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Conference Paper: RET transcriptional regulation by HOXB5 in Hirschsprung’s disease
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TitleRET transcriptional regulation by HOXB5 in Hirschsprung’s disease
 
AuthorsZhu, J
Garcia-Barcelo, MM
Tam, PKH
Lui, VCH
 
Issue Date2012
 
CitationThe 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
 
DC FieldValue
dc.contributor.authorZhu, J
 
dc.contributor.authorGarcia-Barcelo, MM
 
dc.contributor.authorTam, PKH
 
dc.contributor.authorLui, VCH
 
dc.date.accessioned2012-09-20T08:21:40Z
 
dc.date.available2012-09-20T08:21:40Z
 
dc.date.issued2012
 
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.descriptionSession: Genetics of Hirschsprung’s Disease, no. E35
 
dc.descriptionPoster presentation
 
dc.description.uriPoster presentation
 
dc.identifier.citationThe 3rd International Symposium on Development of the Enteric Nervous System: Cells, Signals and Genes, Hong Kong, 25-28 March 2012 [How to Cite?]
 
dc.identifier.hkuros211263
 
dc.identifier.urihttp://hdl.handle.net/10722/165649
 
dc.languageeng
 
dc.relation.ispartofInternational Symposium on Development of the Enteric Nervous System: Cells, Signals and Genes
 
dc.titleRET transcriptional regulation by HOXB5 in Hirschsprung’s disease
 
dc.typeConference_Paper
 
<?xml encoding="utf-8" version="1.0"?>
<item><contributor.author>Zhu, J</contributor.author>
<contributor.author>Garcia-Barcelo, MM</contributor.author>
<contributor.author>Tam, PKH</contributor.author>
<contributor.author>Lui, VCH</contributor.author>
<date.accessioned>2012-09-20T08:21:40Z</date.accessioned>
<date.available>2012-09-20T08:21:40Z</date.available>
<date.issued>2012</date.issued>
<identifier.citation>The 3rd International Symposium on Development of the Enteric Nervous System: Cells, Signals and Genes, Hong Kong, 25-28 March 2012</identifier.citation>
<identifier.uri>http://hdl.handle.net/10722/165649</identifier.uri>
<description>Session: Genetics of Hirschsprung&#8217;s Disease, no. E35</description>
<description>Poster presentation</description>
<description.abstract>Background and Objectives: Hirschsprung&#8217;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&#8217; of luciferase gene and 
MCS+9.7 to the 3&#8217; 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.</description.abstract>
<description.uri>Poster presentation</description.uri>
<language>eng</language>
<relation.ispartof>International Symposium on Development of the Enteric Nervous System: Cells, Signals and Genes</relation.ispartof>
<title>RET transcriptional regulation by HOXB5 in Hirschsprung&#8217;s disease</title>
<type>Conference_Paper</type>
<identifier.hkuros>211263</identifier.hkuros>
</item>