptional and posttranscriptional regulators of gene expression by interacting with transcription factors, splicing regulators, RNA-binding proteins, and microRNAs or by translating into functional polypeptides. Hence, studying their subcellular localization to assess their function is essential. The discovery of more than a million circRNA and increasing evidence of their involvement in development and diseases require a thorough analysis of their subcellular localization linking to their biological functions. Here, we summarize current knowledge of circRNA localization in cells and extracellular vesicles, factors regulating their subcellular localization, and the implications of circRNA localization on their cellular functions. Given the discovery of many circRNAs in all life forms and their implications in pathophysiology, we discuss the challenges in studying circRNA localization and the opportunities for unlocking the mystery of circRNA functions.
Assuntos
RNA Circular , RNA Circular/genética , RNA Circular/metabolismo , Humanos , Animais , RNA/metabolismo , RNA/genética , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , Regulação da Expressão Gênica , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , MicroRNAs/genética , MicroRNAs/metabolismoRESUMO
RATIONALE: Cardiac pacemaker cells (PCs) in the sinoatrial node (SAN) have a distinct gene expression program that allows them to fire automatically and initiate the heartbeat. Although critical SAN transcription factors, including Isl1 (Islet-1), Tbx3 (T-box transcription factor 3), and Shox2 (short-stature homeobox protein 2), have been identified, the cis-regulatory architecture that governs PC-specific gene expression is not understood, and discrete enhancers required for gene regulation in the SAN have not been identified. OBJECTIVE: To define the epigenetic profile of PCs using comparative ATAC-seq (assay for transposase-accessible chromatin with sequencing) and to identify novel enhancers involved in SAN gene regulation, development, and function. METHODS AND RESULTS: We used ATAC-seq on sorted neonatal mouse SAN to compare regions of accessible chromatin in PCs and right atrial cardiomyocytes. PC-enriched assay for transposase-accessible chromatin peaks, representing candidate SAN regulatory elements, were located near established SAN genes and were enriched for distinct sets of TF (transcription factor) binding sites. Among several novel SAN enhancers that were experimentally validated using transgenic mice, we identified a 2.9-kb regulatory element at the Isl1 locus that was active specifically in the cardiac inflow at embryonic day 8.5 and throughout later SAN development and maturation. Deletion of this enhancer from the genome of mice resulted in SAN hypoplasia and sinus arrhythmias. The mouse SAN enhancer also directed reporter activity to the inflow tract in developing zebrafish hearts, demonstrating deep conservation of its upstream regulatory network. Finally, single nucleotide polymorphisms in the human genome that occur near the region syntenic to the mouse enhancer exhibit significant associations with resting heart rate in human populations. CONCLUSIONS: (1) PCs have distinct regions of accessible chromatin that correlate with their gene expression profile and contain novel SAN enhancers, (2) cis-regulation of Isl1 specifically in the SAN depends upon a conserved SAN enhancer that regulates PC development and SAN function, and (3) a corresponding human ISL1 enhancer may regulate human SAN function.
Assuntos
Arritmia Sinusal/metabolismo , Relógios Biológicos , Sequenciamento de Cromatina por Imunoprecipitação , Elementos Facilitadores Genéticos , Frequência Cardíaca , Proteínas com Homeodomínio LIM/metabolismo , Nó Sinoatrial/metabolismo , Fatores de Transcrição/metabolismo , Potenciais de Ação , Animais , Arritmia Sinusal/genética , Arritmia Sinusal/fisiopatologia , Epigênese Genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Humanos , Proteínas com Homeodomínio LIM/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Polimorfismo de Nucleotídeo Único , Nó Sinoatrial/fisiopatologia , Fatores de Tempo , Fatores de Transcrição/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
RATIONALE: The development and function of the pacemaker cardiomyocytes of the sinoatrial node (SAN), the leading pacemaker of the heart, are tightly controlled by a conserved network of transcription factors, including TBX3 (T-box transcription factor 3), ISL1 (ISL LIM homeobox 1), and SHOX2 (short stature homeobox 2). Yet, the regulatory DNA elements (REs) controlling target gene expression in the SAN pacemaker cells have remained undefined. OBJECTIVE: Identification of the regulatory landscape of human SAN-like pacemaker cells and functional assessment of SAN-specific REs potentially involved in pacemaker cell gene regulation. METHODS AND RESULTS: We performed Assay for Transposase-Accessible Chromatin using sequencing on human pluripotent stem cell-derived SAN-like pacemaker cells and ventricle-like cells and identified thousands of putative REs specific for either human cell type. We validated pacemaker cell-specific elements in the SHOX2 and TBX3 loci. CRISPR-mediated homozygous deletion of the mouse ortholog of a noncoding region with candidate pacemaker-specific REs in the SHOX2 locus resulted in selective loss of Shox2 expression from the developing SAN and embryonic lethality. Putative pacemaker-specific REs were identified up to 1 Mbp upstream of TBX3 in a region close to MED13L harboring variants associated with heart rate recovery after exercise. The orthologous region was deleted in mice, which resulted in selective loss of expression of Tbx3 from the SAN and (cardiac) ganglia and in neonatal lethality. Expression of Tbx3 was maintained in other tissues including the atrioventricular conduction system, lungs, and liver. Heterozygous adult mice showed increased SAN recovery times after pacing. The human REs harboring the associated variants robustly drove expression in the SAN of transgenic mouse embryos. CONCLUSIONS: We provided a genome-wide collection of candidate human pacemaker-specific REs, including the loci of SHOX2, TBX3, and ISL1, and identified a link between human genetic variants influencing heart rate recovery after exercise and a variant RE with highly conserved function, driving SAN expression of TBX3.
Assuntos
Relógios Biológicos , Elementos Facilitadores Genéticos , Frequência Cardíaca , Miócitos Cardíacos/metabolismo , Nó Sinoatrial/metabolismo , Proteínas com Domínio T/metabolismo , Potenciais de Ação , Animais , Linhagem Celular , Epigênese Genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Estudo de Associação Genômica Ampla , Humanos , Masculino , Camundongos Transgênicos , Mutação , Proteínas com Domínio T/genética , Peixe-ZebraRESUMO
MEF2C is a member of the highly conserved MEF2 family of transcription factors and is a key regulator of cardiovascular development. In mice, Mef2c is expressed in the developing heart and vasculature, including the endothelium. Loss of Mef2c function in germline knockout mice leads to early embryonic demise and profound developmental abnormalities in the cardiovascular system. Previous attempts to uncover the cause of embryonic lethality by specifically disrupting Mef2c function in the heart or vasculature failed to recapitulate the global Mef2c knockout phenotype and instead resulted in relatively minor defects that did not compromise viability or result in significant cardiovascular defects. However, previous studies examined the requirement of Mef2c in the myocardial and endothelial lineages using Cre lines that begin to be expressed after the expression of Mef2c has already commenced. Here, we tested the requirement of Mef2c in the myocardial and endothelial lineages using conditional knockout approaches in mice with Cre lines that deleted Mef2c prior to onset of its expression in embryonic development. We found that deletion of Mef2c in the early myocardial lineage using Nkx2-5Cre resulted in cardiac and vascular abnormalities that were indistinguishable from the defects in the global Mef2c knockout. In contrast, early deletion of Mef2c in the vascular endothelium using an Etv2::Cre line active prior to the onset of Mef2c expression resulted in viable offspring that were indistinguishable from wild type controls with no overt defects in vascular development, despite nearly complete early deletion of Mef2c in the vascular endothelium. Thus, these studies support the idea that the requirement of MEF2C for vascular development is secondary to its requirement in the heart and suggest that the observed failure in vascular remodeling in Mef2c knockout mice results from defective heart function.
Assuntos
Sistema Cardiovascular/embriologia , Animais , Fenômenos Fisiológicos Cardiovasculares/genética , Endotélio Vascular/anormalidades , Endotélio Vascular/embriologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/genética , Fatores de Transcrição MEF2/deficiência , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/fisiologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Mutantes , Camundongos Transgênicos , Organogênese/genética , Organogênese/fisiologia , GravidezRESUMO
Enhancers frequently contain multiple binding sites for the same transcription factor. These homotypic binding sites often exhibit synergy, whereby the transcriptional output from two or more binding sites is greater than the sum of the contributions of the individual binding sites alone. Although this phenomenon is frequently observed, the mechanistic basis for homotypic binding site synergy is poorly understood. Here, we identify a bona fide cardiac-specific Prkaa2 enhancer that is synergistically activated by homotypic MEF2 binding sites. We show that two MEF2 sites in the enhancer function cooperatively due to bridging of the MEF2C-bound sites by the SAP domain-containing co-activator protein myocardin, and we show that paired sites buffer the enhancer from integration site-dependent effects on transcription in vivo Paired MEF2 sites are prevalent in cardiac enhancers, suggesting that this might be a common mechanism underlying synergy in the control of cardiac gene expression in vivo.
Assuntos
Fatores de Transcrição MEF2/metabolismo , Miocárdio/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Transcrição Gênica , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Elementos Facilitadores Genéticos , Camundongos Transgênicos , Multimerização ProteicaRESUMO
Congenital heart defects are the most common birth defects in humans, and those that affect the proper alignment of the outflow tracts and septation of the ventricles are a highly significant cause of morbidity and mortality in infants. A late differentiating population of cardiac progenitors, referred to as the anterior second heart field (AHF), gives rise to the outflow tract and the majority of the right ventricle and provides an embryological context for understanding cardiac outflow tract alignment and membranous ventricular septal defects. However, the transcriptional pathways controlling AHF development and their roles in congenital heart defects remain incompletely elucidated. Here, we inactivated the gene encoding the transcription factor MEF2C in the AHF in mice. Loss of Mef2c function in the AHF results in a spectrum of outflow tract alignment defects ranging from overriding aorta to double-outlet right ventricle and dextro-transposition of the great arteries. We identify Tdgf1, which encodes a Nodal co-receptor (also known as Cripto), as a direct transcriptional target of MEF2C in the outflow tract via an AHF-restricted Tdgf1 enhancer. Importantly, both the MEF2C and TDGF1 genes are associated with congenital heart defects in humans. Thus, these studies establish a direct transcriptional pathway between the core cardiac transcription factor MEF2C and the human congenital heart disease gene TDGF1. Moreover, we found a range of outflow tract alignment defects resulting from a single genetic lesion, supporting the idea that AHF-derived outflow tract alignment defects may constitute an embryological spectrum rather than distinct anomalies.
Assuntos
Fator de Crescimento Epidérmico/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Glicoproteínas de Membrana/fisiologia , Proteínas de Neoplasias/fisiologia , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Fator de Crescimento Epidérmico/genética , Feminino , Deleção de Genes , Coração/embriologia , Cardiopatias Congênitas/genética , Comunicação Interventricular/genética , Ventrículos do Coração , Humanos , Hibridização In Situ , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/fisiologia , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Morfogênese/genética , Proteínas de Neoplasias/genética , Organogênese , Análise de Sequência de RNA , Distribuição Tecidual , Transcrição Gênica , Transposição dos Grandes Vasos/genéticaRESUMO
Wnt5a, a non-canonical Wnt ligand critical for outflow tract (OFT) morphogenesis, is expressed specifically in second heart field (SHF) progenitors in the caudal splanchnic mesoderm (SpM) near the inflow tract (IFT). Using a conditional Wnt5a gain of function (GOF) allele and Islet1-Cre, we broadly over-expressed Wnt5a throughout the SHF lineage, including the entire SpM between the IFT and OFT. Wnt5a over-expression in Wnt5a null mutants can rescue the cell polarity and actin polymerization defects as well as severe SpM shortening, but fails to rescue OFT shortening. Moreover, Wnt5a over-expression in wild-type background is able to cause OFT shortening. We find that Wnt5a over-expression does not perturb SHF cell proliferation, apoptosis or differentiation, but affects the deployment of SHF cells by causing them to accumulate into a large bulge at the rostral SpM and fail to enter the OFT. Our immunostaining analyses suggest an inverse correlation between cell cohesion and Wnt5a level in the wild-type SpM. Ectopic Wnt5a expression in the rostral SpM of Wn5a-GOF mutants diminishes the upregulation of adherens junction; whereas loss of Wnt5a in Wnt5a null mutants causes premature increase in adherens junction level in the caudal SpM. Over-expression of mouse Wnt5a in Xenopus animal cap cells also reduces C-cadherin distribution on the plasma membrane without affecting its overall protein level, suggesting that Wnt5a may play an evolutionarily conserved role in controlling the cell surface level of cadherin to modulate cell cohesion during tissue morphogenesis. Collectively, our data indicate that restricted expression of Wnt5a in the caudal SpM is essential for normal OFT morphogenesis, and uncover a novel function of spatially regulated cell cohesion by Wnt5a in driving the deployment of SHF cells from the SpM into the OFT.
Assuntos
Miocárdio/citologia , Células-Tronco/citologia , Proteínas Wnt/fisiologia , Animais , Camundongos , Transdução de Sinais , Proteína Wnt-5aRESUMO
Outflow tract (OFT) malformation accounts for â¼30% of human congenital heart defects and manifests frequently in TBX1 haplo-insufficiency associated DiGeorge (22q11.2 deletion) syndrome. OFT myocardium originates from second heart field (SHF) progenitors in the pharyngeal and splanchnic mesoderm (SpM), but how these progenitors are deployed to the OFT is unclear. We find that SHF progenitors in the SpM gradually gain epithelial character and are deployed to the OFT as a cohesive sheet. Wnt5a, a non-canonical Wnt, is expressed specifically in the caudal SpM and may regulate oriented cell intercalation to incorporate SHF progenitors into an epithelial-like sheet, thereby generating the pushing force to deploy SHF cells rostrally into the OFT. Using enhancer trap and Cre transgenes, our lineage tracing experiments show that in Wnt5a null mice, SHF progenitors are trapped in the SpM and fail to be deployed to the OFT efficiently, resulting in a reduction in the inferior OFT myocardial wall and its derivative, subpulmonary myocardium. Concomitantly, the superior OFT and subaortic myocardium are expanded. Finally, in chick embryos, blocking the Wnt5a function in the caudal SpM perturbs polarized elongation of SHF progenitors, and compromises their deployment to the OFT. Collectively, our results highlight a critical role for Wnt5a in deploying SHF progenitors from the SpM to the OFT. Given that Wnt5a is a putative transcriptional target of Tbx1, and the similar reduction of subpulmonary myocardium in Tbx1 mutant mice, our results suggest that perturbing Wnt5a-mediated SHF deployment may be an important pathogenic mechanism contributing to OFT malformations in DiGeorge syndrome.
Assuntos
Síndrome de DiGeorge/genética , Células-Tronco Embrionárias/patologia , Coração/embriologia , Proteínas Wnt/genética , Animais , Embrião de Galinha , Síndrome de DiGeorge/etiologia , Deleção de Genes , Camundongos , Camundongos Knockout , Miocárdio/patologia , Proteína Wnt-5aRESUMO
Planar cell polarity (PCP) signaling is an evolutionarily conserved mechanism that coordinates polarized cell behavior to regulate tissue morphogenesis during vertebrate gastrulation, neurulation and organogenesis. In Xenopus and zebrafish, PCP signaling is activated by non-canonical Wnts such as Wnt11, and detailed understanding of Wnt11 expression has provided important clues on when, where and how PCP may be activated to regulate tissue morphogenesis. To explore the role of Wnt11 in mammalian development, we established a Wnt11 expression and lineage map with high spatial and temporal resolution by creating and analyzing a tamoxifen-inducible Wnt11-CreER BAC (bacterial artificial chromosome) transgenic mouse line. Our short- and long-term lineage tracing experiments indicated that Wnt11-CreER could faithfully recapitulate endogenous Wnt11 expression, and revealed for the first time that cells transiently expressing Wnt11 at early gastrulation were fated to become specifically the progenitors of the entire endoderm. During mid-gastrulation, Wnt11-CreER expressing cells also contribute extensively to the endothelium in both embryonic and extraembryonic compartments, and the endocardium in all chambers of the developing heart. In contrast, Wnt11-CreER expression in the myocardium starts from late-gastrulation, and occurs in three transient, sequential waves: first in the precursors of the left ventricular (LV) myocardium from E7.0 to 8.0; subsequently in the right ventricular (RV) myocardium from E8.0 to 9.0; and finally in the superior wall of the outflow tract (OFT) myocardium from E8.5 to 10.5. These results provide formal genetic proof that the majority of the endocardium and myocardium diverge by mid-gastrulation in the mouse, and suggest a tight spatial and temporal control of Wnt11 expression in the myocardial lineage to coordinate with myocardial differentiation in the first and second heart field progenitors to form the LV, RV and OFT. The insights gained from this study will also guide future investigations to decipher the role of non-canonical Wnt/PCP signaling in endoderm development, vasculogenesis and heart formation.
Assuntos
Linhagem da Célula , Desenvolvimento Embrionário , Proteínas Wnt/metabolismo , Animais , Cromossomos Artificiais Bacterianos/metabolismo , Embrião de Mamíferos/irrigação sanguínea , Embrião de Mamíferos/metabolismo , Endoderma/embriologia , Endoderma/metabolismo , Gastrulação , Integrases/metabolismo , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos Transgênicos , Miocárdio/metabolismo , Linha Primitiva/embriologia , Linha Primitiva/metabolismo , Transgenes , Proteínas Wnt/genéticaRESUMO
Disheveled (Dvl) is a key regulator of both the canonical Wnt and the planar cell polarity (PCP) pathway. Previous genetic studies in mice indicated that outflow tract (OFT) formation requires Dvl1 and 2, but it was unclear which pathway was involved and whether Dvl1/2-mediated signaling was required in the second heart field (SHF) or the cardiac neural crest (CNC) lineage, both of which are critical for OFT development. In this study, we used Dvl1/2 null mice and a set of Dvl2 BAC transgenes that function in a pathway-specific fashion to demonstrate that Dvl1/2-mediated PCP signaling is essential for OFT formation. Lineage-specific gene-ablation further indicated that Dvl1/2 function is dispensable in the CNC, but required in the SHF for OFT lengthening to promote cardiac looping. Mutating the core PCP gene Vangl2 and non-canonical Wnt gene Wnt5a recapitulated the OFT morphogenesis defects observed in Dvl1/2 mutants. Consistent with genetic interaction studies suggesting that Wnt5a signals through the PCP pathway, Dvl1/2 and Wnt5a mutants display aberrant cell packing and defective actin polymerization and filopodia formation specifically in SHF cells in the caudal splanchnic mesoderm (SpM), where Wnt5a and Dvl2 are co-expressed specifically. Our results reveal a critical role of PCP signaling in the SHF during early OFT lengthening and cardiac looping and suggest that a Wnt5aâ Dvl PCP signaling cascade may regulate actin polymerization and protrusive cell behavior in the caudal SpM to promote SHF deployment, OFT lengthening and cardiac looping.
Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Polaridade Celular/fisiologia , Coração/embriologia , Morfogênese/fisiologia , Fosfoproteínas/metabolismo , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Animais , Cromossomos Artificiais Bacterianos/genética , Primers do DNA/genética , Proteínas Desgrenhadas , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Morfogênese/genética , Mutagênese , Proteínas do Tecido Nervoso/genética , Fosfoproteínas/deficiência , Polimerização , Reação em Cadeia da Polimerase em Tempo Real , Transgenes/genética , Proteínas Wnt/genética , Proteína Wnt-5aRESUMO
Brachydactyly type B (BDB1) and Robinow syndrome (RRS) are two skeletal disorders caused by mutations in ROR2, a co-receptor of Wnt5a. Wnt5a/Ror2 can activate multiple branches of non-canonical Wnt signaling, but it is unclear which branch(es) mediates Wnt5a/Ror2 function in limb skeletal development. Here, we provide evidence implicating the planar cell polarity (PCP) pathway as the downstream component of Wnt5a in the limb. We show that a mutation in the mouse PCP gene Vangl2 causes digit defects resembling the clinical phenotypes in BDB1, including loss of phalanges. Halving the dosage of Wnt5a in Vangl2 mutants enhances the severity and penetrance of the digit defects and causes long bone defects reminiscent of RRS, suggesting that Wnt5a and Vangl2 function in the same pathway and disruption of PCP signaling may underlie both BDB1 and RRS. Consistent with a role for PCP signaling in tissue morphogenesis, mutation of Vangl2 alters the shape and dimensions of early limb buds: the width and thickness are increased, whereas the length is decreased. The digit pre-chondrogenic condensates also become wider, thicker and shorter. Interestingly, altered limb bud dimensions in Vangl2 mutants also affect limb growth by perturbing the signaling network that regulates the balance between Fgf and Bmp signaling. Halving the dosage of Bmp4 partially suppresses the loss of phalanges in Vangl2 mutants, supporting the hypothesis that an aberrant increase in Bmp signaling is the cause of the brachydactyly defect. These findings provide novel insight into the signaling mechanisms of Wnt5a/Ror2 and the pathogenesis in BDB1 and RRS.
Assuntos
Deformidades Congênitas dos Membros , Anormalidades Maxilofaciais , Morfogênese/genética , Desenvolvimento Musculoesquelético/genética , Transdução de Sinais/genética , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Animais , Proteína Morfogenética Óssea 4/antagonistas & inibidores , Proteína Morfogenética Óssea 4/genética , Polaridade Celular/fisiologia , Sobrevivência Celular/genética , Dosagem de Genes , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Botões de Extremidades/anormalidades , Deformidades Congênitas dos Membros/embriologia , Deformidades Congênitas dos Membros/genética , Deformidades Congênitas dos Membros/metabolismo , Deformidades Congênitas dos Membros/patologia , Anormalidades Maxilofaciais/embriologia , Anormalidades Maxilofaciais/genética , Anormalidades Maxilofaciais/patologia , Camundongos , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Coluna Vertebral/anormalidades , Coluna Vertebral/embriologia , Coluna Vertebral/patologia , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteína Wnt-5aRESUMO
Circular (circ)RNAs have emerged as novel regulators of gene expression through various mechanisms. However, most publications focus on functional circRNAs regulating target gene expression by interacting with micro (mi)RNAs and acting as competing endogenous RNAs (ceRNAs). Although the theory of miRNA sponging by ceRNAs suggests the inhibition of miRNA activity, many studies are biased toward the selection of miRNAs showing a reverse expression pattern compared with circRNA expression. Although several computational tools and molecular assays have been used to predict and validate the interaction of miRNAs with circRNAs, the actual validation of functional in vivo interactions needs careful consideration of molecular experiments with specific controls. As extensive research is being performed on circRNA, many questions arise on the functional significance of circRNA-miRNA interactions. We hope the critical discussion on the criteria for selecting circRNA-miRNA pairs for functional analysis and providing standard methods for validating circRNA-miRNA interactions will advance our understanding of circRNAs as novel gene regulators. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs Translation > Regulation RNA Methods > RNA Analyses in Cells.
RESUMO
Circular RNAs (circRNAs) are covalently closed RNA molecules generated from precursor RNAs by the head-to-tail backsplicing of exons. Hundreds of studies demonstrated that circRNAs are ubiquitously expressed and regulate cellular events by modulating microRNA (miRNA) and RNA-binding protein (RBP) activities. A few circRNAs are also known to translate into functional polypeptides regulating cellular physiology. All these functions primarily depend on the full-length sequence of the circRNAs. CircRNA backsplice junction sequence is the key to identifying circRNAs and their full-length mature sequence. However, some multi-exonic circRNAs exist in different isoforms sharing identical backsplice junction sequences and are termed circRNA splice variants. Here, we analyzed the previously published HeLa cell RNA-seq datasets to identify circRNA splice variants using the de novo module of the CIRCexplorer2 circRNA annotation pipeline. A subset of circRNAs with splice variants was validated by the circRNA-rolling circle amplification (circRNA-RCA) method. Interestingly, several validated circRNAs were predicted to translate into proteins by the riboCIRC database. Furthermore, polyribosome fractionation followed by quantitative PCR confirmed the association of a subset of circRNAs with polyribosome supporting their protein-coding potential. Finally, bioinformatics analysis of proteins derived from splice variants of circCORO1C and circASPH suggested altered protein sequences and structures that could affect their physiological functions. Together, our study identified novel circRNA splice variants and their potential translation into protein isoforms which may regulate various physiological processes.
Assuntos
MicroRNAs , Biossíntese de Proteínas , RNA Circular , Humanos , Processamento Alternativo/genética , Células HeLa , MicroRNAs/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Circular/genética , RNA Circular/metabolismoRESUMO
Mechanisms underlying distinct specification, commitment, and differentiation phases of cell fate determination remain undefined due to difficulties capturing these processes. Here, we interrogate the activity of ETV2, a transcription factor necessary and sufficient for hematoendothelial differentiation, within isolated fate intermediates. We observe transcriptional upregulation of Etv2 and opening of ETV2-binding sites, indicating new ETV2 binding, in a common cardiac-hematoendothelial progenitor population. Accessible ETV2-binding sites are active at the Etv2 locus but not at other hematoendothelial regulator genes. Hematoendothelial commitment coincides with the activation of a small repertoire of previously accessible ETV2-binding sites at hematoendothelial regulators. Hematoendothelial differentiation accompanies activation of a large repertoire of new ETV2-binding sites and upregulation of hematopoietic and endothelial gene regulatory networks. This work distinguishes specification, commitment, and sublineage differentiation phases of ETV2-dependent transcription and suggests that the shift from ETV2 binding to ETV2-bound enhancer activation, not ETV2 binding to target enhancers, drives hematoendothelial fate commitment.
Assuntos
Células-Tronco Hematopoéticas , Fatores de Transcrição , Diferenciação Celular/genética , Endotélio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Mammals have limited capacity for heart regeneration, whereas zebrafish have extraordinary regeneration abilities. During zebrafish heart regeneration, endothelial cells promote cardiomyocyte cell cycle reentry and myocardial repair, but the mechanisms responsible for promoting an injury microenvironment conducive to regeneration remain incompletely defined. Here, we identify the matrix metalloproteinase Mmp14b as an essential regulator of heart regeneration. We identify a TEAD-dependent mmp14b endothelial enhancer induced by heart injury in zebrafish and mice, and we show that the enhancer is required for regeneration, supporting a role for Hippo signaling upstream of mmp14b. Last, we show that MMP-14 function in mice is important for the accumulation of Agrin, an essential regulator of neonatal mouse heart regeneration. These findings reveal mechanisms for extracellular matrix remodeling that promote heart regeneration.
Assuntos
Células Endoteliais , Peixe-Zebra , Animais , Camundongos , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Proliferação de Células , Regeneração , MamíferosRESUMO
The efficacy and safety of gene-therapy strategies for indications like tissue damage hinge on precision; yet, current methods afford little spatial or temporal control of payload delivery. Here, we find that tissue-regeneration enhancer elements (TREEs) isolated from zebrafish can direct targeted, injury-associated gene expression from viral DNA vectors delivered systemically in small and large adult mammalian species. When employed in combination with CRISPR-based epigenome editing tools in mice, zebrafish TREEs stimulated or repressed the expression of endogenous genes after ischemic myocardial infarction. Intravenously delivered recombinant AAV vectors designed with a TREE to direct a constitutively active YAP factor boosted indicators of cardiac regeneration in mice and improved the function of the injured heart. Our findings establish the application of contextual enhancer elements as a potential therapeutic platform for spatiotemporally controlled tissue regeneration in mammals.
Assuntos
Elementos Facilitadores Genéticos , Terapia Genética , Coração , Infarto do Miocárdio , Miócitos Cardíacos , Regeneração , Animais , Camundongos , Proliferação de Células , Coração/fisiologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/terapia , Miócitos Cardíacos/metabolismo , Peixe-Zebra/genética , Terapia Genética/métodos , Regeneração/genéticaRESUMO
Functional proteins in the cell are translated from the messenger RNA (mRNA) molecules, constituting less than 5% of the cellular transcriptome. The majority of the RNA molecules in the cell are noncoding RNAs, including rRNA, tRNA, snRNA, piRNA, lncRNA, microRNA, and poorly characterized circular RNAs (circRNAs). Recent studies established that circRNAs regulate gene expression by associating with RNA-binding proteins and microRNAs. With the growing understanding of circRNA functions, a subset of circRNAs has been reported to translate into proteins. Interestingly, the presence of Open Reading Frames (ORFs), N6-methyladenosine (m6A) modifications, and internal ribosomal entry sites (IRES) in the circRNA sequences indicate their coding potential through the cap-independent translation initiation mechanism. The purpose of this review is to highlight the mechanism of circRNA translation and the importance of circRNA-encoded proteins (circ-proteins) in cellular physiology and pathology. Here, we discuss the computational and molecular methods currently utilized to systematically identify translatable circRNAs and the functional characterization of the circ-proteins. We foresee that the ongoing and future studies on circRNA translation will uncover the hidden proteome and their therapeutic implications in human health. This article is categorized under: RNA Methods > RNA Analyses in Cells Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs Translation > Mechanisms.
Assuntos
MicroRNAs , RNA Circular , Humanos , Sítios Internos de Entrada Ribossomal , Fases de Leitura Aberta , Proteoma , RNA/genéticaRESUMO
Circular RNAs (circRNAs) are a novel class of covalently closed RNA molecules that recently emerged as a critical regulator of gene expression in development and diseases. Recent research has highlighted the importance of novel circRNAs in the biosynthesis and secretion of insulin from ß-cells of pancreatic islets. However, all circRNAs expressed in pancreatic islets or ß-cells are not readily available in the database. In this study, we analyzed publicly available RNA-sequencing datasets of the pancreatic islets to catalog all circRNAs expressed in pancreatic islets to construct the PanCircBase (https://www.pancircbase.net/) database that provides the following resources: 1) pancreatic islet circRNA annotation details (genomic position, host gene, exon information, splice length, sequence, other database IDs, cross-species conservation), 2) divergent primers for PCR analysis of circRNAs, 3) siRNAs for silencing of target circRNAs, 4) miRNAs associated with circRNAs, 5) possible protein-coding circRNAs and their polypeptides. In summary, this is a comprehensive online resource for exploring circRNA expression and its possible function in pancreatic ß-cells.
RESUMO
Endothelial and erythropoietic lineages arise from a common developmental progenitor. Etv2 is a master transcriptional regulator required for the development of both lineages. However, the mechanisms through which Etv2 initiates the gene-regulatory networks (GRNs) for endothelial and erythropoietic specification and how the two GRNs diverge downstream of Etv2 remain incompletely understood. Here, by analyzing a hypomorphic Etv2 mutant, we demonstrate different threshold requirements for initiation of the downstream GRNs for endothelial and erythropoietic development. We show that Etv2 functions directly in a coherent feedforward transcriptional network for vascular endothelial development, and a low level of Etv2 expression is sufficient to induce and sustain the endothelial GRN. In contrast, Etv2 induces the erythropoietic GRN indirectly via activation of Tal1, which requires a significantly higher threshold of Etv2 to initiate and sustain erythropoietic development. These results provide important mechanistic insight into the divergence of the endothelial and erythropoietic lineages.