RESUMO
Gene regulation by transcription factors (TFs) determines developmental programs and cell identity. Consequently, mutations in TFs can lead to dramatic phenotypes in humans by disrupting gene regulation. To date, the molecular mechanisms that actually cause these phenotypes have been difficult to address experimentally. ChIP-seq, which couples chromatin immunoprecipitation with high-throughput sequencing, allows TF function to be investigated on a genome-wide scale, enabling new approaches for the investigation of gene regulation. Here, we present the application of ChIP-seq to explore the effect of missense mutations in TFs on their genome-wide binding profile. Using a retroviral expression system in chicken mesenchymal stem cells, we elucidated the mechanism underlying a novel missense mutation in HOXD13 (Q317K) associated with a complex hand and foot malformation phenotype. The mutated glutamine (Q) is conserved in most homeodomains, a notable exception being bicoid-type homeodomains that have lysine (K) at this position. Our results show that the mutation results in a shift in the binding profile of the mutant toward a bicoid/PITX1 motif. Gene expression analysis and functional assays using in vivo overexpression studies confirm that the mutation results in a partial conversion of HOXD13 into a TF with bicoid/PITX1 properties. A similar shift was not observed with another mutation, Q317R, which is associated with brachysyndactyly, suggesting that the bicoid/PITX1-shift observed for Q317K might be related to the severe clinical phenotype. The methodology described can be used to investigate a wide spectrum of TFs and mutations that have not previously been amenable to ChIP-seq experiments.
Assuntos
Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Deformidades Congênitas dos Membros/genética , Fatores de Transcrição Box Pareados/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Embrião de Galinha , Imunoprecipitação da Cromatina , Feminino , Perfilação da Expressão Gênica , Genoma Humano , Glutamina/genética , Humanos , Células-Tronco Mesenquimais/metabolismo , Mutação de Sentido Incorreto , Análise de Sequência com Séries de Oligonucleotídeos , Fatores de Transcrição Box Pareados/genética , Fenótipo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genéticaRESUMO
Synpolydactyly (SPD) is a rare congenital limb disorder characterized by syndactyly between the third and fourth fingers and an additional digit in the syndactylous web. In most cases SPD is caused by heterozygous mutations in HOXD13 resulting in the expansion of a N-terminal polyalanine tract. If homozygous, the mutation results in severe shortening of all metacarpals and phalanges with a morphological transformation of metacarpals to carpals. Here, we describe a novel homozygous missense mutation in a family with unaffected consanguineous parents and severe brachydactyly and metacarpal-to-carpal transformation in the affected child. We performed whole exome sequencing on the index patient, followed by Sanger sequencing of parents and patient to investigate cosegregation. The DNA-binding ability of the mutant protein was tested with electrophoretic mobility shift assays. We demonstrate that the c.938C>G (p.313T>R) mutation in the DNA-binding domain of HOXD13 prevents binding to DNA in vitro. Our results show to our knowledge for the first time that a missense mutation in HOXD13 underlies severe brachydactyly with metacarpal-to-carpal transformation. The mutation is non-penetrant in heterozygous carriers. In conjunction with the literature we propose the possibility that the metacarpal-to-carpal transformation results from a homozygous loss of functional HOXD13 protein in humans in combination with an accumulation of non-functional HOXD13 that might be able to interact with other transcription factors in the developing limb.
Assuntos
Braquidactilia/genética , Proteínas de Homeodomínio/genética , Homozigoto , Mutação de Sentido Incorreto , Sindactilia/genética , Fatores de Transcrição/genética , Adulto , Sequência de Bases , Braquidactilia/diagnóstico , Braquidactilia/patologia , Ossos do Carpo/anormalidades , Ossos do Carpo/metabolismo , Pré-Escolar , Consanguinidade , Ensaio de Desvio de Mobilidade Eletroforética , Exoma , Feminino , Expressão Gênica , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Ossos Metacarpais/anormalidades , Ossos Metacarpais/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Linhagem , Sindactilia/diagnóstico , Sindactilia/patologiaRESUMO
Icosahedral capsids of viruses are lattices of defined geometry and homogeneous size. The (quasi-)equivalent organization of their protein building blocks provides, in numerous systems, the binding sites to assemble arrays of viral polypeptides organized with nanometer precision that protrude from the capsid surface. The capsid of bacterial virus (bacteriophage) SPP1 exposes, at its surface, the 6.6-kDa viral polypeptide gp12 that binds to the center of hexamers of the major capsid protein. Gp12 forms an elongated trimer with collagen-like properties. This is consistent with the fold of eight internal GXY repeats of gp12 to build a stable intersubunit triple helix in a prokaryotic setting. The trimer dissociates and unfolds at near physiological temperatures, as reported for eukaryotic collagen. Its structural organization is reacquired within seconds upon cooling. Interaction with the SPP1 capsid hexamers strongly stabilizes gp12, increasing its Tm to 54 °C. Above this temperature, gp12 dissociates from its binding sites and unfolds reversibly. Multivalent binding of gp12 trimers to the capsid is highly cooperative. The capsid lattice also provides a platform to assist folding and association of unfolded gp12 polypeptides. The original physicochemical properties of gp12 offer a thermoswitchable system for multivalent binding of the polypeptide to the SPP1 capsid surface.
Assuntos
Fagos Bacilares/química , Capsídeo/química , Proteínas Estruturais Virais/química , Fagos Bacilares/genética , Fagos Bacilares/metabolismo , Capsídeo/metabolismo , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismoRESUMO
BACKGROUND: The sheep is an important model animal for testing novel fracture treatments and other medical applications. Despite these medical uses and the well known economic and cultural importance of the sheep, relatively little research has been performed into sheep genetics, and DNA sequences are available for only a small number of sheep genes. RESULTS: In this work we have sequenced over 47 thousand expressed sequence tags (ESTs) from libraries developed from healing bone in a sheep model of fracture healing. These ESTs were clustered with the previously available 10 thousand sheep ESTs to a total of 19087 contigs with an average length of 603 nucleotides. We used the newly identified sequences to develop RT-PCR assays for 78 sheep genes and measured differential expression during the course of fracture healing between days 7 and 42 postfracture. All genes showed significant shifts at one or more time points. 23 of the genes were differentially expressed between postfracture days 7 and 10, which could reflect an important role for these genes for the initiation of osteogenesis. CONCLUSION: The sequences we have identified in this work are a valuable resource for future studies on musculoskeletal healing and regeneration using sheep and represent an important head-start for genomic sequencing projects for Ovis aries, with partial or complete sequences being made available for over 5,800 previously unsequenced sheep genes.
Assuntos
Etiquetas de Sequências Expressas , Consolidação da Fratura/genética , Perfilação da Expressão Gênica/métodos , Carneiro Doméstico/genética , Animais , Análise por Conglomerados , Mapeamento de Sequências Contíguas/métodos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Análise de Sequência de DNA/métodosRESUMO
Gp7 is a minor capsid protein of the Bacillus subtilis bacteriophage SPP1. Homologous proteins are found in numerous phages but their function remained unknown. Deletion of gene 7 from the SPP1 genome yielded a mutant phage (SPP1del7) with reduced burst-size. SPP1del7 infections led to normal assembly of virus particles whose morphology, DNA and protein composition was undistinguishable from wild-type virions. However, only approximately 25% of the viral particles that lack gp7 were infectious. SPP1del7 particles caused a reduced depolarization of the B. subtilis membrane in infection assays suggesting a defect in virus genome traffic to the host cell. A higher number of SPP1del7 DNA ejection events led to abortive release of DNA to the culture medium when compared with wild-type infections. DNA ejection in vitro showed that no detectable gp7 is co-ejected with the SPP1 genome and that its presence in the virion correlated with anchoring of released DNA to the phage particle. The release of DNA from wild-type phages was slower than that from SPP1del7 suggesting that gp7 controls DNA exit from the virion. This feature is proposed to play a central role in supporting correct routing of the phage genome from the virion to the cell cytoplasm.
Assuntos
Fagos Bacilares/fisiologia , Bacillus subtilis/virologia , Proteínas do Capsídeo/fisiologia , Fagos Bacilares/genética , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Membrana Celular/fisiologia , Membrana Celular/virologia , DNA Viral/metabolismo , DNA Viral/ultraestrutura , Deleção de Genes , Genoma Viral , Vírion/genética , Vírion/metabolismoRESUMO
The icosahedral procapsid of tailed bacteriophages is composed of a large number of identical subunits and of minor proteins found in a few copies. Proteins present in a very low copy number are targeted to the viral procapsid by an unknown mechanism. Bacteriophage SPP1 procapsids and mature virions contain two copies of gp7 on average. Gp7 forms stable complexes with the SPP1 portal protein gp6. Deletion of the gp6 carboxyl-terminus and the mutation Y467-->C localized in the same region prevent gp6-gp7 complex formation. Gp7 binds double-stranded and single-stranded DNA. Gp6 competes for this interaction, and purified gp6-gp7 complexes do not bind DNA. Procapsid structures assembled in the absence of gp6 or carrying the mutant gp6 Y467-->C lack gp7. The gp6-gp7 interaction thus targets gp7 to the procapsid where the portal protein is localized asymmetrically at a single vertex of the icosahedral structure. The interaction between the two proteins is disrupted during viral assembly. Proteins homologous to gp6 and gp7 are coded by contiguous genes in a variety of phage genomes from Gram-positive bacteria, suggesting that the gp6-gp7 complex is widespread in this group of phages. Transient association with the portal protein, an essential component of tailed bacteriophages and herpes viruses, provides a novel strategy to target minor proteins to the virion structure that might be operative in a large number of viruses.
Assuntos
Fagos Bacilares , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Capsídeo/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Capsídeo/metabolismo , Capsídeo/ultraestrutura , Proteínas do Capsídeo/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Deleção de Genes , Genes Virais , Mutação de Sentido Incorreto/genética , Mapeamento de Interação de Proteínas , Homologia de Sequência , Proteínas Virais/isolamento & purificação , Montagem de Vírus/genética , Montagem de Vírus/fisiologiaRESUMO
Poly-alanine (Ala) tract expansions in transcription factors have been shown to be associated with human birth defects such as malformations of the brain, the digits, and other structures. Expansions of a poly-Ala tract from 15 to 22 (+7)-29 (+14) Ala in Hoxd13, for example, result in the limb malformation synpolydactyly in humans and in mice [synpolydactyly homolog (spdh)]. Here, we show that an increase of the Ala repeat above a certain length (22 Ala) is associated with a shift in the localization of Hoxd13 from nuclear to cytoplasmic, where it forms large amorphous aggregates. We observed similar aggregates for expansion mutations in SOX3, RUNX2 and HOXA13, pointing to a common mechanism. Cytoplasmic aggregation of mutant Hoxd13 protein is influenced by the length of the repeat, the level of expression and the efficacy of degradation by the proteasome. Heat shock proteins Hsp70 and Hsp40 co-localize with the aggregates and activation of the chaperone system by geldanamycin leads to a reduction of aggregate formation. Furthermore, recombinant mutant Hoxd13 protein forms aggregates in vitro demonstrating spontaneous misfolding of the protein. We analyzed the mouse mutant spdh, which harbors a +7 Ala expansion in Hoxd13 similar to the human synpolydactyly mutations, as an in vivo model and were able to show a reduction of mutant Hoxd13 and, in contrast to wt Hoxd13, a primarily cytoplasmic localization of the protein. Our results provide evidence that poly-Ala repeat expansions in transcription factors result in misfolding, degradation and cytoplasmic aggregation of the mutant proteins.