Publicação
Functional analysis of candidate genes affecting Hoxa10 activity
| Resumo: | Hox genes encode transcription factors that control axial patterning in all bilaterians. They are characterized by the presence of a protein motif, the homeodomain (HD), which is responsible for the physical interaction between Hox proteins and their DNA targets. In vertebrates, Hox10 genes have rib-repressing activity, which determines the thoracic to lumbar transition, a functional property not shared by any other Hox protein. Previous work showed that Hox10 functional specificity does not reside in their HD but requires input from other parts of the protein. This includes a conserved motif, known as C1. A yeast two-hybrid screen identified several factors potentially interacting with C1, which could be candidates for Hox10 functional cofactors. Here, we analysed some of those factors, including Grg3, IFT144 and Smad4 for their ability to interact with Hoxa10 in a co-immunoprecipitation assay in cultured cells. These experiments failed to detect interactions between any of these proteins and Hoxa10, thus arguing against them being Hox10 cofactors. In addition, we created a new mutant line for Grg3 and analysed its role in skeletal formation. These analyses revealed that the axial skeleton in general and ribs in particular form in the absence of Grg3, thus reinforcing the conclusion that this protein is not a functional cofactor of Hox10 proteins. In addition to this, we further tested the role of the C1 motif for the rib-repressing function of Hox10 proteins by testing several deletion mutants in transgenic mouse embryos. These experiments indicated that C1 plays a role in Hox10 rib-blocking function. In addition, when the C1 motif was introduced into Hoxb9, the chimeric protein blocked rib formation in transgenic embryos, a property absent from the native Hoxb9 protein. These experiments showed that the C1 motif is also sufficient to promote a rib-repressing function. Surprisingly, these embryos also contained skeletal phenotypes consistent with abnormal segmentation of the paraxial mesoderm. These data suggest that the C1 motif might interact with the segmentation clock opening the possibility that regulation of rib formation might occur by modulating specific features of the segmentation network. |
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| Autores principais: | Mesquita, André Daniel Faustino |
| Assunto: | Hox genes Grg3 Smad4 IFT144 Motivo C1 Teses de mestrado - 2017 |
| Ano: | 2017 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Hox genes encode transcription factors that control axial patterning in all bilaterians. They are characterized by the presence of a protein motif, the homeodomain (HD), which is responsible for the physical interaction between Hox proteins and their DNA targets. In vertebrates, Hox10 genes have rib-repressing activity, which determines the thoracic to lumbar transition, a functional property not shared by any other Hox protein. Previous work showed that Hox10 functional specificity does not reside in their HD but requires input from other parts of the protein. This includes a conserved motif, known as C1. A yeast two-hybrid screen identified several factors potentially interacting with C1, which could be candidates for Hox10 functional cofactors. Here, we analysed some of those factors, including Grg3, IFT144 and Smad4 for their ability to interact with Hoxa10 in a co-immunoprecipitation assay in cultured cells. These experiments failed to detect interactions between any of these proteins and Hoxa10, thus arguing against them being Hox10 cofactors. In addition, we created a new mutant line for Grg3 and analysed its role in skeletal formation. These analyses revealed that the axial skeleton in general and ribs in particular form in the absence of Grg3, thus reinforcing the conclusion that this protein is not a functional cofactor of Hox10 proteins. In addition to this, we further tested the role of the C1 motif for the rib-repressing function of Hox10 proteins by testing several deletion mutants in transgenic mouse embryos. These experiments indicated that C1 plays a role in Hox10 rib-blocking function. In addition, when the C1 motif was introduced into Hoxb9, the chimeric protein blocked rib formation in transgenic embryos, a property absent from the native Hoxb9 protein. These experiments showed that the C1 motif is also sufficient to promote a rib-repressing function. Surprisingly, these embryos also contained skeletal phenotypes consistent with abnormal segmentation of the paraxial mesoderm. These data suggest that the C1 motif might interact with the segmentation clock opening the possibility that regulation of rib formation might occur by modulating specific features of the segmentation network. |
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