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2.
Bioinformatics ; 40(2)2024 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-38366652

RESUMO

SUMMARY: Spatial transcriptomics has changed our way to study tissue structure and cellular organization. However, there are still limitations in its resolution, and most available platforms do not reach a single cell resolution. To address this issue, we introduce SpatialDDLS, a fast neural network-based algorithm for cell type deconvolution of spatial transcriptomics data. SpatialDDLS leverages single-cell RNA sequencing data to simulate mixed transcriptional profiles with predefined cellular composition, which are subsequently used to train a fully connected neural network to uncover cell type diversity within each spot. By comparing it with two state-of-the-art spatial deconvolution methods, we demonstrate that SpatialDDLS is an accurate and fast alternative to the available state-of-the art tools. AVAILABILITY AND IMPLEMENTATION: The R package SpatialDDLS is available via CRAN-The Comprehensive R Archive Network: https://CRAN.R-project.org/package=SpatialDDLS. A detailed manual of the main functionalities implemented in the package can be found at https://diegommcc.github.io/SpatialDDLS.


Assuntos
Algoritmos , Software , Perfilação da Expressão Gênica , Redes Neurais de Computação
3.
Nat Cardiovasc Res ; 2: 2023530-549, 2023 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-37745941

RESUMO

The Notch pathway is a major regulator of endothelial transcriptional specification. Targeting the Notch receptors or Delta-like ligand 4 (Dll4) dysregulates angiogenesis. Here, by analyzing single and compound genetic mutants for all Notch signaling members, we find significant differences in the way ligands and receptors regulate liver vascular homeostasis. Loss of Notch receptors caused endothelial hypermitogenic cell-cycle arrest and senescence. Conversely, Dll4 loss triggered a strong Myc-driven transcriptional switch inducing endothelial proliferation and the tip-cell state. Myc loss suppressed the induction of angiogenesis in the absence of Dll4, without preventing the vascular enlargement and organ pathology. Similarly, inhibition of other pro-angiogenic pathways, including MAPK/ERK and mTOR, had no effect on the vascular expansion induced by Dll4 loss; however, anti-VEGFA treatment prevented it without fully suppressing the transcriptional and metabolic programs. This study shows incongruence between single-cell transcriptional states, vascular phenotypes and related pathophysiology. Our findings also suggest that the vascular structure abnormalization, rather than neoplasms, causes the reported anti-Dll4 antibody toxicity.

5.
Nat Commun ; 14(1): 12, 2023 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-36596811

RESUMO

Here we explored the role of interleukin-1ß (IL-1ß) repressor cytokine, IL-1 receptor antagonist (IL-1rn), in both healthy and abnormal hematopoiesis. Low IL-1RN is frequent in acute myeloid leukemia (AML) patients and represents a prognostic marker of reduced survival. Treatments with IL-1RN and the IL-1ß monoclonal antibody canakinumab reduce the expansion of leukemic cells, including CD34+ progenitors, in AML xenografts. In vivo deletion of IL-1rn induces hematopoietic stem cell (HSC) differentiation into the myeloid lineage and hampers B cell development via transcriptional activation of myeloid differentiation pathways dependent on NFκB. Low IL-1rn is present in an experimental model of pre-leukemic myelopoiesis, and IL-1rn deletion promotes myeloproliferation, which relies on the bone marrow hematopoietic and stromal compartments. Conversely, IL-1rn protects against pre-leukemic myelopoiesis. Our data reveal that HSC differentiation is controlled by balanced IL-1ß/IL-1rn levels under steady-state, and that loss of repression of IL-1ß signaling may underlie pre-leukemic lesion and AML progression.


Assuntos
Leucemia Mieloide Aguda , Receptores de Interleucina-1 , Humanos , Receptores de Interleucina-1/genética , Medula Óssea , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Proliferação de Células , Antígenos CD34
6.
Circulation ; 147(1): 47-65, 2023 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-36325906

RESUMO

BACKGROUND: The complex genetics underlying human cardiac disease is evidenced by its heterogenous manifestation, multigenic basis, and sporadic occurrence. These features have hampered disease modeling and mechanistic understanding. Here, we show that 2 structural cardiac diseases, left ventricular noncompaction (LVNC) and bicuspid aortic valve, can be caused by a set of inherited heterozygous gene mutations affecting the NOTCH ligand regulator MIB1 (MINDBOMB1) and cosegregating genes. METHODS: We used CRISPR-Cas9 gene editing to generate mice harboring a nonsense or a missense MIB1 mutation that are both found in LVNC families. We also generated mice separately carrying these MIB1 mutations plus 5 additional cosegregating variants in the ASXL3, APCDD1, TMX3, CEP192, and BCL7A genes identified in these LVNC families by whole exome sequencing. Histological, developmental, and functional analyses of these mouse models were carried out by echocardiography and cardiac magnetic resonance imaging, together with gene expression profiling by RNA sequencing of both selected engineered mouse models and human induced pluripotent stem cell-derived cardiomyocytes. Potential biochemical interactions were assayed in vitro by coimmunoprecipitation and Western blot. RESULTS: Mice homozygous for the MIB1 nonsense mutation did not survive, and the mutation caused LVNC only in heteroallelic combination with a conditional allele inactivated in the myocardium. The heterozygous MIB1 missense allele leads to bicuspid aortic valve in a NOTCH-sensitized genetic background. These data suggest that development of LVNC is influenced by genetic modifiers present in affected families, whereas valve defects are highly sensitive to NOTCH haploinsufficiency. Whole exome sequencing of LVNC families revealed single-nucleotide gene variants of ASXL3, APCDD1, TMX3, CEP192, and BCL7A cosegregating with the MIB1 mutations and LVNC. In experiments with mice harboring the orthologous variants on the corresponding Mib1 backgrounds, triple heterozygous Mib1 Apcdd1 Asxl3 mice showed LVNC, whereas quadruple heterozygous Mib1 Cep192 Tmx3;Bcl7a mice developed bicuspid aortic valve and other valve-associated defects. Biochemical analysis suggested interactions between CEP192, BCL7A, and NOTCH. Gene expression profiling of mutant mouse hearts and human induced pluripotent stem cell-derived cardiomyocytes revealed increased cardiomyocyte proliferation and defective morphological and metabolic maturation. CONCLUSIONS: These findings reveal a shared genetic substrate underlying LVNC and bicuspid aortic valve in which MIB1-NOTCH variants plays a crucial role in heterozygous combination with cosegregating genetic modifiers.


Assuntos
Doença da Válvula Aórtica Bicúspide , Cardiomiopatias , Cardiopatias Congênitas , Células-Tronco Pluripotentes Induzidas , Humanos , Animais , Camundongos , Cardiopatias Congênitas/complicações , Cardiomiopatias/etiologia , Miócitos Cardíacos , Valva Aórtica/diagnóstico por imagem , Fatores de Transcrição , Proteínas Cromossômicas não Histona
7.
Sci Adv ; 8(28): eabo3583, 2022 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-35857513

RESUMO

Pluripotent cells are a transient population of the mammalian embryo dependent on transcription factors, such as OCT4 and NANOG, which maintain pluripotency while suppressing lineage specification. However, these factors are also expressed during early phases of differentiation, and their role in the transition from pluripotency to lineage specification is largely unknown. We found that pluripotency factors play a dual role in regulating key lineage specifiers, initially repressing their expression and later being required for their proper activation. We show that Oct4 is necessary for activation of HoxB genes during differentiation of embryonic stem cells and in the embryo. In addition, we show that the HoxB cluster is coordinately regulated by OCT4 binding sites located at the 3' end of the cluster. Our results show that core pluripotency factors are not limited to maintaining the precommitted epiblast but are also necessary for the proper deployment of subsequent developmental programs.

9.
Circulation ; 145(14): 1084-1101, 2022 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-35236094

RESUMO

BACKGROUND: In most eukaryotic cells, the mitochondrial DNA (mtDNA) is transmitted uniparentally and present in multiple copies derived from the clonal expansion of maternally inherited mtDNA. All copies are therefore near-identical, or homoplasmic. The presence of >1 mtDNA variant in the same cytoplasm can arise naturally or result from new medical technologies aimed at preventing mitochondrial genetic diseases and improving fertility. The latter is called divergent nonpathologic mtDNA heteroplasmy (DNPH). We hypothesized that DNPH is maladaptive and usually prevented by the cell. METHODS: We engineered and characterized DNPH mice throughout their lifespan using transcriptomic, metabolomic, biochemical, physiologic, and phenotyping techniques. We focused on in vivo imaging techniques for noninvasive assessment of cardiac and pulmonary energy metabolism. RESULTS: We show that DNPH impairs mitochondrial function, with profound consequences in critical tissues that cannot resolve heteroplasmy, particularly cardiac and skeletal muscle. Progressive metabolic stress in these tissues leads to severe pathology in adulthood, including pulmonary hypertension and heart failure, skeletal muscle wasting, frailty, and premature death. Symptom severity is strongly modulated by the nuclear context. CONCLUSIONS: Medical interventions that may generate DNPH should address potential incompatibilities between donor and recipient mtDNA.


Assuntos
Fragilidade , Cardiopatias , Hipertensão Pulmonar , Adulto , Animais , DNA Mitocondrial/genética , Fragilidade/patologia , Cardiopatias/patologia , Heteroplasmia , Humanos , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Camundongos , Mitocôndrias/genética
10.
Development ; 149(6)2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35312773

RESUMO

During development, the heart grows by addition of progenitor cells to the poles of the primordial heart tube. In the zebrafish, Wilms tumor 1 transcription factor a (wt1a) and b (wt1b) genes are expressed in the pericardium, at the venous pole of the heart. From this pericardial layer, the proepicardium emerges. Proepicardial cells are subsequently transferred to the myocardial surface and form the epicardium, covering the myocardium. We found that while wt1a and wt1b expression is maintained in proepicardial cells, it is downregulated in pericardial cells that contributes cardiomyocytes to the developing heart. Sustained wt1b expression in cardiomyocytes reduced chromatin accessibility of specific genomic loci. Strikingly, a subset of wt1a- and wt1b-expressing cardiomyocytes changed their cell-adhesion properties, delaminated from the myocardium and upregulated epicardial gene expression. Thus, wt1a and wt1b act as a break for cardiomyocyte differentiation, and ectopic wt1a and wt1b expression in cardiomyocytes can lead to their transdifferentiation into epicardial-like cells.


Assuntos
Miócitos Cardíacos , Peixe-Zebra , Animais , Regulação da Expressão Gênica no Desenvolvimento , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Pericárdio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas WT1/genética , Proteínas WT1/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
11.
Biol Open ; 8(11)2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31791948

RESUMO

Pluripotency is regulated by a network of transcription factors that maintain early embryonic cells in an undifferentiated state while allowing them to proliferate. NANOG is a critical factor for maintaining pluripotency and its role in primordial germ cell differentiation has been well described. However, Nanog is expressed during gastrulation across all the posterior epiblast, and only later in development is its expression restricted to primordial germ cells. In this work, we unveiled a previously unknown mechanism by which Nanog specifically represses genes involved in anterior epiblast lineage. Analysis of transcriptional data from both embryonic stem cells and gastrulating mouse embryos revealed Pou3f1 expression to be negatively correlated with that of Nanog during the early stages of differentiation. We have functionally demonstrated Pou3f1 to be a direct target of NANOG by using a dual transgene system for the controlled expression of Nanog Use of Nanog null ES cells further demonstrated a role for Nanog in repressing a subset of anterior neural genes. Deletion of a NANOG binding site (BS) located nine kilobases downstream of the transcription start site of Pou3f1 revealed this BS to have a specific role in the regionalization of the expression of this gene in the embryo. Our results indicate an active role of Nanog inhibiting neural regulatory networks by repressing Pou3f1 at the onset of gastrulation.This article has an associated First Person interview with the joint first authors of the paper.

12.
Front Genet ; 10: 978, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31708961

RESUMO

The development of single cell transcriptome sequencing has allowed researchers the possibility to dig inside the role of the individual cell types in a plethora of disease scenarios. It also expands to the whole transcriptome what before was only possible for a few tenths of antibodies in cell population analysis. More importantly, it allows resolving the permanent question of whether the changes observed in a particular bulk experiment are a consequence of changes in cell type proportions or an aberrant behavior of a particular cell type. However, single cell experiments are still complex to perform and expensive to sequence making bulk RNA-Seq experiments yet more common. scRNA-Seq data is proving highly relevant information for the characterization of the immune cell repertoire in different diseases ranging from cancer to atherosclerosis. In particular, as scRNA-Seq becomes more widely used, new types of immune cell populations emerge and their role in the genesis and evolution of the disease opens new avenues for personalized immune therapies. Immunotherapy have already proven successful in a variety of tumors such as breast, colon and melanoma and its value in other types of disease is being currently explored. From a statistical perspective, single-cell data are particularly interesting due to its high dimensionality, overcoming the limitations of the "skinny matrix" that traditional bulk RNA-Seq experiments yield. With the technological advances that enable sequencing hundreds of thousands of cells, scRNA-Seq data have become especially suitable for the application of Machine Learning algorithms such as Deep Learning (DL). We present here a DL based method to enumerate and quantify the immune infiltration in colorectal and breast cancer bulk RNA-Seq samples starting from scRNA-Seq. Our method makes use of a Deep Neural Network (DNN) model that allows quantification not only of lymphocytes as a general population but also of specific CD8+, CD4Tmem, CD4Th and CD4Tregs subpopulations, as well as B-cells and Stromal content. Moreover, the signatures are built from scRNA-Seq data from the tumor, preserving the specific characteristics of the tumor microenvironment as opposite to other approaches in which cells were isolated from blood. Our method was applied to synthetic bulk RNA-Seq and to samples from the TCGA project yielding very accurate results in terms of quantification and survival prediction.

13.
Sci Rep ; 9(1): 4647, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30874584

RESUMO

Adult cardiac progenitor/stem cells (CPC/CSC) are multipotent resident populations involved in cardiac homeostasis and heart repair. Assisted by complementary RNAseq analysis, we defined the fraction of the CPC proteome associable with specific functions by comparison with human bone marrow mesenchymal stem cells (MSC), the reference population for cell therapy, and human dermal fibroblasts (HDF), as a distant reference. Label-free proteomic analysis identified 526 proteins expressed differentially in CPC. iTRAQ analysis confirmed differential expression of a substantial proportion of those proteins in CPC relative to MSC, and systems biology analysis defined a clear overrepresentation of several categories related to enhanced angiogenic potential. The CPC plasma membrane compartment comprised 1,595 proteins, including a minimal signature of 167 proteins preferentially or exclusively expressed by CPC. CDH5 (VE-cadherin),  OX2G (OX-2 membrane glycoprotein; CD200), GPR4 (G protein-coupled receptor 4), CACNG7 (calcium voltage-gated channel auxiliary subunit gamma 7) and F11R (F11 receptor; junctional adhesion molecule A; JAM-A; CD321) were selected for validation. Their differential expression was confirmed both in expanded CPC batches and in early stages of isolation, particularly when compared against cardiac fibroblasts. Among them, GPR4 demonstrated the highest discrimination capacity between all cell lineages analyzed.


Assuntos
Diferenciação Celular/fisiologia , Coração/crescimento & desenvolvimento , Miócitos Cardíacos/metabolismo , Adulto , Antígenos CD , Biomarcadores , Caderinas , Canais de Cálcio , Moléculas de Adesão Celular , Perfilação da Expressão Gênica/métodos , Humanos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Multipotentes/metabolismo , Proteoma/genética , Proteômica/métodos , Receptores de Superfície Celular , Receptores Acoplados a Proteínas G , Transcriptoma/genética
14.
Front Genet ; 10: 1373, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-32117421

RESUMO

[This corrects the article DOI: 10.3389/fgene.2019.00978.].

15.
EMBO Rep ; 19(10)2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30126925

RESUMO

The Myc family of oncogenic transcription factors regulates myriad cellular functions. Myc proteins contain a basic region/helix-loop-helix/leucine zipper domain that mediates DNA binding and heterodimerization with its partner Max. Among the Myc proteins, c-Myc is the most widely expressed and relevant in primary B lymphocytes. There is evidence suggesting that c-Myc can perform some of its functions in the absence of Max in different cellular contexts. However, the functional in vivo interplay between c-Myc and Max during B lymphocyte differentiation is not well understood. Using in vivo and ex vivo models, we show that while c-Myc requires Max in primary B lymphocytes, several key biological processes, such as cell differentiation and DNA replication, can initially progress without the formation of c-Myc/Max heterodimers. We also describe that B lymphocytes lacking Myc, Max, or both show upregulation of signaling pathways associated with the B-cell receptor. These data suggest that c-Myc/Max heterodimers are not essential for the initiation of a subset of important biological processes in B lymphocytes, but are required for fine-tuning the initial response after activation.


Assuntos
Linfócitos B/química , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Diferenciação Celular/genética , Proteínas Proto-Oncogênicas c-myc/genética , Sequência de Aminoácidos/genética , Animais , Linfócitos B/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/química , Replicação do DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Dimerização , Sequências Hélice-Alça-Hélice/genética , Humanos , Zíper de Leucina/genética , Camundongos , Ligação Proteica/genética , Proteínas Proto-Oncogênicas c-myc/química , Ativação Transcricional/genética
16.
Sci Transl Med ; 10(434)2018 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-29593106

RESUMO

Heart failure (HF) is a major health and economic burden in developed countries. It has been proposed that the pathogenesis of HF may involve the action of mitochondria. We evaluate three different mouse models of HF: tachycardiomyopathy, HF with preserved left ventricular (LV) ejection fraction (LVEF), and LV myocardial ischemia and hypertrophy. Regardless of whether LVEF is preserved, our results indicate that the three models share common features: an increase in mitochondrial reactive oxygen species followed by ultrastructural alterations in the mitochondrial cristae and loss of mitochondrial integrity that lead to cardiomyocyte death. We show that the ablation of the mitochondrial protease OMA1 averts cardiomyocyte death in all three murine HF models, and thus loss of OMA1 plays a direct role in cardiomyocyte protection. This finding identifies OMA1 as a potential target for preventing the progression of myocardial damage in HF associated with a variety of etiologies.


Assuntos
Insuficiência Cardíaca/metabolismo , Metaloproteases/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Insuficiência Cardíaca/genética , Masculino , Metaloproteases/genética , Camundongos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Miócitos Cardíacos/metabolismo , Espécies Reativas de Oxigênio/metabolismo
17.
J Exp Med ; 215(3): 761-771, 2018 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-29374026

RESUMO

Activation-induced deaminase (AID) initiates antibody diversification in germinal center (GC) B cells through the deamination of cytosines on immunoglobulin genes. AID can also target other regions in the genome, triggering mutations or chromosome translocations, with major implications for oncogenic transformation. However, understanding the specificity of AID has proved extremely challenging. We have sequenced at very high depth >1,500 genomic regions from GC B cells and identified 275 genes targeted by AID, including 30 of the previously known 35 AID targets. We have also identified the most highly mutated hotspot for AID activity described to date. Furthermore, integrative analysis of the molecular features of mutated genes coupled to machine learning has produced a powerful predictive tool for AID targets. We also have found that base excision repair and mismatch repair back up each other to faithfully repair AID-induced lesions. Finally, our data establish a novel link between AID mutagenic activity and lymphomagenesis.


Assuntos
Citidina Desaminase/genética , Hipermutação Somática de Imunoglobulina/genética , Animais , Sequência de Bases , Dano ao DNA/genética , Reparo de Erro de Pareamento de DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala , Linfoma/genética , Linfoma/patologia , Camundongos , Mutação/genética
18.
Sci Rep ; 7(1): 12490, 2017 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-28970523

RESUMO

Studies in recent years have established that the principal effects in cardiac cell therapy are associated with paracrine/autocrine factors. We combined several complementary techniques to define human cardiac progenitor cell (CPC) secretome constituted by 914 proteins/genes; 51% of these are associated with the exosomal compartment. To define the set of proteins specifically or highly differentially secreted by CPC, we compared human mesenchymal stem cells and dermal fibroblasts; the study defined a group of growth factors, cytokines and chemokines expressed at high to medium levels by CPC. Among them, IL-1, GROa (CXCL1), CXCL6 (GCP2) and IL-8 are examples whose expression was confirmed by most techniques used. ELISA showed that CXCL6 is significantly overexpressed in CPC conditioned medium (CM) (18- to 26-fold) and western blot confirmed expression of its receptors CXCR1 and CXCR2. Addition of anti-CXCL6 completely abolished migration in CPC-CM compared with anti-CXCR2, which promoted partial inhibition, and anti-CXCR1, which was inefficient. Anti-CXCL6 also significantly inhibited CPC CM angiogenic activity. In vivo evaluation also supported a relevant role for angiogenesis. Altogether, these results suggest a notable angiogenic potential in CPC-CM and identify CXCL6 as an important paracrine factor for CPC that signals mainly through CXCR2.


Assuntos
Quimiocina CXCL6/genética , Miocárdio/metabolismo , Neovascularização Fisiológica/genética , Comunicação Parácrina/genética , Proteoma/genética , Receptores de Interleucina-8B/metabolismo , Células-Tronco/metabolismo , Animais , Anticorpos Neutralizantes/farmacologia , Movimento Celular , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Quimiocina CXCL6/antagonistas & inibidores , Quimiocina CXCL6/metabolismo , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Interleucina-1/genética , Interleucina-1/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/citologia , Proteoma/metabolismo , Receptores de Interleucina-8A/antagonistas & inibidores , Receptores de Interleucina-8A/genética , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/antagonistas & inibidores , Receptores de Interleucina-8B/genética , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos
19.
PLoS Genet ; 13(8): e1006985, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28846746

RESUMO

Cardiac progenitors are specified early in development and progressively differentiate and mature into fully functional cardiomyocytes. This process is controlled by an extensively studied transcriptional program. However, the regulatory events coordinating the progression of such program from development to maturation are largely unknown. Here, we show that the genome organizer CTCF is essential for cardiogenesis and that it mediates genomic interactions to coordinate cardiomyocyte differentiation and maturation in the developing heart. Inactivation of Ctcf in cardiac progenitor cells and their derivatives in vivo during development caused severe cardiac defects and death at embryonic day 12.5. Genome wide expression analysis in Ctcf mutant hearts revealed that genes controlling mitochondrial function and protein production, required for cardiomyocyte maturation, were upregulated. However, mitochondria from mutant cardiomyocytes do not mature properly. In contrast, multiple development regulatory genes near predicted heart enhancers, including genes in the IrxA cluster, were downregulated in Ctcf mutants, suggesting that CTCF promotes cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Accordingly, loss of CTCF disrupts gene expression and chromatin interactions as shown by chromatin conformation capture followed by deep sequencing. Furthermore, CRISPR-mediated deletion of an intergenic CTCF site within the IrxA cluster alters gene expression in the developing heart. Thus, CTCF mediates local regulatory interactions to coordinate transcriptional programs controlling transitions in morphology and function during heart development.


Assuntos
Cromatina/genética , Desenvolvimento Embrionário/genética , Ventrículos do Coração/crescimento & desenvolvimento , Coração/crescimento & desenvolvimento , Proteínas Repressoras/genética , Animais , Fator de Ligação a CCCTC , Diferenciação Celular/genética , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Ventrículos do Coração/embriologia , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Organogênese/genética , Regiões Promotoras Genéticas , Ligação Proteica , Ativação Transcricional/genética
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