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This dissertation, Molecular Analysis of the Roles of NRSF in TUBB3 Transcription Control by Yi, Mou, 牟奕, 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: Abstract of thesis entitled Molecular Analysis of the Roles of NRSF in TUBB3 Transcription Control Submitted by Mr Mou Yi For the Degree of Master of Philosophy At the University of Hong Kong In April 2007 The neuronal specific class III, beta tubulin (TUBB3) is one of the earliest neuronal markers during the central nervous system (CNS) development whose presence is associated with terminal mitosis. Previous reports suggested the TUBB3 proximal promoter affected the differentiation stage-specific transcription. However, the known cis-elements in the TUBB3 proximal promoter, like SP1 and activating protein 1 (AP- 1), had no direct effects on tissue-specific transcription and could be because the analysis region was limited to the 5' flanking region. In this study, the TUBB3 gene of rat, mouse and human, including both 5' and 3' 5kb-flanking regions were analyzed by Match program and a common cis-element highly identical to neuronal restrictive silencer element (NRSE) consensus sequence was identified. This NRSE element was conserved in TUBB3 genes of dog, cattle, chimpanzee and rhesus, which shared similar gene structure and the C-terminal isotype sequence to mice, rat and human TUBB3, and suggested that it might be conserved during evolution. The interaction of TUBB3 NRSE and NRSF was examined by electrophoretic mobility shift assay (EMSA). Binding of wild type biotin labeled TUBB3 NRSE could be inhibited by the higher concentration of the unlabeled experiment-confirmed iNRSE (100X), but not unlabeled mutant type (100X), suggesting that mice TUBB3 NRSE could interact with neuron-restrictive silencer factor (NRSF) in vitro. Previous reports suggested NRSE containing region was associated with DNA methylation and indeed TUBB3 NRSE was located in a cytosine--phosphate diester--guanine (CpG) rich region as identified by CpGplot. To evaluate the methylation state in TUBB3 NRSE containing region, both bisulfite genomic sequencing and combined bisulfite restriction assay (COBRA) were performed for DNA of cell lines including N2a, Hepa, C2C12 and adult mouse tissues, including liver, skeletal muscle, heart and testis. In C2C12, Hepa, and N2a, all the CpG positions were methylated whereas liver, skeletal muscle, heart had no DNA methylation and testis had methylation at one position. st In summary, NRSE was a conserved element in the 1 intron of TUBB3, which could interact with NRSF in vitro, and NRSE in TUBB3 was associated with DNA methylation in N2A, C2C12 and Hepa cell lines but not in adult liver, heart and skeletal muscles. The different methylation status suggested that silencing of TUBB3 might involve additional epigenetic modifications. (461 words) ii DOI: 10.5353/th_b3871279 Subjects: TubulinsTranscription factorsGenetic regulationNeurons

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