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1.
Nucleic Acids Res ; 44(6): 2538-53, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-26582913

RESUMO

DPF3 (BAF45c) is a member of the BAF chromatin remodeling complex. Two isoforms have been described, namely DPF3a and DPF3b. The latter binds to acetylated and methylated lysine residues of histones. Here, we elaborate on the role of DPF3a and describe a novel pathway of cardiac gene transcription leading to pathological cardiac hypertrophy. Upon hypertrophic stimuli, casein kinase 2 phosphorylates DPF3a at serine 348. This initiates the interaction of DPF3a with the transcriptional repressors HEY, followed by the release of HEY from the DNA. Moreover, BRG1 is bound by DPF3a, and is thus recruited to HEY genomic targets upon interaction of the two components. Consequently, the transcription of downstream targets such as NPPA and GATA4 is initiated and pathological cardiac hypertrophy is established. In human, DPF3a is significantly up-regulated in hypertrophic hearts of patients with hypertrophic cardiomyopathy or aortic stenosis. Taken together, we show that activation of DPF3a upon hypertrophic stimuli switches cardiac fetal gene expression from being silenced by HEY to being activated by BRG1. Thus, we present a novel pathway for pathological cardiac hypertrophy, whose inhibition is a long-term therapeutic goal for the treatment of the course of heart failure.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Cardiomegalia/genética , Montagem e Desmontagem da Cromatina , Cromatina/química , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Animais , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Diferenciação Celular , Cromatina/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Mioblastos/citologia , Mioblastos/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Proteínas Nucleares/metabolismo , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transcrição Gênica
2.
Hum Mol Genet ; 23(12): 3115-28, 2014 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-24459294

RESUMO

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart disease. Its genetic basis is demonstrated by an increased recurrence risk in siblings and familial cases. However, the majority of TOF are sporadic, isolated cases of undefined origin and it had been postulated that rare and private autosomal variations in concert define its genetic basis. To elucidate this hypothesis, we performed a multilevel study using targeted re-sequencing and whole-transcriptome profiling. We developed a novel concept based on a gene's mutation frequency to unravel the polygenic origin of TOF. We show that isolated TOF is caused by a combination of deleterious private and rare mutations in genes essential for apoptosis and cell growth, the assembly of the sarcomere as well as for the neural crest and secondary heart field, the cellular basis of the right ventricle and its outflow tract. Affected genes coincide in an interaction network with significant disturbances in expression shared by cases with a mutually affected TOF gene. The majority of genes show continuous expression during adulthood, which opens a new route to understand the diversity in the long-term clinical outcome of TOF cases. Our findings demonstrate that TOF has a polygenic origin and that understanding the genetic basis can lead to novel diagnostic and therapeutic routes. Moreover, the novel concept of the gene mutation frequency is a versatile measure and can be applied to other open genetic disorders.


Assuntos
Predisposição Genética para Doença , Variação Genética , Estudo de Associação Genômica Ampla/métodos , Miocárdio/patologia , Tetralogia de Fallot/genética , Tetralogia de Fallot/patologia , Apoptose , Sequência de Bases , Proliferação de Células , Estudos de Coortes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Frequência do Gene , Humanos , Dados de Sequência Molecular , Herança Multifatorial , Mutação , Miocárdio/metabolismo , Análise de Sequência de DNA , Tetralogia de Fallot/sangue
3.
Mol Biosyst ; 8(2): 495-503, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22101280

RESUMO

The adaptation of the cellular network to functional changes, timing and patterning of gene expression is regulated by binding of transcription factors to gene regulatory elements, which in turn depends on co-occurring histone modifications. These two layers influence each other, enabling a further level of regulatory fine-tuning. We analyzed the interdependencies between histone 3 acetylation, histone 3 lysine 4 dimethylation, the transcription factor Srf and the histone acetyltransferase p300 in an in vivo model using chromatin immunoprecipitation in a time-series during cardiac maturation in mouse. We found a strong correlation between the presence of the two histone modifications and binding of Srf and p300. Using linear modeling techniques we could show that each factor contributes individually as well as conjointly to histone 3 acetylation and gene expression, probably aided by accompanying histone 3 lysine 4 dimethylation. We further demonstrate that changes in gene expression during cardiac maturation are attended by changes of the analyzed regulators while revealing a high variability of combinatorial regulation. Finally, we propose a model of Srf-driven gene expression in cardiomyocytes.


Assuntos
Proteína p300 Associada a E1A/metabolismo , Coração/embriologia , Histonas/metabolismo , Fator de Resposta Sérica/metabolismo , Acetilação , Animais , Linhagem Celular , Imunoprecipitação da Cromatina/métodos , DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Metilação , Camundongos , Miócitos Cardíacos/metabolismo , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , RNA Mensageiro/biossíntese , Transcrição Gênica , Ativação Transcricional
4.
PLoS Genet ; 7(2): e1001313, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21379568

RESUMO

The transcriptome, as the pool of all transcribed elements in a given cell, is regulated by the interaction between different molecular levels, involving epigenetic, transcriptional, and post-transcriptional mechanisms. However, many previous studies investigated each of these levels individually, and little is known about their interdependency. We present a systems biology study integrating mRNA profiles with DNA-binding events of key cardiac transcription factors (Gata4, Mef2a, Nkx2.5, and Srf), activating histone modifications (H3ac, H4ac, H3K4me2, and H3K4me3), and microRNA profiles obtained in wild-type and RNAi-mediated knockdown. Finally, we confirmed conclusions primarily obtained in cardiomyocyte cell culture in a time-course of cardiac maturation in mouse around birth. We provide insights into the combinatorial regulation by cardiac transcription factors and show that they can partially compensate each other's function. Genes regulated by multiple transcription factors are less likely differentially expressed in RNAi knockdown of one respective factor. In addition to the analysis of the individual transcription factors, we found that histone 3 acetylation correlates with Srf- and Gata4-dependent gene expression and is complementarily reduced in cardiac Srf knockdown. Further, we found that altered microRNA expression in Srf knockdown potentially explains up to 45% of indirect mRNA targets. Considering all three levels of regulation, we present an Srf-centered transcription network providing on a single-gene level insights into the regulatory circuits establishing respective mRNA profiles. In summary, we show the combinatorial contribution of four DNA-binding transcription factors in regulating the cardiac transcriptome and provide evidence that histone modifications and microRNAs modulate their functional consequence. This opens a new perspective to understand heart development and the complexity cardiovascular disorders.


Assuntos
Redes Reguladoras de Genes , Histonas/metabolismo , MicroRNAs/metabolismo , Miocárdio/metabolismo , Processamento de Proteína Pós-Traducional/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Acetilação , Animais , Sítios de Ligação , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/metabolismo , Humanos , Fatores de Transcrição MEF2 , Camundongos , Fatores de Regulação Miogênica/metabolismo , Ligação Proteica , Reprodutibilidade dos Testes , Fator de Resposta Sérica/metabolismo
5.
Methods ; 50(4): S19-22, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20215016

RESUMO

Quantitative real-time PCR (qPCR) is a frequently used, sensitive and accurate method to study gene expression profiles. However, its throughput was so far limited for routine laboratories to 384 reactions per run based on the limitations of the available instruments. Recently, the LightCycler 1536 Instrument was launched providing a high-throughput solution for qPCR with the analysis of 1536 reactions in approximately 45 min. We assessed the accuracy and sensitivity of this novel technology for the analysis of gene expression profiles in combination with the Innovadyne Nanodrop Express pipetting robot. We compared expression profiles obtained for 42 genes in 71 samples between the Universal ProbeLibrary and the LightCycler 1536 Instrument and SYBR Green I and the ABI PRISM 7900HT system. We found that the results were highly reproducible between both systems. Beside the higher throughput, the advantage of the LightCycler 1536 Instrument was the reduced consumption of reagents and sample material.


Assuntos
Perfilação da Expressão Gênica/instrumentação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação , Perfilação da Expressão Gênica/métodos , Cardiopatias Congênitas/metabolismo , Humanos , Hidrólise , Miocárdio/química , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade
6.
Genes Dev ; 22(17): 2370-84, 2008 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-18765789

RESUMO

Chromatin remodeling and histone modifications facilitate access of transcription factors to DNA by promoting the unwinding and destabilization of histone-DNA interactions. We present DPF3, a new epigenetic key factor for heart and muscle development characterized by a double PHD finger. DPF3 is associated with the BAF chromatin remodeling complex and binds methylated and acetylated lysine residues of histone 3 and 4. Thus, DPF3 may represent the first plant homeodomains that bind acetylated lysines, a feature previously only shown for the bromodomain. During development Dpf3 is expressed in the heart and somites of mouse, chicken, and zebrafish. Morpholino knockdown of dpf3 in zebrafish leads to incomplete cardiac looping and severely reduced ventricular contractility, with disassembled muscular fibers caused by transcriptional deregulation of structural and regulatory proteins. Promoter analysis identified Dpf3 as a novel downstream target of Mef2a. Taken together, DPF3 adds a further layer of complexity to the BAF complex by representing a tissue-specific anchor between histone acetylations as well as methylations and chromatin remodeling. Furthermore, this shows that plant homeodomain proteins play a yet unexplored role in recruiting chromatin remodeling complexes to acetylated histones.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Coração/embriologia , Desenvolvimento Muscular/fisiologia , Fatores de Transcrição/biossíntese , Acetilação , Sequência de Aminoácidos , Animais , Embrião de Galinha , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/genética , Epigênese Genética , Histonas/metabolismo , Humanos , Metilação , Camundongos , Dados de Sequência Molecular , Miocárdio/metabolismo , Fatores de Transcrição/genética , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo
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