published_or_final_version / Biological Sciences / Master / Master of Philosophy

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. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

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The ATP-dependent chromatin remodeling complex SWR1 exchanges a variant histone H2A.Z-H2B dimer for a canonical H2A-H2B dimer at nucleosomes flanking histone-depleted regions, such as promoters. This localization of H2A.Z is conserved throughout eukaryotes. SWR1 is a 1 Mega-Dalton complex containing 14 different polypeptides, including the AAA+ ATPases Rvb1 and Rvb2. Using electron microscopy, we obtained the three-dimensional structure of SWR1 and mapped its major functional components. Our data show that SWR1 contains a single hetero-hexameric Rvb1/2 ring that, together with the catalytic subunit Swr1, brackets two independently assembled multi-subunit modules. We also show that SWR1 undergoes a large conformational change upon engaging a limited region of the nucleosome core particle. Our work suggests an important structural role for the Rvb1/2 ring and a distinct substrate-handling mode by SWR1, thereby providing the first structural framework for understanding the complex dimer-exchange reaction.

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<p> <p>&nbsp / </p> </p> <p align="left">This study utilized large-scale gene expression data to define the regulatory networks of genes expressing in the hippocampus to which multiple disease pathologies may be associated. Specific aims were: ident i fy key regulatory transcription factors (TFs) responsible for observed gene expression patterns, reconstruct transcription regulatory networks, and prioritize likely TFs responsible for anatomically restricted gene expression. Most of the analysis was restricted to the CA3 sub-region of Ammon&rsquo / s horn within the hippocampus. We identified 155 core genes expressing throughout the CA3 sub-region and predicted corresponding TF binding site (TFBS) distributions. Our analysis shows plausible transcription regulatory networks for twelve clusters of co-expressed genes. We demonstrate the validity of the predictions by re-clustering genes based on TFBS distributions and found that genes tend to be correctly assigned to groups of previously identified co-expressing genes with sensitivity of 67.74% and positive predictive value of 100%. Taken together, this study represents one of the first to merge anatomical architecture, expression profiles and transcription regulatory potential on such a large scale in hippocampal sub-anatomy.</p>

<p>This thesis reports on the development of a novel comprehensive database (Dragon Database of Genes Implicated in Esophageal Cancer, DDEC) as an integrated knowledge database aimed at representing a gateway to esophageal cancer related data. More importantly, it illustrates how the biocurated genes in the database may represent a reliable starting point for divulging transcriptional regulation, diagnostic markers and the biology related to esophageal cancer.</p>

<p>This thesis reports on the development of a novel comprehensive database (Dragon Database of Genes Implicated in Esophageal Cancer, DDEC) as an integrated knowledge database aimed at representing a gateway to esophageal cancer related data. More importantly, it illustrates how the biocurated genes in the database may represent a reliable starting point for divulging transcriptional regulation, diagnostic markers and the biology related to esophageal cancer.</p>

The CCAAT/Enhancer Binding Protein Beta (C/EBPβ) is part of the leucine zipper family of transcription factors and is involved in a myriad of processes including cellular proliferation and differentiation. C/EBPβ is expressed as three isoforms (LAP*, LAP, LIP), translated from a single mRNA by a leaky ribosomal scanning mechanism. While LAP* and LAP have activating functions, LIP is recognized as being a repressor of transcription due to its lack of activation domains. Numerous studies have shown that C/EBPβ acetylation state modulates its activity in a promoter-specific manner. For instance, the acetyltransferases GCN5/PCAF and the deacetylase complex mSin3A/HDAC1 regulate C/EBPβ activity on the C/EBPa promoter. GCN5/PCAF-mediated acetylation of C/EBPβ was shown to positively affect its transcriptional activity in a steroid-dependent mechanism via the glucocorticoid receptor (GR). GR relieves HDAC1 association from C/EBPβ by targeting the deacetylase for proteasomal degradation, hence favouring GCN5-mediated acetylation of C/EBPβ and allowing maximum activation capacity to be reached. In order to further elucidate C/EBPβ activation, I sought to characterize the interplay between GCN5 and HDAC1 in regulating C/EBPβ LAP/LIP activity during murine adipogenesis by identifying their binding domain in C/EBPβ. I identified a minimal domain located within regulatory domain 1 (RD1) of C/EBPβ that is required for both GCN5 and HDAC1 binding. Furthermore, the loss of the identified domain in C/EBPβ appears to partially mimic the GR effect, thus giving C/EBPβ a higher basal transcriptional activity that accelerates NIH 3T3 and 3T3 L1 adipogenesis. Moreover, I also showed that the LIP isoform inhibitory mode of action is partially mediated through the mSin3A/HDAC1 repressor complex, which gives LIP an active repressor function. In addition to LIP inhibitory function, I also showed that a cysteine residue located in LAP* negatively regulates its transactivating function during murine adipogenesis. Although RD1 of C/EBPβ has been suggested to act as a negative regulatory domain, I showed that only five residues are responsible for most of its inhibitory effect. Hence, in an attempt to further define sub-domains within RD1, I characterized a new positive regulatory domain at its N-terminal region, which seems to be required for C/EBPβ activity in a promoter-specific manner. In conclusion, this study not only supports previously hypothesized mechanisms by which C/EBPβ is regulated, but it also redefines the contribution of LAP*, LAP and LIP in regulating transcription. Most importantly, the results emphasize the countless possibilities by which C/EBPβ transactivation potential could be modulated during cellular differentiation.