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1.
Cell ; 186(3): 479-496.e23, 2023 02 02.
Artículo en Inglés | MEDLINE | ID: mdl-36736300

RESUMEN

Using four-dimensional whole-embryo light sheet imaging with improved and accessible computational tools, we longitudinally reconstruct early murine cardiac development at single-cell resolution. Nascent mesoderm progenitors form opposing density and motility gradients, converting the temporal birth sequence of gastrulation into a spatial anterolateral-to-posteromedial arrangement. Migrating precardiac mesoderm does not strictly preserve cellular neighbor relationships, and spatial patterns only become solidified as the cardiac crescent emerges. Progenitors undergo a mesenchymal-to-epithelial transition, with a first heart field (FHF) ridge apposing a motile juxta-cardiac field (JCF). Anchored along the ridge, the FHF epithelium rotates the JCF forward to form the initial heart tube, along with push-pull morphodynamics of the second heart field. In Mesp1 mutants that fail to make a cardiac crescent, mesoderm remains highly motile but directionally incoherent, resulting in density gradient inversion. Our practicable live embryo imaging approach defines spatial origins and behaviors of cardiac progenitors and identifies their unanticipated morphological transitions.


Asunto(s)
Corazón , Mesodermo , Ratones , Animales , Diferenciación Celular , Morfogénesis , Embrión de Mamíferos , Mamíferos
2.
Cell ; 185(5): 794-814.e30, 2022 03 03.
Artículo en Inglés | MEDLINE | ID: mdl-35182466

RESUMEN

Congenital heart disease (CHD) is present in 1% of live births, yet identification of causal mutations remains challenging. We hypothesized that genetic determinants for CHDs may lie in the protein interactomes of transcription factors whose mutations cause CHDs. Defining the interactomes of two transcription factors haplo-insufficient in CHD, GATA4 and TBX5, within human cardiac progenitors, and integrating the results with nearly 9,000 exomes from proband-parent trios revealed an enrichment of de novo missense variants associated with CHD within the interactomes. Scoring variants of interactome members based on residue, gene, and proband features identified likely CHD-causing genes, including the epigenetic reader GLYR1. GLYR1 and GATA4 widely co-occupied and co-activated cardiac developmental genes, and the identified GLYR1 missense variant disrupted interaction with GATA4, impairing in vitro and in vivo function in mice. This integrative proteomic and genetic approach provides a framework for prioritizing and interrogating genetic variants in heart disease.


Asunto(s)
Factor de Transcripción GATA4/metabolismo , Cardiopatías Congénitas , Proteínas Nucleares/metabolismo , Oxidorreductasas/metabolismo , Factores de Transcripción , Animales , Cardiopatías Congénitas/genética , Ratones , Mutación , Proteómica , Proteínas de Dominio T Box/genética , Factores de Transcripción/genética
3.
Cell ; 176(4): 816-830.e18, 2019 02 07.
Artículo en Inglés | MEDLINE | ID: mdl-30595451

RESUMEN

The temporal order of DNA replication (replication timing [RT]) is highly coupled with genome architecture, but cis-elements regulating either remain elusive. We created a series of CRISPR-mediated deletions and inversions of a pluripotency-associated topologically associating domain (TAD) in mouse ESCs. CTCF-associated domain boundaries were dispensable for RT. CTCF protein depletion weakened most TAD boundaries but had no effect on RT or A/B compartmentalization genome-wide. By contrast, deletion of three intra-TAD CTCF-independent 3D contact sites caused a domain-wide early-to-late RT shift, an A-to-B compartment switch, weakening of TAD architecture, and loss of transcription. The dispensability of TAD boundaries and the necessity of these "early replication control elements" (ERCEs) was validated by deletions and inversions at additional domains. Our results demonstrate that discrete cis-regulatory elements orchestrate domain-wide RT, A/B compartmentalization, TAD architecture, and transcription, revealing fundamental principles linking genome structure and function.


Asunto(s)
Momento de Replicación del ADN/fisiología , Replicación del ADN/genética , Replicación del ADN/fisiología , Animales , Factor de Unión a CCCTC/genética , Factor de Unión a CCCTC/metabolismo , Cromatina , ADN/genética , Momento de Replicación del ADN/genética , Células Madre Embrionarias , Elementos de Facilitación Genéticos/genética , Mamíferos/genética , Mamíferos/metabolismo , Ratones , Proteínas Represoras/metabolismo , Análisis Espacio-Temporal
4.
Cell ; 175(1): 38-40, 2018 09 20.
Artículo en Inglés | MEDLINE | ID: mdl-30241613

RESUMEN

TAD boundaries are insulators of genomic neighborhoods. In this issue, Sun et al. show that disease-associated tandem repeats are located to TAD boundaries and affect their insulation. The findings have important implications for TAD function and mechanisms underlying diseases such as fragile X syndrome and Huntington's disease.


Asunto(s)
Cromatina , Síndrome del Cromosoma X Frágil/genética , Genoma , Genómica , Humanos , Repeticiones de Microsatélite
5.
Nat Immunol ; 21(5): 513-524, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32284594

RESUMEN

Oxidative stress is a central part of innate immune-induced neurodegeneration. However, the transcriptomic landscape of central nervous system (CNS) innate immune cells contributing to oxidative stress is unknown, and therapies to target their neurotoxic functions are not widely available. Here, we provide the oxidative stress innate immune cell atlas in neuroinflammatory disease and report the discovery of new druggable pathways. Transcriptional profiling of oxidative stress-producing CNS innate immune cells identified a core oxidative stress gene signature coupled to coagulation and glutathione-pathway genes shared between a microglia cluster and infiltrating macrophages. Tox-seq followed by a microglia high-throughput screen and oxidative stress gene network analysis identified the glutathione-regulating compound acivicin, with potent therapeutic effects that decrease oxidative stress and axonal damage in chronic and relapsing multiple sclerosis models. Thus, oxidative stress transcriptomics identified neurotoxic CNS innate immune populations and may enable discovery of selective neuroprotective strategies.


Asunto(s)
Encefalomielitis Autoinmune Experimental/genética , Perfilación de la Expresión Génica/métodos , Microglía/fisiología , Esclerosis Múltiple/genética , Inflamación Neurogénica/genética , Animales , Antioxidantes/uso terapéutico , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Femenino , Redes Reguladoras de Genes , Ensayos Analíticos de Alto Rendimiento , Humanos , Inmunidad Innata , Isoxazoles/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Esclerosis Múltiple/tratamiento farmacológico , Inflamación Neurogénica/tratamiento farmacológico , Estrés Oxidativo , Análisis de Secuencia de ARN , Análisis de la Célula Individual
7.
Cell ; 169(5): 930-944.e22, 2017 May 18.
Artículo en Inglés | MEDLINE | ID: mdl-28525758

RESUMEN

The molecular mechanisms underlying folding of mammalian chromosomes remain poorly understood. The transcription factor CTCF is a candidate regulator of chromosomal structure. Using the auxin-inducible degron system in mouse embryonic stem cells, we show that CTCF is absolutely and dose-dependently required for looping between CTCF target sites and insulation of topologically associating domains (TADs). Restoring CTCF reinstates proper architecture on altered chromosomes, indicating a powerful instructive function for CTCF in chromatin folding. CTCF remains essential for TAD organization in non-dividing cells. Surprisingly, active and inactive genome compartments remain properly segregated upon CTCF depletion, revealing that compartmentalization of mammalian chromosomes emerges independently of proper insulation of TADs. Furthermore, our data support that CTCF mediates transcriptional insulator function through enhancer blocking but not as a direct barrier to heterochromatin spreading. Beyond defining the functions of CTCF in chromosome folding, these results provide new fundamental insights into the rules governing mammalian genome organization.


Asunto(s)
Cromosomas de los Mamíferos/química , Animales , Factor de Unión a CCCTC , Ciclo Celular , Cromatina/metabolismo , Cromosomas de los Mamíferos/genética , Cromosomas de los Mamíferos/metabolismo , Células Madre Embrionarias/metabolismo , Regulación de la Expresión Génica , Ácidos Indolacéticos/farmacología , Ratones , Proteínas Represoras/metabolismo , Transcripción Genética
8.
Cell ; 164(5): 999-1014, 2016 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-26875865

RESUMEN

Transcription factors (TFs) are thought to function with partners to achieve specificity and precise quantitative outputs. In the developing heart, heterotypic TF interactions, such as between the T-box TF TBX5 and the homeodomain TF NKX2-5, have been proposed as a mechanism for human congenital heart defects. We report extensive and complex interdependent genomic occupancy of TBX5, NKX2-5, and the zinc finger TF GATA4 coordinately controlling cardiac gene expression, differentiation, and morphogenesis. Interdependent binding serves not only to co-regulate gene expression but also to prevent TFs from distributing to ectopic loci and activate lineage-inappropriate genes. We define preferential motif arrangements for TBX5 and NKX2-5 cooperative binding sites, supported at the atomic level by their co-crystal structure bound to DNA, revealing a direct interaction between the two factors and induced DNA bending. Complex interdependent binding mechanisms reveal tightly regulated TF genomic distribution and define a combinatorial logic for heterotypic TF regulation of differentiation.


Asunto(s)
Factor de Transcripción GATA4/metabolismo , Proteínas de Homeodominio/metabolismo , Miocardio/citología , Organogénesis , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular , Cristalografía por Rayos X , Embrión de Mamíferos/metabolismo , Proteína Homeótica Nkx-2.5 , Proteínas de Homeodominio/genética , Ratones , Ratones Transgénicos , Modelos Moleculares , Miocardio/metabolismo , Regiones Promotoras Genéticas , Dominios y Motivos de Interacción de Proteínas , Proteínas de Dominio T Box/genética , Factores de Transcripción/genética
9.
Mol Cell ; 83(15): 2624-2640, 2023 08 03.
Artículo en Inglés | MEDLINE | ID: mdl-37419111

RESUMEN

The four-dimensional nucleome (4DN) consortium studies the architecture of the genome and the nucleus in space and time. We summarize progress by the consortium and highlight the development of technologies for (1) mapping genome folding and identifying roles of nuclear components and bodies, proteins, and RNA, (2) characterizing nuclear organization with time or single-cell resolution, and (3) imaging of nuclear organization. With these tools, the consortium has provided over 2,000 public datasets. Integrative computational models based on these data are starting to reveal connections between genome structure and function. We then present a forward-looking perspective and outline current aims to (1) delineate dynamics of nuclear architecture at different timescales, from minutes to weeks as cells differentiate, in populations and in single cells, (2) characterize cis-determinants and trans-modulators of genome organization, (3) test functional consequences of changes in cis- and trans-regulators, and (4) develop predictive models of genome structure and function.


Asunto(s)
Núcleo Celular , Genoma , Genoma/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo
10.
Cell ; 160(6): 1072-86, 2015 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-25768904

RESUMEN

The mechanisms by which transcription factor haploinsufficiency alters the epigenetic and transcriptional landscape in human cells to cause disease are unknown. Here, we utilized human induced pluripotent stem cell (iPSC)-derived endothelial cells (ECs) to show that heterozygous nonsense mutations in NOTCH1 that cause aortic valve calcification disrupt the epigenetic architecture, resulting in derepression of latent pro-osteogenic and -inflammatory gene networks. Hemodynamic shear stress, which protects valves from calcification in vivo, activated anti-osteogenic and anti-inflammatory networks in NOTCH1(+/+), but not NOTCH1(+/-), iPSC-derived ECs. NOTCH1 haploinsufficiency altered H3K27ac at NOTCH1-bound enhancers, dysregulating downstream transcription of more than 1,000 genes involved in osteogenesis, inflammation, and oxidative stress. Computational predictions of the disrupted NOTCH1-dependent gene network revealed regulatory nodes that, when modulated, restored the network toward the NOTCH1(+/+) state. Our results highlight how alterations in transcription factor dosage affect gene networks leading to human disease and reveal nodes for potential therapeutic intervention.


Asunto(s)
Epigénesis Genética , Redes Reguladoras de Genes , Receptor Notch1/genética , Células Endoteliales/metabolismo , Femenino , Haploinsuficiencia , Código de Histonas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Inflamación/metabolismo , Masculino , Osteogénesis , Linaje , Receptor Notch1/metabolismo , Estrés Mecánico , Transcripción Genética
11.
Genes Dev ; 36(11-12): 652-663, 2022 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-35835508

RESUMEN

Congenital heart defects (CHDs) are among the most common birth defects, but their etiology has long been mysterious. In recent decades, the development of a variety of experimental models has led to a greater understanding of the molecular basis of CHDs. In this review, we contrast mouse models of CHD, which maintain the anatomical arrangement of the heart, and human cellular models of CHD, which are more likely to capture human-specific biology but lack anatomical structure. We also discuss the recent development of cardiac organoids, which are a promising step toward more anatomically informative human models of CHD.


Asunto(s)
Cardiopatías Congénitas , Organoides , Animales , Modelos Animales de Enfermedad , Corazón , Cardiopatías Congénitas/genética , Humanos , Ratones
12.
Genes Dev ; 35(21-22): 1401-1402, 2021 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-34725128

RESUMEN

In this issue of Genes & Development, Amândio and colleagues (pp. 1490-1509) dissect the function of a cluster of several CTCF binding sites in the HoxD cluster by iterative deletions in mice. They found additive functions for some, and intriguingly found that some sites function as insulators, while others function as anchors for enhancer-promoter interactions. These functions vary depending on developmental context. The work provides new insights into the roles played by CTCF in regulating developmental patterns and 3D chromatin organization.


Asunto(s)
Cromatina , Elementos de Facilitación Genéticos , Animales , Sitios de Unión/genética , Factor de Unión a CCCTC/genética , Factor de Unión a CCCTC/metabolismo , Cromatina/genética , Elementos de Facilitación Genéticos/genética , Proteínas de Homeodominio , Ratones , Regiones Promotoras Genéticas/genética
13.
Nature ; 602(7895): 129-134, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-35082446

RESUMEN

Differentiation proceeds along a continuum of increasingly fate-restricted intermediates, referred to as canalization1,2. Canalization is essential for stabilizing cell fate, but the mechanisms that underlie robust canalization are unclear. Here we show that the BRG1/BRM-associated factor (BAF) chromatin-remodelling complex ATPase gene Brm safeguards cell identity during directed cardiogenesis of mouse embryonic stem cells. Despite the establishment of a well-differentiated precardiac mesoderm, Brm-/- cells predominantly became neural precursors, violating germ layer assignment. Trajectory inference showed a sudden acquisition of a non-mesodermal identity in Brm-/- cells. Mechanistically, the loss of Brm prevented de novo accessibility of primed cardiac enhancers while increasing the expression of neurogenic factor POU3F1, preventing the binding of the neural suppressor REST and shifting the composition of BRG1 complexes. The identity switch caused by the Brm mutation was overcome by increasing BMP4 levels during mesoderm induction. Mathematical modelling supports these observations and demonstrates that Brm deletion affects cell fate trajectory by modifying saddle-node bifurcations2. In the mouse embryo, Brm deletion exacerbated mesoderm-deleted Brg1-mutant phenotypes, severely compromising cardiogenesis, and reveals an in vivo role for Brm. Our results show that Brm is a compensable safeguard of the fidelity of mesoderm chromatin states, and support a model in which developmental canalization is not a rigid irreversible path, but a highly plastic trajectory.


Asunto(s)
Diferenciación Celular , Linaje de la Célula , Mesodermo/citología , Mesodermo/metabolismo , Miocitos Cardíacos/citología , Factores de Transcripción/metabolismo , Animales , Proteína Morfogenética Ósea 4/metabolismo , Cromatina/genética , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , ADN Helicasas/metabolismo , Embrión de Mamíferos , Epigénesis Genética , Femenino , Regulación de la Expresión Génica , Masculino , Ratones , Miocardio/metabolismo , Neurogénesis , Neuronas/citología , Neuronas/metabolismo , Proteínas Nucleares/metabolismo , Factor 6 de Transcripción de Unión a Octámeros/metabolismo , Fenotipo , Proteínas Represoras/metabolismo , Células Madre/citología , Factores de Tiempo , Factores de Transcripción/deficiencia , Factores de Transcripción/genética
14.
Development ; 151(6)2024 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-38411343

RESUMEN

In the nascent mesoderm, TBXT expression must be precisely regulated to ensure that cells exit the primitive streak and pattern the anterior-posterior axis, but how varying dosage informs morphogenesis is not well understood. In this study, we define the transcriptional consequences of TBXT dosage reduction during early human gastrulation using human induced pluripotent stem cell models of gastrulation and mesoderm differentiation. Multi-omic single-nucleus RNA and single-nucleus ATAC sequencing of 2D gastruloids comprising wild-type, TBXT heterozygous or TBXT null human induced pluripotent stem cells reveal that varying TBXT dosage does not compromise the ability of a cell to differentiate into nascent mesoderm, but instead directly influences the temporal progression of the epithelial-to-mesenchymal transition with wild type transitioning first, followed by TBXT heterozygous and then TBXT null. By differentiating cells into nascent mesoderm in a monolayer format, we further illustrate that TBXT dosage directly impacts the persistence of junctional proteins and cell-cell adhesions. These results demonstrate that epithelial-to-mesenchymal transition progression can be decoupled from the acquisition of mesodermal identity in the early gastrula and shed light on the mechanisms underlying human embryogenesis.


Asunto(s)
Células Madre Pluripotentes Inducidas , Humanos , Mesodermo/metabolismo , Gástrula/metabolismo , Gastrulación/genética , Diferenciación Celular/genética
15.
Cell ; 151(1): 206-20, 2012 Sep 28.
Artículo en Inglés | MEDLINE | ID: mdl-22981692

RESUMEN

Heart development is exquisitely sensitive to the precise temporal regulation of thousands of genes that govern developmental decisions during differentiation. However, we currently lack a detailed understanding of how chromatin and gene expression patterns are coordinated during developmental transitions in the cardiac lineage. Here, we interrogated the transcriptome and several histone modifications across the genome during defined stages of cardiac differentiation. We find distinct chromatin patterns that are coordinated with stage-specific expression of functionally related genes, including many human disease-associated genes. Moreover, we discover a novel preactivation chromatin pattern at the promoters of genes associated with heart development and cardiac function. We further identify stage-specific distal enhancer elements and find enriched DNA binding motifs within these regions that predict sets of transcription factors that orchestrate cardiac differentiation. Together, these findings form a basis for understanding developmentally regulated chromatin transitions during lineage commitment and the molecular etiology of congenital heart disease.


Asunto(s)
Epigénesis Genética , Redes Reguladoras de Genes , Miocardio/citología , Animales , Diferenciación Celular , Cromatina/metabolismo , Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos , Corazón/embriología , Humanos , Ratones , Factores de Transcripción/metabolismo , Transcriptoma
16.
Mol Cell ; 76(3): 412-422.e5, 2019 11 07.
Artículo en Inglés | MEDLINE | ID: mdl-31522988

RESUMEN

The function of the CCCTC-binding factor (CTCF) in the organization of the genome has become an important area of investigation, but the mechanisms by which CTCF dynamically contributes to genome organization are not clear. We previously discovered that CTCF binds to large numbers of endogenous RNAs, promoting its self-association. In this regard, we now report two independent features that disrupt CTCF association with chromatin: inhibition of transcription and disruption of CTCF-RNA interactions through mutations of 2 of its 11 zinc fingers that are not required for CTCF binding to its cognate DNA site: zinc finger 1 (ZF1) or zinc finger 10 (ZF10). These mutations alter gene expression profiles as CTCF mutants lose their ability to form chromatin loops and thus the ability to insulate chromatin domains and to mediate CTCF long-range genomic interactions. Our results point to the importance of CTCF-mediated RNA interactions as a structural component of genome organization.


Asunto(s)
Factor de Unión a CCCTC/metabolismo , Cromatina/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , ARN/metabolismo , Animales , Sitios de Unión , Factor de Unión a CCCTC/química , Factor de Unión a CCCTC/genética , Línea Celular , Cromatina/química , Cromatina/genética , Ratones , Mutación , Conformación de Ácido Nucleico , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , ARN/química , ARN/genética , Relación Estructura-Actividad , Transcripción Genética , Dedos de Zinc
17.
Development ; 150(9)2023 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-36994838

RESUMEN

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.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Epigenómica , Miocitos Cardíacos , Animales , Ratones , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/fisiología , Regulación del Desarrollo de la Expresión Génica , Mesodermo/metabolismo , Miocitos Cardíacos/metabolismo , Factores de Transcripción/metabolismo
18.
Cell ; 142(3): 375-86, 2010 Aug 06.
Artículo en Inglés | MEDLINE | ID: mdl-20691899

RESUMEN

The reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) raises the possibility that a somatic cell could be reprogrammed to an alternative differentiated fate without first becoming a stem/progenitor cell. A large pool of fibroblasts exists in the postnatal heart, yet no single "master regulator" of direct cardiac reprogramming has been identified. Here, we report that a combination of three developmental transcription factors (i.e., Gata4, Mef2c, and Tbx5) rapidly and efficiently reprogrammed postnatal cardiac or dermal fibroblasts directly into differentiated cardiomyocyte-like cells. Induced cardiomyocytes expressed cardiac-specific markers, had a global gene expression profile similar to cardiomyocytes, and contracted spontaneously. Fibroblasts transplanted into mouse hearts one day after transduction of the three factors also differentiated into cardiomyocyte-like cells. We believe these findings demonstrate that functional cardiomyocytes can be directly reprogrammed from differentiated somatic cells by defined factors. Reprogramming of endogenous or explanted fibroblasts might provide a source of cardiomyocytes for regenerative approaches.


Asunto(s)
Diferenciación Celular , Fibroblastos/citología , Miocardio/citología , Miocitos Cardíacos/citología , Animales , Separación Celular , Fibroblastos/metabolismo , Factor de Transcripción GATA4/metabolismo , Perfilación de la Expresión Génica , Factores de Transcripción MEF2 , Ratones , Contracción Muscular , Miocitos Cardíacos/metabolismo , Factores Reguladores Miogénicos/metabolismo , Proteínas de Dominio T Box/metabolismo
19.
Circulation ; 146(10): 770-787, 2022 09 06.
Artículo en Inglés | MEDLINE | ID: mdl-35938400

RESUMEN

BACKGROUND: GATA4 (GATA-binding protein 4), a zinc finger-containing, DNA-binding transcription factor, is essential for normal cardiac development and homeostasis in mice and humans, and mutations in this gene have been reported in human heart defects. Defects in alternative splicing are associated with many heart diseases, yet relatively little is known about how cell type- or cell state-specific alternative splicing is achieved in the heart. Here, we show that GATA4 regulates cell type-specific splicing through direct interaction with RNA and the spliceosome in human induced pluripotent stem cell-derived cardiac progenitors. METHODS: We leveraged a combination of unbiased approaches including affinity purification of GATA4 and mass spectrometry, enhanced cross-linking with immunoprecipitation, electrophoretic mobility shift assays, in vitro splicing assays, and unbiased transcriptomic analysis to uncover GATA4's novel function as a splicing regulator in human induced pluripotent stem cell-derived cardiac progenitors. RESULTS: We found that GATA4 interacts with many members of the spliceosome complex in human induced pluripotent stem cell-derived cardiac progenitors. Enhanced cross-linking with immunoprecipitation demonstrated that GATA4 also directly binds to a large number of mRNAs through defined RNA motifs in a sequence-specific manner. In vitro splicing assays indicated that GATA4 regulates alternative splicing through direct RNA binding, resulting in functionally distinct protein products. Correspondingly, knockdown of GATA4 in human induced pluripotent stem cell-derived cardiac progenitors resulted in differential alternative splicing of genes involved in cytoskeleton organization and calcium ion import, with functional consequences associated with the protein isoforms. CONCLUSIONS: This study shows that in addition to its well described transcriptional function, GATA4 interacts with members of the spliceosome complex and regulates cell type-specific alternative splicing via sequence-specific interactions with RNA. Several genes that have splicing regulated by GATA4 have functional consequences and many are associated with dilated cardiomyopathy, suggesting a novel role for GATA4 in achieving the necessary cardiac proteome in normal and stress-responsive conditions.


Asunto(s)
Factor de Transcripción GATA4 , Células Madre Pluripotentes Inducidas , Empalme Alternativo , Animales , Factor de Transcripción GATA4/genética , Factor de Transcripción GATA4/metabolismo , Corazón , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Miocitos Cardíacos/metabolismo , ARN/genética , ARN/metabolismo
20.
Development ; 147(21)2020 10 21.
Artículo en Inglés | MEDLINE | ID: mdl-33087326

RESUMEN

The heart is an essential organ with a fascinating developmental biology. It is also one of the organs that is most often affected in human disease, either during development or in postnatal life. Over the last few decades, insights into the development of the heart have led to fundamental new concepts in gene regulation, but also to genetic and mechanistic insights into congenital heart defects. In more recent years, the lessons learned from studying heart development have been applied to interrogating regeneration of the diseased heart, exemplifying the importance of understanding the mechanistic underpinnings that lead to the development of an organ.


Asunto(s)
Cardiopatías Congénitas/embriología , Corazón/embriología , Animales , Cromatina/metabolismo , Corazón/fisiopatología , Cardiopatías Congénitas/fisiopatología , Humanos , Regeneración , Factores de Transcripción/metabolismo
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