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Conference Paper: RET transcriptional regulation by HOXB5 in Hirschsprung’s disease

TitleRET transcriptional regulation by HOXB5 in Hirschsprung’s disease
Authors
Issue Date2012
Citation
The 3rd International Symposium on Development of the Enteric Nervous System: Cells, Signals and Genes, Hong Kong, 25-28 March 2012 How to Cite?
AbstractBackground and Objectives: Hirschsprung’s disease (HSCR) is the major enteric nervous system anomaly affecting newborns with the highest incidence in Asians. HSCR is a congenital complex genetic disorder characterised by a lack of enteric ganglia along a variable length of the intestine. The receptor tyrosine kinase gene (RET) is the major HSCR gene and its expression is crucial for ENS development. The human homeobox B5, HOXB5, has an important role in the development of enteric neural crest cells, and perturbation of Hoxb5 signaling causes HSCR phenotypes in mice. To investigate the roles of HOXB5 in the regulation of RET expression and in the aetiology of HSCR, I sought to (i) elucidate the underlying mechanisms that HOXB5 mediates RET expression, and (ii) to examine the interactions between HOXB5 and other transcription factors implicated in RET expression and HSCR. Methods: Luciferase assay was applied to study the trans-activity of HOXB5 from both promoter and intron regions. The RET promoter luciferase reporter was constructed as previously reported and the RET enhancer mini-gene was constructed by ligating the RET promoter region (-1205 to +196 or -177 to +196) to the 5’ of luciferase gene and MCS+9.7 to the 3’ of luciferase gene. EMSA assay and ChIP assay were introduced to examine the binding activity of HOXB5 to the putative binding sites, which were predicted by in silico analysis. Furthermore, we also utilized IP and Co-IP assay to study the protein-protein interaction between HOXB5 and other transcription factors. Results: In this study, we demonstrate human HOXB5 binds to the promoter and regulates RET expression. HOXB5 and NKX2-1 form a protein complex and mediate RET expression in a synergistic manner. In contrast, HOXB5 cooperates in an additive manner with SOX10, PAX3 and PHOX2B in trans-activation of RET promoter. In silico analysis, EMSA and ChIP analysis showed that HOXB5 binds to another HSCR-implicated MCS+9.7 enhancer in Intron 1 of RET. Specific binding of HOXB5 was abolished by introducing a deletion of core binding sequence or sequence variant in MCS+9.7 region. Luciferase assay indicated that alternative alleles at several SNPs significantly reduced the trans-activity of HOXB5 from the RET mini-gene. Conclusion: Our data suggest that HOXB5 in coordination with other transcription factors mediates RET expression. Therefore, defects in cis- or trans-regulation of RET by HOXB5 could lead to reduction of RET expression and contribute to the manifestation of the HSCR phenotype.
DescriptionSession: Genetics of Hirschsprung’s Disease, no. E35
Poster presentation
???metadata.dc.description.uri???Poster presentation
Persistent Identifierhttp://hdl.handle.net/10722/165649

 

DC FieldValueLanguage
dc.contributor.authorZhu, Jen_US
dc.contributor.authorGarcia-Barcelo, MMen_US
dc.contributor.authorTam, PKHen_US
dc.contributor.authorLui, VCHen_US
dc.date.accessioned2012-09-20T08:21:40Z-
dc.date.available2012-09-20T08:21:40Z-
dc.date.issued2012en_US
dc.identifier.citationThe 3rd International Symposium on Development of the Enteric Nervous System: Cells, Signals and Genes, Hong Kong, 25-28 March 2012en_US
dc.identifier.urihttp://hdl.handle.net/10722/165649-
dc.descriptionSession: Genetics of Hirschsprung’s Disease, no. E35-
dc.descriptionPoster presentation-
dc.description.abstractBackground and Objectives: Hirschsprung’s disease (HSCR) is the major enteric nervous system anomaly affecting newborns with the highest incidence in Asians. HSCR is a congenital complex genetic disorder characterised by a lack of enteric ganglia along a variable length of the intestine. The receptor tyrosine kinase gene (RET) is the major HSCR gene and its expression is crucial for ENS development. The human homeobox B5, HOXB5, has an important role in the development of enteric neural crest cells, and perturbation of Hoxb5 signaling causes HSCR phenotypes in mice. To investigate the roles of HOXB5 in the regulation of RET expression and in the aetiology of HSCR, I sought to (i) elucidate the underlying mechanisms that HOXB5 mediates RET expression, and (ii) to examine the interactions between HOXB5 and other transcription factors implicated in RET expression and HSCR. Methods: Luciferase assay was applied to study the trans-activity of HOXB5 from both promoter and intron regions. The RET promoter luciferase reporter was constructed as previously reported and the RET enhancer mini-gene was constructed by ligating the RET promoter region (-1205 to +196 or -177 to +196) to the 5’ of luciferase gene and MCS+9.7 to the 3’ of luciferase gene. EMSA assay and ChIP assay were introduced to examine the binding activity of HOXB5 to the putative binding sites, which were predicted by in silico analysis. Furthermore, we also utilized IP and Co-IP assay to study the protein-protein interaction between HOXB5 and other transcription factors. Results: In this study, we demonstrate human HOXB5 binds to the promoter and regulates RET expression. HOXB5 and NKX2-1 form a protein complex and mediate RET expression in a synergistic manner. In contrast, HOXB5 cooperates in an additive manner with SOX10, PAX3 and PHOX2B in trans-activation of RET promoter. In silico analysis, EMSA and ChIP analysis showed that HOXB5 binds to another HSCR-implicated MCS+9.7 enhancer in Intron 1 of RET. Specific binding of HOXB5 was abolished by introducing a deletion of core binding sequence or sequence variant in MCS+9.7 region. Luciferase assay indicated that alternative alleles at several SNPs significantly reduced the trans-activity of HOXB5 from the RET mini-gene. Conclusion: Our data suggest that HOXB5 in coordination with other transcription factors mediates RET expression. Therefore, defects in cis- or trans-regulation of RET by HOXB5 could lead to reduction of RET expression and contribute to the manifestation of the HSCR phenotype.-
dc.description.uriPoster presentation-
dc.languageengen_US
dc.relation.ispartofInternational Symposium on Development of the Enteric Nervous System: Cells, Signals and Genesen_US
dc.titleRET transcriptional regulation by HOXB5 in Hirschsprung’s diseaseen_US
dc.typeConference_Paperen_US
dc.identifier.emailZhu, J: jiangzhu@hku.hken_US
dc.identifier.emailGarcia-Barcelo, MM: mmgarcia@hku.hken_US
dc.identifier.emailTam, PKH: paultam@hku.hken_US
dc.identifier.emailLui, VCH: vchlui@hku.hken_US
dc.identifier.authorityGarcia-Barcelo, MM=rp00445en_US
dc.identifier.authorityTam, PKH=rp00060en_US
dc.identifier.authorityLui, VCH=rp00363en_US
dc.identifier.hkuros211263en_US

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