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1.
Cell ; 175(5): 1228-1243.e20, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30392959

RESUMO

Genetic drivers of cancer can be dysregulated through epigenetic modifications of DNA. Although the critical role of DNA 5-methylcytosine (5mC) in the regulation of transcription is recognized, the functions of other non-canonical DNA modifications remain obscure. Here, we report the identification of novel N6-methyladenine (N6-mA) DNA modifications in human tissues and implicate this epigenetic mark in human disease, specifically the highly malignant brain cancer glioblastoma. Glioblastoma markedly upregulated N6-mA levels, which co-localized with heterochromatic histone modifications, predominantly H3K9me3. N6-mA levels were dynamically regulated by the DNA demethylase ALKBH1, depletion of which led to transcriptional silencing of oncogenic pathways through decreasing chromatin accessibility. Targeting the N6-mA regulator ALKBH1 in patient-derived human glioblastoma models inhibited tumor cell proliferation and extended the survival of tumor-bearing mice, supporting this novel DNA modification as a potential therapeutic target for glioblastoma. Collectively, our results uncover a novel epigenetic node in cancer through the DNA modification N6-mA.


Assuntos
Adenina/análogos & derivados , Neoplasias Encefálicas/patologia , Metilação de DNA , Glioblastoma/patologia , Adenina/análise , Adenina/química , Adulto , Idoso , Homólogo AlkB 1 da Histona H2a Dioxigenase/antagonistas & inibidores , Homólogo AlkB 1 da Histona H2a Dioxigenase/genética , Homólogo AlkB 1 da Histona H2a Dioxigenase/metabolismo , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidade , Hipóxia Celular , Criança , Epigenômica , Feminino , Glioblastoma/metabolismo , Glioblastoma/mortalidade , Heterocromatina/metabolismo , Histonas/metabolismo , Humanos , Estimativa de Kaplan-Meier , Masculino , Camundongos , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína Supressora de Tumor p53/metabolismo
2.
Nature ; 583(7817): 625-630, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32669713

RESUMO

The recent discovery of N6-methyladenine (N6-mA) in mammalian genomes suggests that it may serve as an epigenetic regulatory mechanism1. However, the biological role of N6-mA and the molecular pathways that exert its function remain unclear. Here we show that N6-mA has a key role in changing the epigenetic landscape during cell fate transitions in early development. We found that N6-mA is upregulated during the development of mouse trophoblast stem cells, specifically at regions of stress-induced DNA double helix destabilization (SIDD)2-4. Regions of SIDD are conducive to topological stress-induced unpairing of the double helix and have critical roles in organizing large-scale chromatin structures3,5,6. We show that the presence of N6-mA reduces the in vitro interactions by more than 500-fold between SIDD and SATB1, a crucial chromatin organizer that interacts with SIDD regions. Deposition of N6-mA also antagonizes SATB1 function in vivo by preventing its binding to chromatin. Concordantly, N6-mA functions at the boundaries between euchromatin and heterochromatin to restrict the spread of euchromatin. Repression of SIDD-SATB1 interactions mediated by N6-mA is essential for gene regulation during trophoblast development in cell culture models and in vivo. Overall, our findings demonstrate an unexpected molecular mechanism for N6-mA function via SATB1, and reveal connections between DNA modification, DNA secondary structures and large chromatin domains in early embryonic development.


Assuntos
Adenina/análogos & derivados , DNA/química , DNA/metabolismo , Desenvolvimento Embrionário , Proteínas de Ligação à Região de Interação com a Matriz/antagonistas & inibidores , Adenina/metabolismo , Animais , Pareamento de Bases , Desenvolvimento Embrionário/genética , Eucromatina/genética , Eucromatina/metabolismo , Feminino , Humanos , Masculino , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Camundongos , Células-Tronco/citologia , Células-Tronco/metabolismo , Termodinâmica , Trofoblastos/citologia
3.
Proc Natl Acad Sci U S A ; 120(1): e2209062120, 2023 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-36577070

RESUMO

Hematopoietic stem and progenitor cells (HSPCs) are a heterogeneous group of cells with expansion, differentiation, and repopulation capacities. How HSPCs orchestrate the stemness state with diverse lineage differentiation at steady condition or acute stress remains largely unknown. Here, we show that zebrafish mutants that are deficient in an epigenetic regulator Atf7ip or Setdb1 methyltransferase undergo excessive myeloid differentiation with impaired HSPC expansion, manifesting a decline in T cells and erythroid lineage. We find that Atf7ip regulates hematopoiesis through Setdb1-mediated H3K9me3 modification and chromatin remodeling. During hematopoiesis, the interaction of Atf7ip and Setdb1 triggers H3K9me3 depositions in hematopoietic regulatory genes including cebpß and cdkn1a, preventing HSPCs from loss of expansion and premature differentiation into myeloid lineage. Concomitantly, loss of Atf7ip or Setdb1 derepresses retrotransposons that instigate the viral sensor Mda5/Rig-I like receptor (RLR) signaling, leading to stress-driven myelopoiesis and inflammation. We find that ATF7IP or SETDB1 depletion represses human leukemic cell growth and induces myeloid differentiation with retrotransposon-triggered inflammation. These findings establish that Atf7ip/Setdb1-mediated H3K9me3 deposition constitutes a genome-wide checkpoint that impedes the myeloid potential and maintains HSPC stemness for diverse blood cell production, providing unique insights into potential intervention in hematological malignancy.


Assuntos
Células-Tronco Hematopoéticas , Histona-Lisina N-Metiltransferase , Peixe-Zebra , Animais , Humanos , Diferenciação Celular , Linhagem da Célula , Hematopoese , Células-Tronco Hematopoéticas/patologia , Histona-Lisina N-Metiltransferase/genética , Inflamação/patologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
4.
Development ; 147(18)2020 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-32988975

RESUMO

Teleost zebrafish and neonatal mammalian hearts exhibit the remarkable capacity to regenerate through dedifferentiation and proliferation of pre-existing cardiomyocytes (CMs). Although many mitogenic signals that stimulate zebrafish heart regeneration have been identified, transcriptional programs that restrain injury-induced CM renewal are incompletely understood. Here, we report that mutations in gridlock (grl; also known as hey2), encoding a Hairy-related basic helix-loop-helix transcriptional repressor, enhance CM proliferation and reduce fibrosis following damage. In contrast, myocardial grl induction blunts CM dedifferentiation and regenerative responses to heart injury. RNA sequencing analyses uncover Smyd2 lysine methyltransferase (KMT) as a key transcriptional target repressed by Grl. Reduction in Grl protein levels triggered by injury induces smyd2 expression at the wound myocardium, enhancing CM proliferation. We show that Smyd2 functions as a methyltransferase and modulates the Stat3 methylation and phosphorylation activity. Inhibition of the KMT activity of Smyd2 reduces phosphorylated Stat3 at cardiac wounds, suppressing the elevated CM proliferation in injured grl mutant hearts. Our findings establish an injury-specific transcriptional repression program in governing CM renewal during heart regeneration, providing a potential strategy whereby silencing Grl repression at local regions might empower regeneration capacity to the injured mammalian heart.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Coração/fisiologia , Lisina/genética , Metiltransferases/genética , Regeneração/genética , Transcrição Gênica/genética , Vertebrados/genética , Proteínas de Peixe-Zebra/genética , Animais , Animais Recém-Nascidos , Diferenciação Celular/genética , Proliferação de Células/genética , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Fosforilação/genética , Fator de Transcrição STAT3/genética , Transdução de Sinais/genética , Peixe-Zebra/genética
5.
J Cell Physiol ; 237(6): 2632-2643, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-34927727

RESUMO

Prostaglandin (PG) signaling regulates a wide variety of physiological and pathological processes, including body temperature, cardiovascular homeostasis, reproduction, and inflammation. Recent studies have revealed that PGs play pivotal roles in embryo development, ciliogenesis, and organ formation. Prostaglandin E2 (PGE2) and its receptor EP4 modulate ciliogenesis by increasing the anterograde intraflagellar transport. Many G-protein-coupled receptors (GPCRs) including EP4 are localized in cilia for modulating cAMP signaling under various conditions. During development, PGE2 signaling regulates embryogenesis, hepatocyte differentiation, hematopoiesis, and kidney formation. Prostaglandins are also essential for skeletal muscle repair. This review outlines recent advances in understanding the functions and mechanisms of prostaglandin signaling in ciliogenesis, embryo development, and organ formation.


Assuntos
Dinoprostona , Prostaglandinas , Cílios , Desenvolvimento Embrionário/genética , Transdução de Sinais
6.
Genome Res ; 2019 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-31831591

RESUMO

Genome editing by the well-established CRISPR/Cas9 technology has greatly facilitated our understanding of many biological processes. However, a complete whole-genome knockout for any species or model organism has rarely been achieved. Here, we performed a systematic knockout of all the genes (1333) on Chromosome 1 in zebrafish, successfully mutated 1029 genes, and generated 1039 germline-transmissible alleles corresponding to 636 genes. Meanwhile, by high-throughput bioinformatics analysis, we found that sequence features play pivotal roles in effective gRNA targeting at specific genes of interest, while the success rate of gene targeting positively correlates with GC content of the target sites. Moreover, we found that nearly one-fourth of all mutants are related to human diseases, and several representative CRISPR/Cas9-generated mutants are described here. Furthermore, we tried to identify the underlying mechanisms leading to distinct phenotypes between genetic mutants and antisense morpholino-mediated knockdown embryos. Altogether, this work has generated the first chromosome-wide collection of zebrafish genetic mutants by the CRISPR/Cas9 technology, which will serve as a valuable resource for the community, and our bioinformatics analysis also provides some useful guidance to design gene-specific gRNAs for successful gene editing.

7.
Nature ; 532(7599): 329-33, 2016 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-27027282

RESUMO

It has been widely accepted that 5-methylcytosine is the only form of DNA methylation in mammalian genomes. Here we identify N(6)-methyladenine as another form of DNA modification in mouse embryonic stem cells. Alkbh1 encodes a demethylase for N(6)-methyladenine. An increase of N(6)-methyladenine levels in Alkbh1-deficient cells leads to transcriptional silencing. N(6)-methyladenine deposition is inversely correlated with the evolutionary age of LINE-1 transposons; its deposition is strongly enriched at young (<1.5 million years old) but not old (>6 million years old) L1 elements. The deposition of N(6)-methyladenine correlates with epigenetic silencing of such LINE-1 transposons, together with their neighbouring enhancers and genes, thereby resisting the gene activation signals during embryonic stem cell differentiation. As young full-length LINE-1 transposons are strongly enriched on the X chromosome, genes located on the X chromosome are also silenced. Thus, N(6)-methyladenine developed a new role in epigenetic silencing in mammalian evolution distinct from its role in gene activation in other organisms. Our results demonstrate that N(6)-methyladenine constitutes a crucial component of the epigenetic regulation repertoire in mammalian genomes.


Assuntos
Adenina/análogos & derivados , Metilação de DNA , Epigênese Genética/genética , Células-Tronco Embrionárias Murinas/metabolismo , Adenina/metabolismo , Homólogo AlkB 1 da Histona H2a Dioxigenase , Animais , Diferenciação Celular/genética , Elementos de DNA Transponíveis/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/deficiência , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Elementos Facilitadores Genéticos/genética , Evolução Molecular , Inativação Gênica , Elementos Nucleotídeos Longos e Dispersos/genética , Mamíferos/genética , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Regulação para Cima/genética , Cromossomo X/genética , Cromossomo X/metabolismo
8.
Curr Cardiol Rep ; 24(10): 1425-1438, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35925512

RESUMO

PURPOSE OF REVIEW: Cardiovascular diseases are the leading cause of death worldwide, largely due to the limited regenerative capacity of the adult human heart. In contrast, teleost zebrafish hearts possess natural regeneration capacity by proliferation of pre-existing cardiomyocytes after injury. Hearts of mice can regenerate if injured in a few days after birth, which coincides with the transient capacity for cardiomyocyte proliferation. This review tends to elaborate the roles and mechanisms of Wnt/ß-catenin signaling in heart development and regeneration in mammals and non-mammalian vertebrates. RECENT FINDINGS: Studies in zebrafish, mice, and human embryonic stem cells demonstrate the binary effect for Wnt/ß-catenin signaling during heart development. Both Wnts and Wnt antagonists are induced in multiple cell types during cardiac development and injury repair. In this review, we summarize composites of the Wnt signaling pathway and their different action routes, followed by the discussion of their involvements in cardiac specification, proliferation, and patterning. We provide overviews about canonical and non-canonical Wnt activity during heart homeostasis, remodeling, and regeneration. Wnt/ß-catenin signaling exhibits biphasic and antagonistic effects on cardiac specification and differentiation depending on the stage of embryogenesis. Inhibition of Wnt signaling is beneficial for cardiac wound healing and functional recovery after injury. Understanding of the roles and mechanisms of Wnt signaling pathway in injured animal hearts will contribute to the development of potential therapeutics for human diseased hearts.


Assuntos
Coração , Via de Sinalização Wnt , Adulto , Animais , Diferenciação Celular , Coração/crescimento & desenvolvimento , Humanos , Camundongos , Miócitos Cardíacos , Regeneração , Via de Sinalização Wnt/fisiologia , Peixe-Zebra , beta Catenina/metabolismo
9.
Int J Mol Sci ; 23(22)2022 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-36430661

RESUMO

cAMP-PKA signaling plays a pivotal role in melanin synthesis and melanosome transport by responding to the binding of the α-melanocyte-stimulating hormone (α-MSH) to melanocortin-1 receptor (MC1R). Adenylate cyclases (ADCYs) are the enzymes responsible for the synthesis of cAMP from ATP, which comprises nine transmembrane isoforms (ADCYs 1-9) and one soluble adenylate cyclase (ADCY 10) in mammals. However, little is known about which and how ADCY isoforms regulate melanocyte generation, melanin biosynthesis, and melanosome transport in vivo. In this study, we have generated a series of single and double mutants of Adcy isoforms in zebrafish. Among them, adcy3a-/- and adcy5-/- double mutants cause defects in melanosome dispersion but do not impair melanoblast differentiation and melanocyte regeneration during the embryonic or larval stages. Activation of PKA, the main effector of cAMP signaling, significantly ameliorates the defects in melanosome dispersion in adcy3a-/- and adcy5-/- double mutants. Mechanistically, Adcy3a and Adcy5 regulate melanosome dispersion by activating kinesin-1 while inhibiting cytoplasmic dynein-1. In adult zebrafish, Adcy3a and Adcy5 participate in the regulation of the expression of microphthalmia transcription factor (Mitfa) and melanin synthesis enzymes Tyr, Dct, and Trp1b. The deletion of Adcy3a and Adcy5 inhibits melanin production and reduces pigmented melanocyte numbers, causing a defect in establishing adult melanocyte stripes. Hence, our studies demonstrate that Adcy3a and Adcy5 play essential but redundant functions in mediating α-MSH-MC1R/cAMP-PKA signaling for regulating melanin synthesis and melanosome dispersion.


Assuntos
Melanossomas , Peixe-Zebra , Animais , Melanossomas/metabolismo , Peixe-Zebra/genética , Melaninas/metabolismo , alfa-MSH/metabolismo , Melanócitos/metabolismo , Receptor Tipo 1 de Melanocortina/genética , Receptor Tipo 1 de Melanocortina/metabolismo , Mamíferos
10.
Genome Res ; 28(7): 1067-1078, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29764913

RESUMO

N6-Methyladenine (m6dA) has been discovered as a novel form of DNA methylation prevalent in eukaryotes; however, methods for high-resolution mapping of m6dA events are still lacking. Single-molecule real-time (SMRT) sequencing has enabled the detection of m6dA events at single-nucleotide resolution in prokaryotic genomes, but its application to detecting m6dA in eukaryotic genomes has not been rigorously examined. Herein, we identified unique characteristics of eukaryotic m6dA methylomes that fundamentally differ from those of prokaryotes. Based on these differences, we describe the first approach for mapping m6dA events using SMRT sequencing specifically designed for the study of eukaryotic genomes and provide appropriate strategies for designing experiments and carrying out sequencing in future studies. We apply the novel approach to study two eukaryotic genomes. For green algae, we construct the first complete genome-wide map of m6dA at single-nucleotide and single-molecule resolution. For human lymphoblastoid cells (hLCLs), it was necessary to integrate SMRT sequencing data with independent sequencing data. The joint analyses suggest putative m6dA events are enriched in the promoters of young full-length LINE-1 elements (L1s), but call for validation by additional methods. These analyses demonstrate a general method for rigorous mapping and characterization of m6dA events in eukaryotic genomes.


Assuntos
Eucariotos/genética , Genoma/genética , Linhagem Celular , Mapeamento Cromossômico/métodos , Metilação de DNA/genética , Humanos , Células Procarióticas/metabolismo , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNA/métodos
11.
Int J Mol Sci ; 22(1)2020 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-33383974

RESUMO

Pseudoxanthoma elasticum (PXE), caused by ABCC6/MRP6 mutation, is a heritable multisystem disorder in humans. The progressive clinical manifestations of PXE are accompanied by ectopic mineralization in various connective tissues. However, the pathomechanisms underlying the PXE multisystem disorder remains obscure, and effective treatment is currently available. In this study, we generated zebrafish abcc6a mutants using the transcription activator-like effector nuclease (TALEN) technique. In young adult zebrafish, abcc6a is expressed in the eyes, heart, intestine, and other tissues. abcc6a mutants exhibit extensive calcification in the ocular sclera and Bruch's membrane, recapitulating part of the PXE manifestations. Mutations in abcc6a upregulate extracellular matrix (ECM) genes, leading to fibrotic heart with reduced cardiomyocyte number. We found that abcc6a mutation reduced levels of both vitamin K and pyrophosphate (PPi) in the serum and diverse tissues. Vitamin K administration increased the gamma-glutamyl carboxylated form of matrix gla protein (cMGP), alleviating ectopic calcification and fibrosis in vertebrae, eyes, and hearts. Our findings contribute to a comprehensive understanding of PXE pathophysiology from zebrafish models.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Calcinose/genética , Calcinose/patologia , Fibrose/genética , Fibrose/patologia , Proteínas de Peixe-Zebra/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Olho/metabolismo , Olho/patologia , Predisposição Genética para Doença , Mutação , Miocárdio/metabolismo , Miocárdio/patologia , Vitamina K/metabolismo , Vitamina K/farmacologia , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
12.
Biochem Biophys Res Commun ; 510(2): 230-235, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30685088

RESUMO

Photoreceptor cells are highly specialized sensory neurons capable of visual phototransduction. The connecting cilia in the retinal photoreceptors link the inner segment to the outer segment and mediate the transport of opsins in vision. Although our previous study demonstrates that Prostaglandin E2 (PGE2) signaling is required for ciliogenesis in zebrafish, its roles in retinal ciliogenesis and photoreceptor cell development remain unknown. Here, we investigated the function of prostaglandin signaling in retina and photoreceptor cell development. We generated zebrafish mutations in prostaglandin endoperoxide synthase 1 (PTGS1) and prostaglandin endoperoxide synthase 2 (PTGS2), two rate-limiting enzymes responsible for prostaglandin production. We found that ptgs2b knockdown in ptgs1-/-ptgs2a-/- double mutants significantly reduced the length of connecting cilia and resulted in severe defects in photoreceptor cell differentiation. Furthermore, mutation in PGE2 transporter Leakytail (Lkt)/ATP-binding cassette transporter 4 (ABCC4) exhibited loss of connecting cilia and outer segment in photoreceptor cells, leading to mislocalization of opsins in the cell bodies of photoreceptors. Together, our findings suggest that PGE2 production and transport are crucial for connecting cilia formation and photoreceptor cell development.


Assuntos
Dinoprostona/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Proteínas de Peixe-Zebra/genética , Animais , Diferenciação Celular , Cílios/metabolismo , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 2/genética , Mutação , Transdução de Sinais , Peixe-Zebra
13.
Circulation ; 134(24): 1991-2007, 2016 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-27803037

RESUMO

BACKGROUND: Myocardial infarction is one of the leading causes of morbidity and mortality worldwide, triggering irreversible myocardial cell damage and heart failure. The role of low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) as coreceptors of the Wnt/ß-catenin pathway in the adult heart remain unknown. Insulin-like growth factor binding protein 4 and dickkopf-related protein 1 (Dkk1) are 2 secreted LRP5/6 binding proteins that play a crucial role in heart development through preventing Wnt/ß-catenin pathway activation. However, their roles in the adult heart remain unexplored. METHODS: To understand the role of LRP5/6 and ß-catenin in the adult heart, we constructed conditional cardiomyocyte-specific LRP5/6 and ß-catenin knockout mice and induced surgical myocardial infarction. We also directly injected recombinant proteins of insulin-like growth factor binding protein 4 and Dkk1 into the heart immediately following myocardial infarction to further examine the mechanisms through which these proteins regulate LRP5/6 and ß-catenin. RESULTS: Deletion of LRP5/6 promoted cardiac ischemic insults. Conversely, deficiency of ß-catenin, a downstream target of LRP5/6, was beneficial in ischemic injury. It is interesting to note that although both insulin-like growth factor binding protein 4 and Dkk1 are secreted Wnt/ß-catenin pathway inhibitors, insulin-like growth factor binding protein 4 protected the ischemic heart by inhibiting ß-catenin, whereas Dkk1 enhanced the injury response mainly through inducing LRP5/6 endocytosis and degradation. CONCLUSIONS: Our findings reveal previously unidentified dual roles of LRP5/6 involved in the cardiomyocyte response to ischemic injury. These findings suggest new therapeutic strategies in ischemic heart disease by fine-tuning LRP5/6 and ß-catenin signaling within the Wnt/ß-catenin pathway.


Assuntos
Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Isquemia Miocárdica/patologia , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo , Animais , Dano ao DNA/efeitos dos fármacos , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Histonas/metabolismo , Peróxido de Hidrogênio/toxicidade , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/uso terapêutico , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/antagonistas & inibidores , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/antagonistas & inibidores , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/prevenção & controle , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/metabolismo , beta Catenina/antagonistas & inibidores , beta Catenina/genética
14.
Biotechnol Lett ; 39(7): 1069-1077, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28353145

RESUMO

OBJECTIVES: To better understand the molecular mechanisms of regeneration and explore the potential signalling pathways as therapeutic targets for heart attacks. RESULTS: After treatment with the MEK inhibitor AZD6244 upon cardiac injury, the core members in MAPK/ERK signalling-mek and erk-demonstrate elevated expression, and these proteins are deposited at the injury site in zebrafish. pERK is also induced in non-cardiomyocytes near the injury site. Furthermore, the induced expression of a dominant-negative form of MEK1 inhibits zebrafish cardiac regeneration, characterized by increased cardiac fibrosis (a hallmark of regenerative failure), reduced or delayed production of regenerative myocardium, and migration of FLI1+ endothelial cells, without direct inhibition of cardiomyocyte proliferation. CONCLUSION: Appropriate activation of MAPK/ERK signalling is essential for zebrafish cardiac regeneration.


Assuntos
Coração/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Regeneração , Transdução de Sinais , Peixe-Zebra/fisiologia , Animais , Benzimidazóis/administração & dosagem , Inibidores de Proteínas Quinases/administração & dosagem
15.
Int J Mol Sci ; 18(2)2017 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-28230770

RESUMO

Vascular endothelial growth factor A (Vegfa) signaling regulates cardiovascular development. However, the cellular mechanisms of Vegfa signaling in early cardiogenesis remain poorly understood. The present study aimed to understand the differential functions and mechanisms of Vegfa signaling in cardiac development. A loss-of-function approach was utilized to study the effect of Vegfa signaling in cardiogenesis. Both morphants and mutants for vegfaa display defects in cardiac looping and chamber formation, especially the ventricle. Vegfa regulates the heart morphogenesis in a dose-dependent manner. Furthermore, the initial fusion of the bilateral myocardium population is delayed rather than endocardium. The results demonstrate that Vegfa signaling plays a direct impact on myocardium fusion, indicating that it is the initial cause of the heart defects. The heart morphogenesis is regulated by Vegfa in a dose-dependent manner, and later endocardium defects may be secondary to impaired myocardium-endocardium crosstalk.


Assuntos
Coração/embriologia , Miocárdio/metabolismo , Organogênese/genética , Fator A de Crescimento do Endotélio Vascular/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Proliferação de Células , Regulação da Expressão Gênica no Desenvolvimento , Morfogênese/genética , Mutação , Miócitos Cardíacos/metabolismo , Fenótipo , Fator A de Crescimento do Endotélio Vascular/deficiência , Fator A de Crescimento do Endotélio Vascular/metabolismo
16.
Zhonghua Yu Fang Yi Xue Za Zhi ; 51(6): 533-538, 2017 Jun 06.
Artigo em Chinês | MEDLINE | ID: mdl-28592099

RESUMO

Objectives: To explore the association between the polymorphism of persistent obesity and genetic variations in the LEP (human leptin gene, LEP) and LEPR (leptin receptor gene, LEPR) genes and different molecular subtypes of breast cancer. Methods: All 703 female patients of breast cancer diagnosed by histopathology in the Sichuan Cancer Hospital or the West China Hospital, excluding patients with metastatic breast cancer or mental disease, were selected as cases from April 2014 to May 2015. At the same time, 805 healthy women received physical examination in medical examination center of Sichuan People Hospital or Shuangliu maternal and child health care hospital, excluding those with therioma, breast disease, and mental disease, were enrolled in control group. A uniform questionnaire was used to collect general information including demographic characteristic, reproductive history height, weight, and so on. And the obesity status in recent 10 years was judged. Time of Flight Mass Spectrometer was used to determine the genotypes of LEP rs7799039, LEPR rs1137100 and LEPR rs1137101, while the multinomial logistic regression analysis was conducted to estimate the effect of risk factors related to breast cancer in different molecular subtypes; and then, the association between polymorphism of persistent obesity, the LEP, LEPR genes and breast cancer of different molecular subtypes was analyzed by binary logistic regression models. Results: The average age of controls was (48.98±8.83) years old, while the age of cases of TNBC, Luminal A, Luminal B, and HER-2+ were (51.43±11.33), (49.94±10.10), (49.73±9.38), (50.50±9.04) years old, respectively. The frequency of genotype LEP rs7799039, LEPR rs1137100 and LEPR rs1137101 in control group was separately 74.8%(1 157/1 546), 83.6%(1 339/1 602) and 88.4%(1 416/1 602); while 77.6% (1 074/1 384), 82.4% (1 155/1 402) and 87.9% (1 232/1 402) respectively in case group. Compared with non-persistent obesity subjects, the persistent obesity ones showed an increased risk in TNBC (OR=3.58, 95%CI: 1.90-6.72), Luminal A (OR=2.65, 95%CI: 1.35-5.21) and Luminal B (OR=1.90, 95%CI: 1.26-2.89) breast cancer. LEP rs7799039-AA was relevant with the upward risk of Luminal B independently (OR=1.30, 95%CI: 1.00-1.69). Besides, persistent obesity was found to have a combined effect on Luminal B (ß=3.34, 95% CI: 1.00-11.12) with LEPR rs1137101-GG. Conclusion: Persistent obesity could increase the potential risk of TNBC, Luminal A and Luminal B breast cancer. Women who were suffered from persistent obesity with a genotype of LEPR rs1137101-GG were more susceptible to Luminal B breast cancer.


Assuntos
Neoplasias da Mama/genética , Leptina/genética , Obesidade/genética , Polimorfismo Genético , Receptores para Leptina/genética , China , Feminino , Predisposição Genética para Doença , Variação Genética , Genótipo , Humanos , Modelos Logísticos , Polimorfismo de Nucleotídeo Único , Fatores de Risco
17.
Biochem Biophys Res Commun ; 472(4): 637-42, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26966072

RESUMO

P-21 activated kinases, or PAKs, are serine-threonine kinases that play important roles in diverse heart functions include heart development, cardiovascular development and function in a range of models; however, the mechanisms by which PAKs mediate heart regeneration are unknown. Here, we demonstrate that PAK2 and PAK4 expression is induced in cardiomyocytes and vessels, respectively, following zebrafish heart injury. Inhibition of PAK2 and PAK4 using a specific small molecule inhibitor impedes cardiomyocyte proliferation/dedifferentiation and cardiovascular regeneration, respectively. Cdc42 is specifically expressed in the ventricle and may function upstream of PAK2 but not PAK4 under normal conditions and that cardiomyocyte proliferentation during heart regeneration relies on Rac1-mediated activation of Pak2. Our results indicate that PAKs play a key role in heart regeneration.


Assuntos
Coração/fisiologia , Regeneração , Transdução de Sinais , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Ativação Enzimática , Traumatismos Cardíacos/metabolismo , Traumatismos Cardíacos/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Proteína cdc42 de Ligação ao GTP/metabolismo
18.
Circ Res ; 112(4): 606-17, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23283723

RESUMO

RATIONALE: Mutagenesis screening is a powerful genetic tool for probing biological mechanisms underlying vertebrate development and human diseases. However, the increased colony management efforts in vertebrates impose a significant challenge for identifying genes affecting a particular organ, such as the heart, especially those exhibiting adult phenotypes on depletion. OBJECTIVE: We aim to develop a facile approach that streamlines colony management efforts via enriching cardiac mutants, which enables us to screen for adult phenotypes. METHODS AND RESULTS: The transparency of the zebrafish embryos enabled us to score 67 stable transgenic lines generated from an insertional mutagenesis screen using a transposon-based protein trapping vector. Fifteen lines with cardiac monomeric red fluorescent protein reporter expression were identified. We defined the molecular nature for 10 lines and bred them to homozygosity, which led to the identification of 1 embryonic lethal, 1 larval lethal, and 1 adult recessive mutant exhibiting cardiac hypertrophy at 1 year of age. Further characterization of these mutants uncovered an essential function of methionine adenosyltransferase II, α a (mat2aa) in cardiogenesis, an essential function of mitochondrial ribosomal protein S18B (mrps18b) in cardiac mitochondrial homeostasis, as well as a function of DnaJ (Hsp40) homolog, subfamily B, member 6b (dnajb6b) in adult cardiac hypertrophy. CONCLUSIONS: We demonstrate that transposon-based gene trapping is an efficient approach for identifying both embryonic and adult recessive mutants with cardiac expression. The generation of a zebrafish insertional cardiac mutant collection shall facilitate the annotation of a vertebrate cardiac genome, as well as enable heart-based adult screens.


Assuntos
Cardiomegalia/genética , Perfilação da Expressão Gênica , Genes Recessivos , Testes Genéticos/métodos , Mutagênese Insercional , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Cruzamento , Elementos de DNA Transponíveis/genética , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Genes Letais , Genes Reporter , Vetores Genéticos/genética , Genótipo , Coração/embriologia , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Especificidade de Órgãos , Fenótipo , Peixe-Zebra/embriologia , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/fisiologia , Proteína Vermelha Fluorescente
19.
Proc Natl Acad Sci U S A ; 109(38): 15389-94, 2012 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-22908272

RESUMO

Conditional mutations are essential for determining the stage- and tissue-specific functions of genes. Here we achieve conditional mutagenesis in zebrafish using FT1, a gene-trap cassette that can be stably inverted by both Cre and Flp recombinases. We demonstrate that intronic insertions in the gene-trapping orientation severely disrupt the expression of the host gene, whereas intronic insertions in the neutral orientation do not significantly affect host gene expression. Cre- and Flp-mediated recombination switches the orientation of the gene-trap cassette, permitting conditional rescue in one orientation and conditional knockout in the other. To illustrate the utility of this system we analyzed the functional consequence of intronic FT1 insertion in supv3l1, a gene encoding a mitochondrial RNA helicase. Global supv311 mutants have impaired mitochondrial function, embryonic lethality, and agenesis of the liver. Conditional rescue of supv311 expression in hepatocytes specifically corrected the liver defects. To test whether the liver function of supv311 is required for viability we used Flp-mediated recombination in the germline to generate a neutral allele at the locus. Subsequently, tissue-specific expression of Cre conditionally inactivated the targeted locus. Hepatocyte-specific inactivation of supv311 caused liver degeneration, growth retardation, and juvenile lethality, a phenotype that was less severe than the global disruption of supv311. Thus, supv311 is required in multiple tissues for organismal viability. Our mutagenesis approach is very efficient and could be used to generate conditional alleles throughout the zebrafish genome. Furthermore, because FT1 is based on the promiscuous Tol2 transposon, it should be applicable to many organisms.


Assuntos
Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Alelos , Animais , DNA Nucleotidiltransferases/metabolismo , Elementos de DNA Transponíveis , Hepatócitos/citologia , Integrases/metabolismo , Fígado/metabolismo , Fígado/patologia , Mitocôndrias/enzimologia , Modelos Genéticos , Mutagênese , Mutagênicos , Mutação , Fenótipo , Reação em Cadeia da Polimerase/métodos , RNA Helicases/metabolismo , Recombinação Genética
20.
Genet Mol Res ; 14(4): 11975-93, 2015 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-26505345

RESUMO

Small heat shock proteins (sHSPs) are essential for the plant's normal development and stress responses, especially the heat stress response. The information regarding sHSP genes in Chinese cabbage (Brassica rapa ssp pekinensis) is sparse, hence we performed a genome-wide analysis to identify sHSP genes in this species. We identified 26 non-redundant sHSP genes distributed on all chromosomes, except chromosome A7, with one additional sHSP gene identified from an expressed sequence tag library. Chinese cabbage was found to contain more sHSP genes than Arabidopsis. The 27 sHSP genes were classified into 11 subfamilies. We identified 22 groups of sHSP syntenic orthologous genes between Chinese cabbage and Arabidopsis. In addition, eight groups of paralogous genes were uncovered in Chinese cabbage. Protein structures of the 27 Chinese cabbage sHSPs were modeled using Phyre2, which revealed that all of them contain several conserved ß strands across different subfamilies. In general, gene structure was conserved within each subfamily between Chinese cabbage and Arabidopsis, except for peroxisome sHSP. Analysis of promoter motifs showed that most sHSP genes contain heat shock elements or variants. We also found that biased gene loss has occurred during the evolution of the sHSP subfamily in Chinese cabbage. Expression analysis indicated that the greatest transcript abundance of most Chinese cabbage sHSP genes was found in siliques and early cotyledon embryos. Thus, genome-wide identification and characterization of sHSP genes is a first and important step in the investigation of sHSPs in Chinese cabbage.


Assuntos
Brassica rapa/genética , Genes de Plantas , Proteínas de Choque Térmico/genética , Proteínas de Plantas/genética , Sequência de Bases , Cromossomos de Plantas/genética , Proteínas de Choque Térmico/metabolismo , Dados de Sequência Molecular
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