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1.
Hum Mol Genet ; 23(6): 1551-62, 2014 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-24179176

RESUMO

Myotonic dystrophy (DM) is a multi-system neuromuscular disorder for which there is no treatment. We have developed a medium throughput phenotypic assay, based on the identification of nuclear foci in DM patient cell lines using in situ hybridization and high-content imaging to screen for potentially useful therapeutic compounds. A series of further assays based on molecular features of DM have also been employed. Two compounds that reduce and/or remove nuclear foci have been identified, Ro 31-8220 and chromomycin A3. Ro 31-8220 is a PKC inhibitor, previously shown to affect the hyperphosphorylation of CELF1 and ameliorate the cardiac phenotype in a DM1 mouse model. We show that the same compound eliminates nuclear foci, reduces MBNL1 protein in the nucleus, affects ATP2A1 alternative splicing and reduces steady-state levels of CELF1 protein. We demonstrate that this effect is independent of PKC activity and conclude that this compound may be acting on alternative kinase targets within DM pathophysiology. Understanding the activity profile for this compound is key for the development of targeted therapeutics in the treatment of DM.


Assuntos
Núcleo Celular/efeitos dos fármacos , Cromomicina A3/farmacologia , Indóis/farmacologia , Distrofia Miotônica/patologia , Proteínas de Ligação a RNA/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Processamento Alternativo , Animais , Proteínas CELF1 , Núcleo Celular/patologia , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Biblioteca de Peptídeos , Proteínas de Ligação a RNA/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Transdução de Sinais/efeitos dos fármacos , Peixe-Zebra
2.
Nat Genet ; 37(4): 423-8, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15735645

RESUMO

Atrial septal defect is one of the most common forms of congenital heart malformation. We identified a new locus linked with atrial septal defect on chromosome 14q12 in a large family with dominantly inherited atrial septal defect. The underlying mutation is a missense substitution, I820N, in alpha-myosin heavy chain (MYH6), a structural protein expressed at high levels in the developing atria, which affects the binding of the heavy chain to its regulatory light chain. The cardiac transcription factor TBX5 strongly regulates expression of MYH6, but mutant forms of TBX5, which cause Holt-Oram syndrome, do not. Morpholino knock-down of expression of the chick MYH6 homolog eliminates the formation of the atrial septum without overtly affecting atrial chamber formation. These data provide evidence for a link between a transcription factor, a structural protein and congenital heart disease.


Assuntos
Miosinas Cardíacas/genética , Comunicação Interatrial/genética , Mutação de Sentido Incorreto , Cadeias Pesadas de Miosina/genética , Proteínas com Domínio T/genética , Adulto , Substituição de Aminoácidos , Animais , Miosinas Cardíacas/metabolismo , Embrião de Galinha , Criança , Pré-Escolar , Feminino , Ligação Genética , Comunicação Interatrial/embriologia , Humanos , Recém-Nascido , Masculino , Dados de Sequência Molecular , Cadeias Pesadas de Miosina/metabolismo , Linhagem , Proteínas com Domínio T/química
3.
Hum Mol Genet ; 17(2): 256-65, 2008 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-17947298

RESUMO

Atrial septal defect (ASD) is one of the most frequent congenital heart defects (CHDs) with a variable phenotypic effect depending on the size of the septal shunt. We identified two pedigrees comprising 20 members segregating isolated autosomal dominant secundum ASD. By genetic mapping, we identified the gene-encoding alpha-cardiac actin (ACTC1), which is essential for cardiac contraction, as the likely candidate. A mutation screen of the coding regions of ACTC1 revealed a founder mutation predicting an M123V substitution in affected individuals of both pedigrees. Functional analysis of ACTC1 with an M123V substitution shows a reduced affinity for myosin, but with retained actomyosin motor properties. We also screened 408 sporadic patients with CHDs and identified a case with ASD and a 17-bp deletion in ACTC1 predicting a non-functional protein. Morpholino (MO) knockdown of ACTC1 in chick embryos produces delayed looping and reduced atrial septa, supporting a developmental role for this protein. The combined results indicate, for the first time, that ACTC1 mutations or reduced ACTC1 levels may lead to ASD without signs of cardiomyopathy.


Assuntos
Actinas/genética , Comunicação Interatrial/genética , Comunicação Interatrial/metabolismo , Actinas/química , Actinas/metabolismo , Substituição de Aminoácidos , Animais , Embrião de Galinha , Pré-Escolar , Feminino , Deleção de Genes , Coração/embriologia , Humanos , Lactente , Masculino , Mutagênese Sítio-Dirigida , Miosinas/metabolismo , Linhagem
4.
Mol Cell Biol ; 29(8): 2205-18, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19204083

RESUMO

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.


Assuntos
Miosinas Cardíacas/genética , Coração/crescimento & desenvolvimento , Fatores de Regulação Miogênica/metabolismo , Cadeias Pesadas de Miosina/genética , Proteínas com Domínio T/metabolismo , Animais , Linhagem Celular , Embrião não Mamífero , Humanos , Mutação de Sentido Incorreto , Fatores de Regulação Miogênica/fisiologia , Regiões Promotoras Genéticas , Ratos , Proteínas com Domínio T/genética , Proteínas com Domínio T/fisiologia , Transfecção , Peixe-Zebra
5.
Hum Genet ; 120(4): 487-99, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16927100

RESUMO

Myotonic dystrophy (DM) is a dominantly inherited neuromuscular disorder characterised by muscle weakness and wasting. There are two forms of DM; both of which are caused by the expansion of repeated DNA sequences. DM1 is associated with a CTG repeat located in the 3' untranslated region of a gene, DMPK and DM2 with a tetranucleotide repeat expansion, CCTG, located in the first intron of a different gene, ZNF9. Recent data suggest a dominant RNA gain-of-function mechanism underlying DM, as transcripts containing either CUG or CCUG repeat expansions accumulate as foci in the nuclei of DM1 and DM2 cells respectively, where they exert a toxic effect, sequestering specific RNA binding proteins such as Muscleblind, which leads to splicing defects and the disruption of normal cellular functions. Z-band disruption is a well-known histological feature of DM1 muscle, which has also been reported in Muscleblind deficient flies. In order to determine whether there is a common molecular basis for this abnormality we have examined the alternative splicing pattern of transcripts that encode proteins associated with the Z-band in both organisms. Our results demonstrate that the missplicing of ZASP/LDB3 leads to the expression of an isoform in DM1 patient muscle, which is not present in normal controls, nor in other myopathies. Furthermore the Drosophila homologue, CG30084, is also misspliced, in Muscleblind deficient flies. Another Z-band transcript, alpha actinin, is misspliced in mbl mutant flies, but not in DM1 patient samples. These results point to similarities but subtle differences in the molecular breakdown of Z-band structures in flies and DM patients and emphasise the relevance of Muscleblind proteins in DM pathophysiology.


Assuntos
Processamento Alternativo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Musculares/genética , Distrofia Miotônica/genética , Proteínas Nucleares/genética , Actinina/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Sequência de Aminoácidos , Animais , Modelos Animais de Doenças , Proteínas de Drosophila/deficiência , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Dados de Sequência Molecular , Músculo Esquelético/embriologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Mutação , Distrofia Miotônica/embriologia , Distrofia Miotônica/patologia , Proteínas Nucleares/deficiência , Isoformas de Proteínas/genética , RNA/genética , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica
6.
Chromosoma ; 110(8): 511-8, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12068968

RESUMO

There has been debate over the mechanisms that control the copy number of transposable elements in the genome of Drosophila melanogaster. Target sites in D. melanogaster populations are occupied at low frequencies, suggesting that there is some form of selection acting against transposable elements. Three main theories have been proposed to explain how selection acts against transposable elements: insertions of a copy of a transposable element are selected against; chromosomal rearrangements caused by ectopic exchange between element copies are selected against; or the process of transposition itself is selected against. The three theories give different predictions for the pattern of transposable element insertions in the chromosomes of D. melanogaster. We analysed the abundance of six LTR (long terminal repeat) retrotransposons on the X and fourth chromosomes of multiple strains of D. melanogaster, which we compare with the predictions of each theory. The data suggest that no one theory can account for the insertion patterns of all six retrotransposons. Comparing our results with earlier work using these transposable element families, we find a significant correlation between studies in the particular model of copy number regulation supported by the proportion of elements on the X for the different transposable element families. This suggests that different retrotransposon families are regulated by different mechanisms.


Assuntos
Drosophila melanogaster/genética , Dosagem de Genes , Retroelementos , Sequências Repetidas Terminais , Animais , Genoma , Seleção Genética , Cromossomo X
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