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Characterization of the Hoxa2 binding site in dual specificity tyrosine kinase 4 (Dyrk4) and high temperature requirement factor A 3 (HtrA3) genesYan, Xiaoyu 02 May 2008
Hox proteins are evolutionarily conserved transcription factors that control important developmental pathways in morphogenesis of the embryo. The Hoxa2 gene is expressed in the developing central nervous system in rhombomeres 2 to 7 and affects cellular differentiation. Few target genes of Hoxa2 protein have been identified so far and its mechanisms of regulating gene expression remain elusive. Previous work in our laboratory isolated Hoxa2 protein binding sequences from the E18 mouse spinal cord and hindbrain tissues using chromatin immunoprecipitation (ChIP). All isolated DNA fragments contain conserved GATG motifs. Sequence analysis revealed that one fragment belongs to the high temperature requirement factor A 3 (HtrA3) gene and another fragment belongs to the Dual specificity tyrosine kinase 4 (Dyrk4) gene. In this study, direct binding of Hoxa2 protein to the HtrA3 and Dyrk4 fragments was confirmed by electrophoretic mobility shift assays (EMSA). Site-directed mutagenesis and EMSA studies revealed that Hoxa2 protein binds to the multiple GATG motifs within these fragments. HtrA3 fragment also repressed luciferase gene expression in transient transfection and luciferase assays. Mutation of the DNA fragment showed that the repressive activity was affected by the GATG motifs, suggesting Hoxa2 protein regulated gene expression by binding to the GATG motif in the cis-regulatory element. In contrast to the inhibitory activity of Hoxa2 protein, a Hoxa2-VP16 fusion protein (Hoxa2 fused with an activation domain of a virion protein from herpes simplex virus) transactivates the luciferase expression by binding to GATG sites. RT-PCR and immunohistochemistry analysis revealed an upregulation of HtrA3 expression in Hoxa2-/- mice. This observation correlates with the inhibitory role of Hoxa2 protein acting upon the HtrA3 fragment in luciferase assays. Our data suggest that HtrA3 is a direct in vivo downstream target of Hoxa2 protein and support the activity regulation model in which Hox proteins selectively regulate target genes through occupation of multiple monomer binding sites.
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Characterization of the Hoxa2 binding site in dual specificity tyrosine kinase 4 (Dyrk4) and high temperature requirement factor A 3 (HtrA3) genesYan, Xiaoyu 02 May 2008 (has links)
Hox proteins are evolutionarily conserved transcription factors that control important developmental pathways in morphogenesis of the embryo. The Hoxa2 gene is expressed in the developing central nervous system in rhombomeres 2 to 7 and affects cellular differentiation. Few target genes of Hoxa2 protein have been identified so far and its mechanisms of regulating gene expression remain elusive. Previous work in our laboratory isolated Hoxa2 protein binding sequences from the E18 mouse spinal cord and hindbrain tissues using chromatin immunoprecipitation (ChIP). All isolated DNA fragments contain conserved GATG motifs. Sequence analysis revealed that one fragment belongs to the high temperature requirement factor A 3 (HtrA3) gene and another fragment belongs to the Dual specificity tyrosine kinase 4 (Dyrk4) gene. In this study, direct binding of Hoxa2 protein to the HtrA3 and Dyrk4 fragments was confirmed by electrophoretic mobility shift assays (EMSA). Site-directed mutagenesis and EMSA studies revealed that Hoxa2 protein binds to the multiple GATG motifs within these fragments. HtrA3 fragment also repressed luciferase gene expression in transient transfection and luciferase assays. Mutation of the DNA fragment showed that the repressive activity was affected by the GATG motifs, suggesting Hoxa2 protein regulated gene expression by binding to the GATG motif in the cis-regulatory element. In contrast to the inhibitory activity of Hoxa2 protein, a Hoxa2-VP16 fusion protein (Hoxa2 fused with an activation domain of a virion protein from herpes simplex virus) transactivates the luciferase expression by binding to GATG sites. RT-PCR and immunohistochemistry analysis revealed an upregulation of HtrA3 expression in Hoxa2-/- mice. This observation correlates with the inhibitory role of Hoxa2 protein acting upon the HtrA3 fragment in luciferase assays. Our data suggest that HtrA3 is a direct in vivo downstream target of Hoxa2 protein and support the activity regulation model in which Hox proteins selectively regulate target genes through occupation of multiple monomer binding sites.
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