Molecular Characterization of the Mouse Cytoglobin

This dissertation, "Molecular Characterization of the Mouse Cytoglobin" by Kwok-fai, Joseph, Chow, 周國輝, 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 CHARACTERIZATION OF THE MOUSE CYTOGLOBIN Submitted by Chow Kwok Fai Joseph For the degree of Doctor of Philosophy at The University of Hong Kong in July 2006 Cytoglobin (Cygb) is a hexacoordinated globin which belongs to the globin family which also consists of myoglobin, hemoglobin, and neuroglobin. Phylogenetic analysis has revealed that Cygb shared a common phylogenetic ancestry with myoglobin which diverged from an ancient globin 450 million years ago (mya). Cygb is found to be expressed in most tissues and at low levels in neuronal tissues. Cytoglobin is demonstrated to be up-regulated upon hypoxia, in liver fibrosis and is related to cancer. Recent evidence suggests that Cygb is more likely to be an anti-oxidant to protect against reactive oxygen species. However, the exact physiological role has yet to be defined. In this study, we analyzed the genomic DNA region flanking the 5'and 3'end of the mouse Cygb gene by in silico methods, and attempted to identify the promoter region and the distal regulatory element of the mouse Cygb gene experimentally. 5' unidirectional deletion assays demonstrated that the proximal promoter elements of mouse Cygb gene were located within the region of -519bp to -396bp upstream to the translation start site. Site-directed mutagenesis showed that mutations at the Sp motifs at -111bp, -181bp and -202bp remarkably decreased the promoter activities by 69.9%, 74.8%, and 44.6% in NIH/3T3 cells and by 54.9%, 58% and 41.7% in Hela cells respectively. These Sp1 and Sp3 binding motifs were further confirmed by gel shift assays. Over expression of Sp1 and Sp3 in cell cultures revealed that the Sp proteins have an additive effect on the expression of the mouse Cygb gene promoter. An increase of 7.8 to 15.5 folds of promoter activities were observed in NIH/3T3 cells co-transfected with different amounts of Sp expression plasmids. We have mapped DNase I hypersensitive sites (HSs) encompassing 90 kilobases of the Cygb locus in the fetal mouse liver and brain at 14.5 days. The results showed that several HSs were sensitive to the DNase I digestion. These sites were located at +73.1kb, +72.1kb, +61.3kb, +50.1kb, +29.3kb and -18.4kb with respect to the translation start site of the mouse Cygb gene. This result demonstrated that an open chromatin conformation was involved during the activation of the Cygb locus. Using the chromatin conformation capture (3C) technique, we demonstrated that the mouse Cygb gene interacts with its flanking region and neigbouring genes within the 240kbp locus during transcriptional activation. The 3C results showed that the St6galnac2 and Rik genes located upstream of the Cygb gene were in close spatial proximity, indicating that looping or formation of the active chromatin hub (ACH) may be involved in the transcriptional regulation of Cygb gene. In summary, our results pinpointed that Cygb gene expression is up-regulated by transcription factors, Sp1 and Sp3. Distal regulatory regions located at -31.2kbp to -42.6kbp upstream of Cygb gene may directly interact with its promoter to form a holocomplex, the ACH, prior to the activation of the gene. DOI: 10.5353/th_b3830029 Subjects: Globin Genomes Mice - Molecular aspects