RESUMO
TBX5 plays a critical role in heart and forelimb development. Mutations in TBX5 cause Holt-Oram syndrome, an autosomal dominant condition that affects the formation of the heart and upper-limb. Several studies have provided significant insight into the role of TBX5 in cardiogenesis; however, how TBX5 activity is regulated by other factors is still unknown. Here we report that histone acetyltransferases KAT2A and KAT2B associate with TBX5 and acetylate it at Lys339. Acetylation potentiates its transcriptional activity and is required for nuclear retention. Morpholino-mediated knockdown of kat2a and kat2b transcripts in zebrafish severely perturb heart and limb development, mirroring the tbx5a knockdown phenotype. The phenotypes found in MO-injected embryos were also observed when we introduced mutations in the kat2a or kat2b genes using the CRISPR-Cas system. These studies highlight the importance of KAT2A and KAT2B modulation of TBX5 and their impact on heart and limb development.
Assuntos
Extremidades/embriologia , Coração/embriologia , Histona Acetiltransferases/metabolismo , Proteínas com Domínio T/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Acetilação , Sequência de Aminoácidos , Nadadeiras de Animais/embriologia , Animais , Sistemas CRISPR-Cas/genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Regulação para Baixo/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Coração/efeitos dos fármacos , Histona Acetiltransferases/genética , Morfolinos/farmacologia , Fenótipo , Proteínas com Domínio T/química , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
TBX5 is a T-box family transcription factor that regulates heart and forelimb development in vertebrates and functional deficiencies in this protein result in Holt-Oram syndrome. Recently, we have shown that acetylation of TBX5 potentiates its activity and is important for heart and limb development. Here we report that class II histone deacetylases HDAC4 and HDAC5 associate with TBX5 and repress its role in cardiac gene transcription. Both HDAC4 and HDAC5 deacetylate TBX5, which promotes its relocation to the cytoplasm and HDAC4 antagonizes the physical association and functional cooperation between TBX5 and MEF2C. We also show that protein kinase D1 (PRKD1) relieves the HDAC4/5-mediated repression of TBX5. Thus, this study reveals a novel interaction of HDAC4/5 and PRKD1 in the regulation of TBX5 transcriptional activity.
Assuntos
Histona Desacetilases/metabolismo , Proteína Quinase C/metabolismo , Proteínas Repressoras/metabolismo , Proteínas com Domínio T/metabolismo , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Acetilação , Animais , Células COS , Chlorocebus aethiops , Clonagem Molecular , Citoplasma/metabolismo , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/patologia , Comunicação Interatrial/genética , Comunicação Interatrial/patologia , Deformidades Congênitas das Extremidades Inferiores/genética , Deformidades Congênitas das Extremidades Inferiores/patologia , Fatores de Transcrição MEF2/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/isolamento & purificação , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas com Domínio T/genética , Transcrição Gênica , Transfecção , Deformidades Congênitas das Extremidades Superiores/genética , Deformidades Congênitas das Extremidades Superiores/patologiaRESUMO
Myotonic dystrophy (DM; also known as dystrophia myotonica) is an autosomal dominant disorder that affects the heart, eyes, brain and endocrine system, but the predominant symptoms are neuromuscular, with progressive muscle weakness and wasting. DM presents in two forms, DM1 and DM2, both of which are caused by nucleotide repeat expansions: CTG in the DMPK gene for DM1 and CCTG in ZNF9 (CNBP) for DM2. Previous studies have shown that the mutant mRNAs containing the transcribed CUG or CCUG repeats are retained within the nuclei of cells from individuals with DM, where they bind and sequester the muscleblind-like proteins MBNL1, MBNL2 and MBNL3. It has been proposed that the sequestration of these proteins plays a key role in determining the classic features of DM. However, the functions of each of the three MBNL genes are not completely understood. We have generated a zebrafish knockdown model in which we demonstrate that a lack of mbnl2 function causes morphological abnormalities at the eye, heart, brain and muscle levels, supporting an essential role for mbnl2 during embryonic development. Major features of DM are replicated in our model, including muscle defects and splicing abnormalities. We found that the absence of mbnl2 causes disruption to the organization of myofibrils in skeletal and heart muscle of zebrafish embryos, and a reduction in the amount of both slow and fast muscle fibres. Notably, our findings included altered splicing patterns of two transcripts whose expression is also altered in DM patients: clcn1 and tnnt2. The studies described herein provide broader insight into the functions of MBNL2. They also lend support to the hypothesis that the sequestration of this protein is an important determinant in DM pathophysiology, and imply a direct role of MBNL2 in splicing regulation of specific transcripts, which, when altered, contributes to the DM phenotype.
Assuntos
Distrofia Miotônica/patologia , Proteínas de Peixe-Zebra/deficiência , Peixe-Zebra/metabolismo , Processamento Alternativo/efeitos dos fármacos , Processamento Alternativo/genética , Animais , Sequência de Bases , Osso e Ossos/anormalidades , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Embrião não Mamífero/anormalidades , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Embrião não Mamífero/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Dados de Sequência Molecular , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Miocárdio/metabolismo , Miocárdio/patologia , Distrofia Miotônica/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
TBX5 is a transcription factor which plays important roles in the development of the heart and upper limbs. Mutations in this gene produce the inherited disorder Holt-Oram syndrome. Here, we report a physical interaction between TBX5 and MEF2C leading to a synergistic activation of the alpha-cardiac myosin heavy chain (MYH6). Mutants of TBX5, TBX5G80R, and TBX5R279X that produce severe cardiac phenotypes impair the synergy. Using fluorescence resonance energy transfer, we demonstrate the interaction of TBX5 and MEF2C in living cells. We also show that they physically associate through their DNA-binding domains to form a complex on the MYH6 promoter. Morpholino-mediated knockdowns of Tbx5 and Mef2c in zebrafish suggest that the genetic interaction of these proteins is not only required for MYH6 expression but also essential for the early stages of heart development and survival. This is the first report of a functional interaction between a T-box protein and a MADS box factor that may be crucial in cardiomyocyte differentiation.