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Conference Paper: RET transcriptional regulation by HOXB5 in Hirschsprung’s disease
Title | RET transcriptional regulation by HOXB5 in Hirschsprung’s disease |
---|---|
Authors | |
Issue Date | 2012 |
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? |
Abstract | Background 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. |
Description | Session - Genetics of Hirschsprung’s Disease: no. E35 Poster Presentation |
Persistent Identifier | http://hdl.handle.net/10722/165649 |
DC Field | Value | Language |
---|---|---|
dc.contributor.author | Zhu, J | en_US |
dc.contributor.author | Garcia-Barcelo, MM | en_US |
dc.contributor.author | Tam, PKH | en_US |
dc.contributor.author | Lui, VCH | en_US |
dc.date.accessioned | 2012-09-20T08:21:40Z | - |
dc.date.available | 2012-09-20T08:21:40Z | - |
dc.date.issued | 2012 | en_US |
dc.identifier.citation | The 3rd International Symposium on Development of the Enteric Nervous System: Cells, Signals and Genes, Hong Kong, 25-28 March 2012 | en_US |
dc.identifier.uri | http://hdl.handle.net/10722/165649 | - |
dc.description | Session - Genetics of Hirschsprung’s Disease: no. E35 | - |
dc.description | Poster Presentation | - |
dc.description.abstract | Background 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.language | eng | en_US |
dc.relation.ispartof | ENS 2012 Meeting | en_US |
dc.title | RET transcriptional regulation by HOXB5 in Hirschsprung’s disease | en_US |
dc.type | Conference_Paper | en_US |
dc.identifier.email | Zhu, J: jiangzhu@hku.hk | en_US |
dc.identifier.email | Garcia-Barcelo, MM: mmgarcia@hku.hk | en_US |
dc.identifier.email | Tam, PKH: paultam@hku.hk | en_US |
dc.identifier.email | Lui, VCH: vchlui@hku.hk | en_US |
dc.identifier.authority | Garcia-Barcelo, MM=rp00445 | en_US |
dc.identifier.authority | Tam, PKH=rp00060 | en_US |
dc.identifier.authority | Lui, VCH=rp00363 | en_US |
dc.identifier.hkuros | 211263 | en_US |