Jiang Zhu

This dissertation, "RET Transcriptional Regulation by HOXB5 in Hirschsprung's Disease" by 朱江, Jiang, Zhu, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Hirschsprung's disease (HSCR) is the major enteric nervous system anomaly affecting newborns with high incidence in Asians. HSCR is a congenital complex genetic disorder characterized 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 cis-elements in the promoter and intron of RET gene are crucial for RET expression. Abnormal RET expression leading to insufficient RET activity causes defective development of the enteric nervous system and is implicated in the pathogenesis of the Hirschsprung's disease. The human homeobox B5, HOXB5, has an important role in the development of enteric neural crest cells, and perturbation of HOXB5 signaling causes reduced RET expression and 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 including SOX10 and NKX2-1 that have been implicated in RET expression and HSCR. In this study, I demonstrated that HOXB5 binds to the RET promoter and regulates RET expression. HOXB5 and NKX2-1 forma protein complex and mediate RET expression in a synergistic manner. In contrast, HOXB5 cooperates in an additive manner with SOX10in trans-activation from RET promoter. ChIP assay further revealed that HOXB5 and NKX2-1 interact with the same chromatin region proximate to the transcription start site of RET, suggesting that these two factors may interact with each other and regulate the transcription of RET. In silico analysis, EMSA and ChIP analysis showed that HOXB5 also binds to an enhancer element (MCS+9.7)in the intron 1 of RET gene, and HSCR-associated SNPs have been identified in this enhancer element. To further access the HOXB5 trans-activity onMCS+9.7, RET mini-gene was constructed by ligating the RET promoter to the 5'and MCS+9.7 to the 3'of a luciferase gene. Luciferase assay indicated that MCS+9.7 enhances the HOXB5 trans-activation from the RET promoter. In addition, previously identified HSCR-associated SNPs inintron 1 markedly reduce the HOXB5 trans-activation from the RET mini-gene. Moreover, the result of IP-LC-MS/MS indicated that HOXB5 could form protein-protein complexes with nuclear proteins involved in the transcription initiation of genes with TATA-less promoter. This evidence suggested that HOXB5 may cooperate with other activators or co-factors in the remodeling of chromatin conformation, local histone modification and recruitment of essential transcription factors for RNA Polymerase II based transcription from TATA-less promoter, such as RET. My data indicated 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 a reduction of RET expression and contribute to the manifestation of the HSCR phenotype. DOI: 10.5353/th_b4979919 Subjects: Homeobox genes Protein-tyrosine kinase Hirschsprung's disease - Genetic aspects Genetic transcription - Regulation

RET TRANSCRIPTIONAL REGULATION

Dajun Guo

Yeol Je Cho

Special Interest Categories: Pure and applied mathematics, physics, optimisation and control, mechanics and engineering, nonlinear programming, economics, finance, transportation and elasticity. The usual method used in studying nonlinear problems such as topological method, variational method and others are generally only suited to the nonlinear problems with continuity and compactness. However, a lots of the problems appeared in theory and applications have no continuity and compactness, For example, differential equations and integral equations in infinite dimensional spaces, various equations defined on unbounded region are generally having no compactness. The problems can been divided into three types as follows: (1) Without using compact conditions but only using some inequalities related to some ordering, the existence and uniqueness of the fixed point for increasing operators, decreasing operators and mixed monotone operators, and the convergence of the iterative sequence are obtained. Also, these results have been used to nonlinear integral equations defined on unbounded regions. (2) Without using continuity conditions but only using a very relaxed weakly compact conditions, some new fixed point theorem of increasing operators are obtained. We have applied these results to nonlinear equations with discontinuous terms. (3) They systemly use the partial ordering methods to nonlinear integro-differential equations (include impulsive type) in Banach space.

Jiang Zhu

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