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1.
Circulation ; 149(15): 1205-1230, 2024 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-38189150

RESUMO

BACKGROUND: The relationship between heart failure (HF) and atrial fibrillation (AF) is clear, with up to half of patients with HF progressing to AF. The pathophysiological basis of AF in the context of HF is presumed to result from atrial remodeling. Upregulation of the transcription factor FOG2 (friend of GATA2; encoded by ZFPM2) is observed in human ventricles during HF and causes HF in mice. METHODS: FOG2 expression was assessed in human atria. The effect of adult-specific FOG2 overexpression in the mouse heart was evaluated by whole animal electrophysiology, in vivo organ electrophysiology, cellular electrophysiology, calcium flux, mouse genetic interactions, gene expression, and genomic function, including a novel approach for defining functional transcription factor interactions based on overlapping effects on enhancer noncoding transcription. RESULTS: FOG2 is significantly upregulated in the human atria during HF. Adult cardiomyocyte-specific FOG2 overexpression in mice caused primary spontaneous AF before the development of HF or atrial remodeling. FOG2 overexpression generated arrhythmia substrate and trigger in cardiomyocytes, including calcium cycling defects. We found that FOG2 repressed atrial gene expression promoted by TBX5. FOG2 bound a subset of GATA4 and TBX5 co-bound genomic locations, defining a shared atrial gene regulatory network. FOG2 repressed TBX5-dependent transcription from a subset of co-bound enhancers, including a conserved enhancer at the Atp2a2 locus. Atrial rhythm abnormalities in mice caused by Tbx5 haploinsufficiency were rescued by Zfpm2 haploinsufficiency. CONCLUSIONS: Transcriptional changes in the atria observed in human HF directly antagonize the atrial rhythm gene regulatory network, providing a genomic link between HF and AF risk independent of atrial remodeling.


Assuntos
Fibrilação Atrial , Remodelamento Atrial , Insuficiência Cardíaca , Humanos , Camundongos , Animais , Fibrilação Atrial/genética , Redes Reguladoras de Genes , Cálcio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Átrios do Coração , Insuficiência Cardíaca/genética , Genômica , Fator de Transcrição GATA4/genética
2.
Circulation ; 148(21): 1705-1722, 2023 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-37772400

RESUMO

BACKGROUND: Conotruncal defects due to developmental abnormalities of the outflow tract (OFT) are an important cause of cyanotic congenital heart disease. Dysregulation of transcriptional programs tuned by NKX2-5 (NK2 homeobox 5), GATA6 (GATA binding protein 6), and TBX1 (T-box transcription factor 1) have been implicated in abnormal OFT morphogenesis. However, there remains no consensus on how these transcriptional programs function in a unified gene regulatory network within the OFT. METHODS: We generated mice harboring a 226-nucleotide deletion of a highly conserved cardiac enhancer containing 2 GATA-binding sites located ≈9.4 kb upstream of the transcription start site of Nkx2-5 (Nkx2-5∆enh) using CRISPR-Cas9 gene editing and assessed phenotypes. Cardiac defects in Nkx2-5∆enh/∆enh mice were structurally characterized using histology and scanning electron microscopy, and physiologically assessed using electrocardiography, echocardiography, and optical mapping. Transcriptome analyses were performed using RNA sequencing and single-cell RNA sequencing data sets. Endogenous GATA6 interaction with and activity on the NKX2-5 enhancer was studied using chromatin immunoprecipitation sequencing and transposase-accessible chromatin sequencing in human induced pluripotent stem cell-derived cardiomyocytes. RESULTS: Nkx2-5∆enh/∆enh mice recapitulated cyanotic conotruncal defects seen in patients with NKX2-5, GATA6, and TBX1 mutations. Nkx2-5∆enh/∆enh mice also exhibited defects in right Purkinje fiber network formation, resulting in right bundle-branch block. Enhancer deletion reduced embryonic Nkx2-5 expression selectively in the right ventricle and OFT of mutant hearts, indicating that enhancer activity is localized to the anterior second heart field. Transcriptional profiling of the mutant OFT revealed downregulation of important genes involved in OFT rotation and septation, such as Tbx1, Pitx2, and Sema3c. Endogenous GATA6 interacted with the highly conserved enhancer in human induced pluripotent stem cell-derived cardiomyocytes and in wild-type mouse hearts. We found critical dose dependency of cardiac enhancer accessibility on GATA6 gene dosage in human induced pluripotent stem cell-derived cardiomyocytes. CONCLUSIONS: Our results using human and mouse models reveal an essential gene regulatory network of the OFT that requires an anterior second heart field enhancer to link GATA6 with NKX2-5-dependent rotation and septation gene programs.


Assuntos
Células-Tronco Pluripotentes Induzidas , Fatores de Transcrição , Humanos , Camundongos , Animais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Homeodomínio/genética , Redes Reguladoras de Genes , Proteína Homeobox Nkx-2.5/genética , Proteína Homeobox Nkx-2.5/metabolismo , Camundongos Transgênicos , Células-Tronco Pluripotentes Induzidas/metabolismo , Coração , Miócitos Cardíacos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento
3.
Circulation ; 147(10): 824-840, 2023 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-36524479

RESUMO

BACKGROUND: Brugada syndrome (BrS) is an inherited arrhythmia syndrome caused by loss-of-function variants in the cardiac sodium channel gene SCN5A (sodium voltage-gated channel alpha subunit 5) in ≈20% of subjects. We identified a family with 4 individuals diagnosed with BrS harboring the rare G145R missense variant in the cardiac transcription factor TBX5 (T-box transcription factor 5) and no SCN5A variant. METHODS: We generated induced pluripotent stem cells (iPSCs) from 2 members of a family carrying TBX5-G145R and diagnosed with Brugada syndrome. After differentiation to iPSC-derived cardiomyocytes (iPSC-CMs), electrophysiologic characteristics were assessed by voltage- and current-clamp experiments (n=9 to 21 cells per group) and transcriptional differences by RNA sequencing (n=3 samples per group), and compared with iPSC-CMs in which G145R was corrected by CRISPR/Cas9 approaches. The role of platelet-derived growth factor (PDGF)/phosphoinositide 3-kinase (PI3K) pathway was elucidated by small molecule perturbation. The rate-corrected QT (QTc) interval association with serum PDGF was tested in the Framingham Heart Study cohort (n=1893 individuals). RESULTS: TBX5-G145R reduced transcriptional activity and caused multiple electrophysiologic abnormalities, including decreased peak and enhanced "late" cardiac sodium current (INa), which were entirely corrected by editing G145R to wild-type. Transcriptional profiling and functional assays in genome-unedited and -edited iPSC-CMs showed direct SCN5A down-regulation caused decreased peak INa, and that reduced PDGF receptor (PDGFRA [platelet-derived growth factor receptor α]) expression and blunted signal transduction to PI3K was implicated in enhanced late INa. Tbx5 regulation of the PDGF axis increased arrhythmia risk due to disruption of PDGF signaling and was conserved in murine model systems. PDGF receptor blockade markedly prolonged normal iPSC-CM action potentials and plasma levels of PDGF in the Framingham Heart Study were inversely correlated with the QTc interval (P<0.001). CONCLUSIONS: These results not only establish decreased SCN5A transcription by the TBX5 variant as a cause of BrS, but also reveal a new general transcriptional mechanism of arrhythmogenesis of enhanced late sodium current caused by reduced PDGF receptor-mediated PI3K signaling.


Assuntos
Síndrome de Brugada , Humanos , Camundongos , Animais , Fosfatidilinositol 3-Quinases/metabolismo , Fenótipo , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Sódio/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo
4.
Nucleic Acids Res ; 50(16): e91, 2022 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-35640613

RESUMO

Analyzing single-cell transcriptomes promises to decipher the plasticity, heterogeneity, and rapid switches in developmental cellular state transitions. Such analyses require the identification of gene markers for semi-stable transition states. However, there are nontrivial challenges such as unexplainable stochasticity, variable population sizes, and alternative trajectory constructions. By advancing current tipping-point theory-based models with feature selection, network decomposition, accurate estimation of correlations, and optimization, we developed BioTIP to overcome these challenges. BioTIP identifies a small group of genes, called critical transition signal (CTS), to characterize regulated stochasticity during semi-stable transitions. Although methods rooted in different theories converged at the same transition events in two benchmark datasets, BioTIP is unique in inferring lineage-determining transcription factors governing critical transition. Applying BioTIP to mouse gastrulation data, we identify multiple CTSs from one dataset and validated their significance in another independent dataset. We detect the established regulator Etv2 whose expression change drives the haemato-endothelial bifurcation, and its targets together in CTS across three datasets. After comparing to three current methods using six datasets, we show that BioTIP is accurate, user-friendly, independent of pseudo-temporal trajectory, and captures significantly interconnected and reproducible CTSs. We expect BioTIP to provide great insight into dynamic regulations of lineage-determining factors.


Assuntos
Linhagem da Célula , Análise de Célula Única , Fatores de Transcrição , Transcriptoma , Animais , Gástrula/citologia , Marcadores Genéticos , Camundongos , Fatores de Transcrição/genética
5.
Dev Dyn ; 252(4): 483-494, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36495293

RESUMO

BACKGROUND: Frem1 has been linked to human face shape variation, dysmorphology, and malformation, but little is known about its regulation and biological role in facial development. RESULTS: During midfacial morphogenesis in mice, we observed Frem1 expression in the embryonic growth centers that form the median upper lip, nose, and palate. Expansive spatial gradients of Frem1 expression in the cranial neural crest cell (cNCC) mesenchyme of these tissues suggested transcriptional regulation by a secreted morphogen. Accordingly, Frem1 expression paralleled that of the conserved Sonic Hedgehog (Shh) target gene Gli1 in the cNCC mesenchyme. Suggesting direct transcriptional regulation by Shh signaling, we found that Frem1 expression is induced by SHH ligand stimulation or downstream pathway activation in cNCCs and observed GLI transcription factor binding at the Frem1 transcriptional start site during midfacial morphogenesis. Finally, we found that FREM1 is sufficient to induce cNCC proliferation in a concentration-dependent manner and that Shh pathway antagonism reduces Frem1 expression during pathogenesis of midfacial hypoplasia. CONCLUSIONS: By demonstrating that the Shh signaling pathway regulates Frem1 expression in cNCCs, these findings provide novel insight into the mechanisms underlying variation in midfacial morphogenesis.


Assuntos
Proteínas Hedgehog , Crista Neural , Camundongos , Animais , Humanos , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Morfogênese/genética , Transdução de Sinais/fisiologia , Mesoderma/metabolismo , Proteínas da Matriz Extracelular/metabolismo
6.
Semin Cell Dev Biol ; 118: 94-106, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34144893

RESUMO

Congenital Heart Disease (CHD), malformations of the heart present at birth, is the most common class of life-threatening birth defect (Hoffman (1995) [1], Gelb (2004) [2], Gelb (2014) [3]). A major research challenge is to elucidate the genetic determinants of CHD and mechanistically link CHD ontogeny to a molecular understanding of heart development. Although the embryonic origins of CHD are unclear in most cases, dysregulation of cardiovascular lineage specification, patterning, proliferation, migration or differentiation have been described (Olson (2004) [4], Olson (2006) [5], Srivastava (2006) [6], Dunwoodie (2007) [7], Bruneau (2008) [8]). Cardiac differentiation is the process whereby cells become progressively more dedicated in a trajectory through the cardiac lineage towards mature cardiomyocytes. Defects in cardiac differentiation have been linked to CHD, although how the complex control of cardiac differentiation prevents CHD is just beginning to be understood. The stages of cardiac differentiation are highly stereotyped and have been well-characterized (Kattman et al. (2011) [9], Wamstad et al. (2012) [10], Luna-Zurita et al. (2016) [11], Loh et al. (2016) [12], DeLaughter et al. (2016) [13]); however, the developmental and molecular mechanisms that promote or delay the transition of a cell through these stages have not been as deeply investigated. Tight temporal control of progenitor differentiation is critically important for normal organ size, spatial organization, and cellular physiology and homeostasis of all organ systems (Raff et al. (1985) [14], Amthor et al. (1998) [15], Kopan et al. (2014) [16]). This review will focus on the action of signaling pathways in the control of cardiomyocyte differentiation timing. Numerous signaling pathways, including the Wnt, Fibroblast Growth Factor, Hedgehog, Bone Morphogenetic Protein, Insulin-like Growth Factor, Thyroid Hormone and Hippo pathways, have all been implicated in promoting or inhibiting transitions along the cardiac differentiation trajectory. Gaining a deeper understanding of the mechanisms controlling cardiac differentiation timing promises to yield insights into the etiology of CHD and to inform approaches to restore function to damaged hearts.


Assuntos
Miócitos Cardíacos/metabolismo , Diferenciação Celular , Humanos , Transdução de Sinais
7.
Development ; 147(3)2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-31915147

RESUMO

Identification of cell type-specific cis-regulatory elements (CREs) is crucial for understanding development and disease, although identification of functional regulatory elements remains challenging. We hypothesized that context-specific CREs could be identified by context-specific non-coding RNA (ncRNA) profiling, based on the observation that active CREs produce ncRNAs. We applied ncRNA profiling to identify rod and cone photoreceptor CREs from wild-type and mutant mouse retinas, defined by presence or absence, respectively, of the rod-specific transcription factor (TF) NrlNrl-dependent ncRNA expression strongly correlated with epigenetic profiles of rod and cone photoreceptors, identified thousands of candidate rod- and cone-specific CREs, and identified motifs for rod- and cone-specific TFs. Colocalization of NRL and the retinal TF CRX correlated with rod-specific ncRNA expression, whereas CRX alone favored cone-specific ncRNA expression, providing quantitative evidence that heterotypic TF interactions distinguish cell type-specific CRE activity. We validated the activity of novel Nrl-dependent ncRNA-defined CREs in developing cones. This work supports differential ncRNA profiling as a platform for the identification of cell type-specific CREs and the discovery of molecular mechanisms underlying TF-dependent CRE activity.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteínas do Olho/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Transcrição Gênica/genética , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas do Olho/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Camundongos Knockout , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Transativadores/genética , Transativadores/metabolismo , Transcriptoma
8.
Proc Natl Acad Sci U S A ; 117(27): 15712-15723, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32561646

RESUMO

The mechanisms used by embryos to pattern tissues across their axes has fascinated developmental biologists since the founding of embryology. Here, using single-cell technology, we interrogate complex patterning defects and define a Hedgehog (Hh)-fibroblast growth factor (FGF) signaling axis required for anterior mesoderm lineage development during gastrulation. Single-cell transcriptome analysis of Hh-deficient mesoderm revealed selective deficits in anterior mesoderm populations, culminating in defects to anterior embryonic structures, including the pharyngeal arches, heart, and anterior somites. Transcriptional profiling of Hh-deficient mesoderm during gastrulation revealed disruptions to both transcriptional patterning of the mesoderm and FGF signaling for mesoderm migration. Mesoderm-specific Fgf4/Fgf8 double-mutants recapitulated anterior mesoderm defects and Hh-dependent GLI transcription factors modulated enhancers at FGF gene loci. Cellular migration defects during gastrulation induced by Hh pathway antagonism were mitigated by the addition of FGF4 protein. These findings implicate a multicomponent signaling hierarchy activated by Hh ligands from the embryonic node and executed by FGF signals in nascent mesoderm to control anterior mesoderm patterning.


Assuntos
Fator 4 de Crescimento de Fibroblastos/genética , Fator 8 de Crescimento de Fibroblasto/genética , Gastrulação/genética , Proteína GLI1 em Dedos de Zinco/genética , Animais , Padronização Corporal/genética , Linhagem da Célula/genética , Embrião de Galinha , Fatores de Crescimento de Fibroblastos/genética , Gástrula/crescimento & desenvolvimento , Gástrula/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas Hedgehog/genética , Mesoderma/crescimento & desenvolvimento , Mesoderma/metabolismo , Camundongos , Transdução de Sinais/genética , Análise de Célula Única , Transcriptoma/genética
9.
Circ Res ; 127(3): e94-e106, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32290757

RESUMO

RATIONALE: The heartbeat is organized by the cardiac conduction system (CCS), a specialized network of cardiomyocytes. Patterning of the CCS into atrial node versus ventricular conduction system (VCS) components with distinct physiology is essential for the normal heartbeat. Distinct node versus VCS physiology has been recognized for more than a century, but the molecular basis of this regional patterning is not well understood. OBJECTIVE: To study the genetic and genomic mechanisms underlying node versus VCS distinction and investigate rhythm consequences of failed VCS patterning. METHODS AND RESULTS: Using mouse genetics, we found that the balance between T-box transcriptional activator, Tbx5, and T-box transcriptional repressor, Tbx3, determined the molecular and functional output of VCS myocytes. Adult VCS-specific removal of Tbx5 or overexpression of Tbx3 re-patterned the fast VCS into slow, nodal-like cells based on molecular and functional criteria. In these cases, gene expression profiling showed diminished expression of genes required for VCS-specific fast conduction but maintenance of expression of genes required for nodal slow conduction physiology. Action potentials of Tbx5-deficient VCS myocytes adopted nodal-specific characteristics, including increased action potential duration and cellular automaticity. Removal of Tbx5 in vivo precipitated inappropriate depolarizations in the atrioventricular (His)-bundle associated with lethal ventricular arrhythmias. TBX5 bound and directly activated cis-regulatory elements at fast conduction channel genes required for fast physiological characteristics of the VCS action potential, defining the identity of the adult VCS. CONCLUSIONS: The CCS is patterned entirely as a slow, nodal ground state, with a T-box dependent, physiologically dominant, fast conduction network driven specifically in the VCS. Disruption of the fast VCS gene regulatory network allowed nodal physiology to emerge, providing a plausible molecular mechanism for some lethal ventricular arrhythmias.


Assuntos
Arritmias Cardíacas/metabolismo , Nó Atrioventricular/metabolismo , Ventrículos do Coração/metabolismo , Proteínas com Domínio T/metabolismo , Transcrição Gênica , Potenciais de Ação , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatologia , Nó Atrioventricular/fisiopatologia , Padronização Corporal , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Frequência Cardíaca , Ventrículos do Coração/fisiopatologia , Humanos , Masculino , Camundongos Knockout , Proteínas com Domínio T/deficiência , Proteínas com Domínio T/genética , Fatores de Tempo
10.
Circ Res ; 127(1): 34-50, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32717170

RESUMO

Genome-wide association studies have uncovered over a 100 genetic loci associated with atrial fibrillation (AF), the most common arrhythmia. Many of the top AF-associated loci harbor key cardiac transcription factors, including PITX2, TBX5, PRRX1, and ZFHX3. Moreover, the vast majority of the AF-associated variants lie within noncoding regions of the genome where causal variants affect gene expression by altering the activity of transcription factors and the epigenetic state of chromatin. In this review, we discuss a transcriptional regulatory network model for AF defined by effector genes in Genome-wide association studies loci. We describe the current state of the field regarding the identification and function of AF-relevant gene regulatory networks, including variant regulatory elements, dose-sensitive transcription factor functionality, target genes, and epigenetic states. We illustrate how altered transcriptional networks may impact cardiomyocyte function and ionic currents that impact AF risk. Last, we identify the need for improved tools to identify and functionally test transcriptional components to define the links between genetic variation, epigenetic gene regulation, and atrial function.


Assuntos
Fibrilação Atrial/genética , Epigênese Genética , Redes Reguladoras de Genes , Animais , Fibrilação Atrial/metabolismo , Loci Gênicos , Humanos , Transcriptoma
11.
Circ Res ; 127(2): e28-e43, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32347164

RESUMO

RATIONALE: ZO-1 (Zona occludens 1), encoded by the tight junction protein 1 (TJP1) gene, is a regulator of paracellular permeability in epithelia and endothelia. ZO-1 interacts with the actin cytoskeleton, gap, and adherens junction proteins and localizes to intercalated discs in cardiomyocytes. However, the contribution of ZO-1 to cardiac physiology remains poorly defined. OBJECTIVE: We aim to determine the role of ZO-1 in cardiac function. METHODS AND RESULTS: Inducible cardiomyocyte-specific Tjp1 deletion mice (Tjp1fl/fl; Myh6Cre/Esr1*) were generated by crossing the Tjp1 floxed mice and Myh6Cre/Esr1* transgenic mice. Tamoxifen-induced loss of ZO-1 led to atrioventricular (AV) block without changes in heart rate, as measured by ECG and ex vivo optical mapping. Mice with tamoxifen-induced conduction system-specific deletion of Tjp1 (Tjp1fl/fl; Hcn4CreERt2) developed AV block while tamoxifen-induced conduction system deletion of Tjp1 distal to the AV node (Tjp1fl/fl; Kcne1CreERt2) did not demonstrate conduction defects. Western blot and immunostaining analyses of AV nodes showed that ZO-1 loss decreased Cx (connexin) 40 expression and intercalated disc localization. Consistent with the mouse model study, immunohistochemical staining showed that ZO-1 is abundantly expressed in the human AV node and colocalizes with Cx40. Ventricular conduction was not altered despite decreased localization of ZO-1 and Cx43 at the ventricular intercalated disc and modestly decreased left ventricular ejection fraction, suggesting ZO-1 is differentially required for AV node and ventricular conduction. CONCLUSIONS: ZO-1 is a key protein responsible for maintaining appropriate AV node conduction through maintaining gap junction protein localization.


Assuntos
Nó Atrioventricular/metabolismo , Frequência Cardíaca , Proteína da Zônula de Oclusão-1/metabolismo , Animais , Nó Atrioventricular/fisiologia , Conexina 43/genética , Conexina 43/metabolismo , Conexinas/genética , Conexinas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Proteína da Zônula de Oclusão-1/genética , Proteína alfa-5 de Junções Comunicantes
12.
PLoS Genet ; 15(5): e1007711, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31120883

RESUMO

Dominant mutations of Gata4, an essential cardiogenic transcription factor (TF), were known to cause outflow tract (OFT) defects in both human and mouse, but the underlying molecular mechanism was not clear. In this study, Gata4 haploinsufficiency in mice was found to result in OFT defects including double outlet right ventricle (DORV) and ventricular septum defects (VSDs). Gata4 was shown to be required for Hedgehog (Hh)-receiving progenitors within the second heart field (SHF) for normal OFT alignment. Restored cell proliferation in the SHF by knocking-down Pten failed to rescue OFT defects, suggesting that additional cell events under Gata4 regulation is important. SHF Hh-receiving cells failed to migrate properly into the proximal OFT cushion, which is associated with abnormal EMT and cell proliferation in Gata4 haploinsufficiency. The genetic interaction of Hh signaling and Gata4 is further demonstrated to be important for OFT development. Gata4 and Smo double heterozygotes displayed more severe OFT abnormalities including persistent truncus arteriosus (PTA). Restoration of Hedgehog signaling renormalized SHF cell proliferation and migration, and rescued OFT defects in Gata4 haploinsufficiency. In addition, there was enhanced Gata6 expression in the SHF of the Gata4 heterozygotes. The Gata4-responsive repressive sites were identified within 1kbp upstream of the transcription start site of Gata6 by both ChIP-qPCR and luciferase reporter assay. These results suggested a SHF regulatory network comprising of Gata4, Gata6 and Hh-signaling for OFT development.


Assuntos
Fator de Transcrição GATA4/genética , Fator de Transcrição GATA6/genética , Proteínas Hedgehog/genética , Receptor Smoothened/genética , Obstrução do Fluxo Ventricular Externo/genética , Septo Interventricular/metabolismo , Animais , Movimento Celular , Proliferação de Células , Embrião de Mamíferos , Fator de Transcrição GATA4/metabolismo , Fator de Transcrição GATA6/metabolismo , Regulação da Expressão Gênica , Haploinsuficiência , Proteínas Hedgehog/metabolismo , Heterozigoto , Humanos , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Transdução de Sinais , Receptor Smoothened/metabolismo , Tronco Arterial/anormalidades , Tronco Arterial/metabolismo , Obstrução do Fluxo Ventricular Externo/metabolismo , Obstrução do Fluxo Ventricular Externo/patologia , Septo Interventricular/patologia
13.
PLoS Comput Biol ; 16(2): e1007119, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32040509

RESUMO

Long noncoding RNAs (lncRNAs) localize in the cell nucleus and influence gene expression through a variety of molecular mechanisms. Chromatin-enriched RNAs (cheRNAs) are a unique class of lncRNAs that are tightly bound to chromatin and putatively function to locally cis-activate gene transcription. CheRNAs can be identified by biochemical fractionation of nuclear RNA followed by RNA sequencing, but until now, a rigorous analytic pipeline for nuclear RNA-seq has been lacking. In this study, we survey four computational strategies for nuclear RNA-seq data analysis and develop a new pipeline, Tuxedo-ch, which outperforms other approaches. Tuxedo-ch assembles a more complete transcriptome and identifies cheRNA with higher accuracy than other approaches. We used Tuxedo-ch to analyze benchmark datasets of K562 cells and further characterize the genomic features of intergenic cheRNA (icheRNA) and their similarity to enhancer RNAs (eRNAs). We quantify the transcriptional correlation of icheRNA and adjacent genes and show that icheRNA is more positively associated with neighboring gene expression than eRNA or cap analysis of gene expression (CAGE) signals. We also explore two novel genomic associations of cheRNA, which indicate that cheRNAs may function to promote or repress gene expression in a context-dependent manner. IcheRNA loci with significant levels of H3K9me3 modifications are associated with active enhancers, consistent with the hypothesis that enhancers are derived from ancient mobile elements. In contrast, antisense cheRNA (as-cheRNA) may play a role in local gene repression, possibly through local RNA:DNA:DNA triple-helix formation.


Assuntos
Núcleo Celular/genética , Cromatina/metabolismo , Regulação da Expressão Gênica , RNA/genética , Análise de Sequência de RNA/métodos , Animais , Biologia Computacional , Elementos Facilitadores Genéticos , Humanos , RNA Mensageiro/genética
14.
Proc Natl Acad Sci U S A ; 115(48): E11349-E11358, 2018 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-30429326

RESUMO

Biomechanical cues dynamically control major cellular processes, but whether genetic variants actively participate in mechanosensing mechanisms remains unexplored. Vascular homeostasis is tightly regulated by hemodynamics. Exposure to disturbed blood flow at arterial sites of branching and bifurcation causes constitutive activation of vascular endothelium contributing to atherosclerosis, the major cause of coronary artery disease (CAD) and ischemic stroke (IS). Conversely, unidirectional flow promotes quiescent endothelium. Genome-wide association studies (GWAS) have identified chromosome 1p32.2 as strongly associated with CAD/IS; however, the causal mechanism related to this locus remains unknown. Using statistical analyses, assay of transposase accessible chromatin with whole-genome sequencing (ATAC-seq), H3K27ac/H3K4me2 ChIP with whole-genome sequencing (ChIP-seq), and CRISPR interference in human aortic endothelial cells (HAECs), our results demonstrate that rs17114036, a common noncoding polymorphism at 1p32.2, is located in an endothelial enhancer dynamically regulated by hemodynamics. CRISPR-Cas9-based genome editing shows that rs17114036-containing region promotes endothelial quiescence under unidirectional shear stress by regulating phospholipid phosphatase 3 (PLPP3). Chromatin accessibility quantitative trait locus (caQTL) mapping using HAECs from 56 donors, allelic imbalance assay from 7 donors, and luciferase assays demonstrate that CAD/IS-protective allele at rs17114036 in PLPP3 intron 5 confers increased endothelial enhancer activity. ChIP-PCR and luciferase assays show that CAD/IS-protective allele at rs17114036 creates a binding site for transcription factor Krüppel-like factor 2 (KLF2), which increases the enhancer activity under unidirectional flow. These results demonstrate that a human SNP contributes to critical endothelial mechanotransduction mechanisms and suggest that human haplotypes and related cis-regulatory elements provide a previously unappreciated layer of regulatory control in cellular mechanosensing mechanisms.


Assuntos
Isquemia Encefálica/genética , Cromossomos Humanos Par 1/genética , Doença da Artéria Coronariana/genética , Células Endoteliais/fisiologia , Variação Genética , Acidente Vascular Cerebral/genética , Alelos , Velocidade do Fluxo Sanguíneo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/fisiopatologia , Cromatina/genética , Cromatina/metabolismo , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/fisiopatologia , Estudo de Associação Genômica Ampla , Hemodinâmica , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Mecanotransdução Celular , Polimorfismo de Nucleotídeo Único , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/fisiopatologia
15.
Proc Natl Acad Sci U S A ; 115(45): E10615-E10624, 2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30352852

RESUMO

Codevelopment of the lungs and heart underlies key evolutionary innovations in the transition to terrestrial life. Cardiac specializations that support pulmonary circulation, including the atrial septum, are generated by second heart field (SHF) cardiopulmonary progenitors (CPPs). It has been presumed that transcription factors required in the SHF for cardiac septation, e.g., Tbx5, directly drive a cardiac morphogenesis gene-regulatory network. Here, we report instead that TBX5 directly drives Wnt ligands to initiate a bidirectional signaling loop between cardiopulmonary mesoderm and the foregut endoderm for endodermal pulmonary specification and, subsequently, atrial septation. We show that Tbx5 is required for pulmonary specification in mice and amphibians but not for swim bladder development in zebrafish. TBX5 is non-cell-autonomously required for pulmonary endoderm specification by directly driving Wnt2 and Wnt2b expression in cardiopulmonary mesoderm. TBX5 ChIP-sequencing identified cis-regulatory elements at Wnt2 sufficient for endogenous Wnt2 expression domains in vivo and required for Wnt2 expression in precardiac mesoderm in vitro. Tbx5 cooperated with Shh signaling to drive Wnt2b expression for lung morphogenesis. Tbx5 haploinsufficiency in mice, a model of Holt-Oram syndrome, caused a quantitative decrement of mesodermal-to-endodermal Wnt signaling and subsequent endodermal-to-mesodermal Shh signaling required for cardiac morphogenesis. Thus, Tbx5 initiates a mesoderm-endoderm-mesoderm signaling loop in lunged vertebrates that provides a molecular basis for the coevolution of pulmonary and cardiac structures required for terrestrial life.


Assuntos
Evolução Molecular , Coração/embriologia , Pulmão/embriologia , Proteínas com Domínio T/genética , Proteína Wnt2/genética , Animais , Elementos Facilitadores Genéticos , Perfilação da Expressão Gênica , Camundongos , Camundongos Mutantes , Transdução de Sinais , Transcrição Gênica , Peixe-Zebra/embriologia
16.
Proc Natl Acad Sci U S A ; 114(8): E1422-E1431, 2017 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-28167794

RESUMO

GATA4, an essential cardiogenic transcription factor, provides a model for dominant transcription factor mutations in human disease. Dominant GATA4 mutations cause congenital heart disease (CHD), specifically atrial and atrioventricular septal defects (ASDs and AVSDs). We found that second heart field (SHF)-specific Gata4 heterozygote embryos recapitulated the AVSDs observed in germline Gata4 heterozygote embryos. A proliferation defect of SHF atrial septum progenitors and hypoplasia of the dorsal mesenchymal protrusion, rather than anlage of the atrioventricular septum, were observed in this model. Knockdown of the cell-cycle repressor phosphatase and tensin homolog (Pten) restored cell-cycle progression and rescued the AVSDs. Gata4 mutants also demonstrated Hedgehog (Hh) signaling defects. Gata4 acts directly upstream of Hh components: Gata4 activated a cis-regulatory element at Gli1 in vitro and occupied the element in vivo. Remarkably, SHF-specific constitutive Hh signaling activation rescued AVSDs in Gata4 SHF-specific heterozygous knockout embryos. Pten expression was unchanged in Smoothened mutants, and Hh pathway genes were unchanged in Pten mutants, suggesting pathway independence. Thus, both the cell-cycle and Hh-signaling defects caused by dominant Gata4 mutations were required for CHD pathogenesis, suggesting a combinatorial model of disease causation by transcription factor haploinsufficiency.


Assuntos
Proliferação de Células/fisiologia , Fator de Transcrição GATA4/metabolismo , Coração/fisiologia , Proteínas Hedgehog/metabolismo , Animais , Ciclo Celular/fisiologia , Septos Cardíacos/metabolismo , Camundongos , Miocárdio/metabolismo , Transdução de Sinais/fisiologia , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo
18.
PLoS Genet ; 12(4): e1005963, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27058611

RESUMO

Congenital heart disease (CHD) has a complex genetic etiology, and recent studies suggest that high penetrance de novo mutations may account for only a small fraction of disease. In a multi-institutional cohort surveyed by exome sequencing, combining analysis of 987 individuals (discovery cohort of 59 affected trios and 59 control trios, and a replication cohort of 100 affected singletons and 533 unaffected singletons) we observe variation at novel and known loci related to a specific cardiac malformation the atrioventricular septal defect (AVSD). In a primary analysis, by combining developmental coexpression networks with inheritance modeling, we identify a de novo mutation in the DNA binding domain of NR1D2 (p.R175W). We show that p.R175W changes the transcriptional activity of Nr1d2 using an in vitro transactivation model in HUVEC cells. Finally, we demonstrate previously unrecognized cardiovascular malformations in the Nr1d2tm1-Dgen knockout mouse. In secondary analyses we map genetic variation to protein-interaction networks suggesting a role for two collagen genes in AVSD, which we corroborate by burden testing in a second replication cohort of 100 AVSDs and 533 controls (p = 8.37e-08). Finally, we apply a rare-disease inheritance model to identify variation in genes previously associated with CHD (ZFPM2, NSD1, NOTCH1, VCAN, and MYH6), cardiac malformations in mouse models (ADAM17, CHRD, IFT140, PTPRJ, RYR1 and ATE1), and hypomorphic alleles of genes causing syndromic CHD (EHMT1, SRCAP, BBS2, NOTCH2, and KMT2D) in 14 of 59 trios, greatly exceeding variation in control trios without CHD (p = 9.60e-06). In total, 32% of trios carried at least one putatively disease-associated variant across 19 loci,suggesting that inherited and de novo variation across a heterogeneous group of loci may contribute to disease risk.


Assuntos
Defeitos dos Septos Cardíacos/genética , Animais , Feminino , Heterozigoto , Homozigoto , Humanos , Masculino , Camundongos , Camundongos Knockout , Mutação , Linhagem
19.
Hum Mol Genet ; 25(14): 3011-3028, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27340223

RESUMO

Atrioventricular septal defects (AVSDs) are a common severe form of congenital heart disease (CHD). In this study we identified deleterious non-synonymous mutations in two cilia genes, Dnah11 and Mks1, in independent N-ethyl-N-nitrosourea-induced mouse mutant lines with heritable recessive AVSDs by whole-exome sequencing. Cilia are required for left/right body axis determination and second heart field (SHF) Hedgehog (Hh) signaling, and we find that cilia mutations affect these requirements differentially. Dnah11avc4 did not disrupt SHF Hh signaling and caused AVSDs only concurrently with heterotaxy, a left/right axis abnormality. In contrast, Mks1avc6 disrupted SHF Hh signaling and caused AVSDs without heterotaxy. We performed unbiased whole-genome SHF transcriptional profiling and found that cilia motility genes were not expressed in the SHF whereas cilia structural and signaling genes were highly expressed. SHF cilia gene expression predicted the phenotypic concordance between AVSDs and heterotaxy in mice and humans with cilia gene mutations. A two-step model of cilia action accurately predicted the AVSD/heterotaxyu phenotypic expression pattern caused by cilia gene mutations. We speculate that cilia gene mutations contribute to both syndromic and non-syndromic AVSDs in humans and provide a model that predicts the phenotypic consequences of specific cilia gene mutations.


Assuntos
Dineínas do Axonema/genética , Cílios/genética , Defeitos dos Septos Cardíacos/genética , Proteínas/genética , Animais , Dineínas do Axonema/biossíntese , Padronização Corporal/genética , Cílios/efeitos dos fármacos , Modelos Animais de Doenças , Etilnitrosoureia/toxicidade , Exoma/genética , Regulação da Expressão Gênica , Coração/fisiopatologia , Defeitos dos Septos Cardíacos/patologia , Proteínas Hedgehog/biossíntese , Proteínas Hedgehog/genética , Humanos , Camundongos , Mutação , Transdução de Sinais/genética
20.
Proc Natl Acad Sci U S A ; 112(16): E2020-9, 2015 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-25848000

RESUMO

Zfp57 is a maternal-zygotic effect gene that maintains genomic imprinting. Here we report that Zfp57 mutants exhibited a variety of cardiac defects including atrial septal defect (ASD), ventricular septal defect (VSD), thin myocardium, and reduced trabeculation. Zfp57 maternal-zygotic mutant embryos displayed more severe phenotypes with higher penetrance than the zygotic ones. Cardiac progenitor cells exhibited proliferation and differentiation defects in Zfp57 mutants. ZFP57 is a master regulator of genomic imprinting, so the DNA methylation imprint was lost in embryonic heart without ZFP57. Interestingly, the presence of imprinted DLK1, a target of ZFP57, correlated with NOTCH1 activation in cardiac cells. These results suggest that ZFP57 may modulate NOTCH signaling during cardiac development. Indeed, loss of ZFP57 caused loss of NOTCH1 activation in embryonic heart with more severe loss observed in the maternal-zygotic mutant. Maternal and zygotic functions of Zfp57 appear to play redundant roles in NOTCH1 activation and cardiomyocyte differentiation. This serves as an example of a maternal effect that can influence mammalian organ development. It also links genomic imprinting to NOTCH signaling and particular developmental functions.


Assuntos
Coração/embriologia , Receptores Notch/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Zigoto/metabolismo , Animais , Animais Recém-Nascidos , Proteínas de Ligação ao Cálcio , Diferenciação Celular , Proliferação de Células , Regulação para Baixo , Embrião de Mamíferos/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Impressão Genômica , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/metabolismo , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Modelos Biológicos , Mutação , Miócitos Cardíacos/patologia , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Células-Tronco/citologia , Fatores de Transcrição/metabolismo
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