[Formulae and special characters can only be approximated. Please see the pdf version of this abstract for an accurate reproduction.] Tumor necrosis factor (TNF) is a pleiotropic cytokine that mediates a long list of immunological and pathophysiological processes. TNF is produced by a wide variety of cells including immune and non-immune cells, however in most cell types TNF is not expressed prior to stimulation. The function of TNF is mediated via its trimeric domain by binding to TNF receptors that are found on most types of cells, especially of the haematopoietic systems, hence transpiring its effects on a wide variety of cells and organ systems. The cytotoxic (apoptosis) and pro-inflammatory (differentiation, proliferation and activation) functions of TNF are protective but can also result in pathological or deleterious consequences. A biallelic G to A transition polymorphism in the promoter region of TNF at nucleotide position 308 from the transcription start site is suggested to be involved in differential transcriptional regulation of TNF expression. The high TNF producing 308A allele is associated with susceptibility to or worse outcome of many infectious diseases in addition to autoimmune and other pathophysiological conditions. A previous study in our laboratory observed a selective affinity towards the polymorphic 308A allele by an EMSA protein(s) complex, named E. Several other protein complexes were found along with complex E and one of them was identified as Sp1. The identification of complex E was unsuccessful but it was hypothesized to play a major role as transcriptional activator in 308A allele individuals hence transpiring its effect in various pathophysiological states. In this study, the EMSA complexes observed in the TNF promoter region between nucleotides 322 to 283, encompassing the 308 polymorphism, is characterized. EMSA using mutated oligonucleotides mapped the binding sites of complexes B, C, D and E. TRANSFAC database search in addition to previous work revealed the identity of complex C as Sp1 but the rest of complexes remained unknown. Moreover, in contrast to our previous study, the protein(s) in the complex E was found to preferentially bind 308G nucleotide hence posing as a transcriptional repressor, resulting in decreased production state of TNF in 308G allele individuals than 308A allele individuals. In order to characterize putative transcription factors binding to the promoter region, first the biochemical characteristics such as the effects of temperature, salts and cations on DNA binding ability of EMSA complexes were studied. EMSA complexes B, C, DI and E required cations, probably Zn+2, to bind DNA. By optimizing a technique that couples EMSA with SDS-PAGE, the molecular weight of C, DI and E was determined. A novel technique that couples EMSA with IEF determined the pI of complexes B, C, D, DI and E. Although a commonly used technique of identifying unknown DNA-binding protein of interest, Yeast One-Hybrid assay, did not identify complex E, the novel identification method involving chromatography, two-dimensional electrophoresis, EMSA, mass spectrometry and database interrogation successfully identified TNF EMSA complex E as transcription factor Ying Yang 1 (YY1). Supershift EMSA confirmed complex E as YY1. In addition, the supershift assay showed presence of Sp1 and Sp3 in complex C. Similarly, complex DI is identified as Sp3. The novel method in identifying DNA-binding proteins is particularly useful as this technique allows identification of protein seen in EMSA without the need of extensive identification process. YY1 binds to a 6 base pair sequence, 5? TTGAGG 3?, from nt 295 to 290 of TNF promoter. The loss of affinity in 308A allele is caused by transition of underlined G nucleotide to A. The determined and described molecular weight of YY1 in literature is 60 kDa while the theoretical weight is 45 kDa. Both the determined and theoretical pI of YY1 is 5.8. YY1 is a multifunctional transcription factor implicated in both positive and negative regulation of gene expression as well as in initiation of transcription. It is ubiquitously expressed in growing, differentiated, and growth-arrested cells. Although future experiment is yet to establish in vivo presence of YY1 in TNF promoter, our study so far provides convincing evidence that the putative transcription factor that has selective affinity towards 308G allele is indeed YY1.
| Identifer | oai:union.ndltd.org:ADTP/221016 |
| Date | January 2003 |
| Creators | Woo, Andrew Jonghan |
| Publisher | University of Western Australia. School of Biomedical and Chemical Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright Andrew Jonghan Woo, http://www.itpo.uwa.edu.au/UWA-Computer-And-Software-Use-Regulations.html |
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