In silico and functional analyses of the iron metabolism pathway

Thesis (PhD)--Stellenbosch University, 2013.ENGLISH ABSTRACT: Iron is an essential micronutrient that is an absolute requirement for correct cellular function in all eukaryotic organisms. However, ferrous iron has the ability to catalyze the formation of potentially toxic reactive oxygen species and regulation of iron metabolism is therefore of critical importance. Currently, there is little known about the co-ordinated regulation of the plethora of genes coding for proteins involved in this biochemical pathway, with the exception of the well characterized post-transcriptional IRE/IRP system. Regulation of gene expression in eukaryotic organisms is a highly intricate process. Transcriptional regulation is the first step and is controlled by the presence of specific cis-regulatory regions (cis-motifs), residing within the promoter region of genes, and the functional interactions between the products of specific regulatory genes (transcription factors) and these cismotifs. A combinatorial bioinformatic and functional approach was designed and utilized in this study for the analysis of the promoter architecture of genes of the iron metabolic pathway. The upstream non-coding region (~2 kb) of 18 genes (ACO1, CP, CYBRD1, FTH1, FTL, HAMP, HEPH, HFE, HFE2, HMOX1, IREB2, LTF, SLC11A2, SLC40A1, STEAP3, TF, TFRC, TFR2), known to be involved in the iron metabolism pathway, was subjected to computational analyses to identify regions of conserved nucleotide identity utilizing specific software tools. A subset of nine (CYBRD1, FTH1, HAMP, HFE, HFE2, HMOX1, IREB2, LTF, TFRC) of the genes were found to contain a genomic region that demonstrated over 75% sequence identity between the genes of interest. This conserved region (CR) is approximately 140 bp in size and was identified in each of the promoters of the nine genes. The CR was subjected to further detailed examination with comparative algorithms from different software for motif detection. Four specific cis-motifs were discovered within the CR, which were found to be in the same genomic position and orientation in each of the CR-containing genes. In silico prediction of putative transcription factor binding sites revealed the presence of numerous binding motifs of interest that could credibly be associated with a biological function in this pathway, including a novel MTF-1 binding site in five of the genes of interest. Validation of the bioinformatic predictions was performed in order to fully assess the relevance of the results in an in vitro setting. Luciferase reporter constructs for the nine CRcontaining genes were designed containing: 1) the 2 kb promoter, 2) a 1.86 kb promoter with the CR removed and 3) the 140 bp CR element. The expression levels of these three reporter gene constructs were monitored with a dual-luciferase reporter assay under standard culture conditions and simulated iron overload conditions in two different mammalian cell lines. Results of the luciferase assays indicate that the CR promoter constructs displayed statistically significant variation in expression values when compared to the untreated control constructs. Further, the CR appears to mediate transcriptional regulatory effects via an iron-independent mechanism. It is therefore apparent that the bioinformatic predictions were shown to be functionally relevant in this study and warrant further investigation. Results of these experiments represent a unique and comprehensive overview of novel transcriptional control elements of the iron metabolic pathway. The findings of this study strengthen the hypothesis that genes with similar promoter architecture, and involved in a common pathway, may be co-regulated. In addition, the combinatorial strategy employed in this study has applications in alternate pathways, and could serve as a refined approach for the prediction and study of regulatory targets in non-coding genomic DNA.AFRIKAANSE OPSOMMING: Yster is ‘n noodsaaklike mikrovoedingstof wat ‘n vereiste is vir korrekte sellulêre funksie in alle eukariotiese organismes. Yster (II) of Fe2+ het egter die vermoë om die vorming van potensiële toksies reaktiewe suurstof spesies te kataliseer en dus is die regulasie van die yster metaboliese padweg van kardinale belang. Tans is daar beperkte inligting oor koördineerde regulasie van die gene, en dus proteïene waarvoor dit kodeer, in hierdie padweg. ‘n Uitsondering is die goed gekarakteriseerde na-transkripsionele “IRE/IRP” sisteem. Regulasie van geenuitdrukking in eukariotiese organismes is ‘n ingewikkelde proses. Transkripsionele regulasie is die eerste stap en word beheer deur die teenwoordigheid van spesefieke cis-regulatoriese elemente (cis-motiewe), geleë in die promotor area van gene, en die funksionele interaksies wat plaasvind tussen die produkte van spesifieke regulatoriese faktore (of transkripsie faktore) en hierdie cis-motiewe. ‘n Gekombineerde bioinformatiese en funksionele benadering was ontwerp en daarna gebruik in dié studie vir die analise van die promotor argitektuur van gene wat ‘n rol speel in die yster metaboliese padweg. Die stroomop nie-koderende streek (~2 kb) van 18 gene (ACO1, CP, CYBRD1, FTH1, FTL, HAMP, HEPH, HFE, HFE2, HMOX1, IREB2, LTF, SCL11A2, SLC40A1, STEAP3, TF, TFRC, TFR2), bekend vir hul betrokkenheid in die yster metabolisme padweg, was bloodgestel aan bioinformatiese analises om die streke van konservering te identifiseer met die hulp van spesifieke sagteware. Slegs nege (CYBRD1, FTH1, HAMP, HFE, HFE2, HMOX1, IREB2, LTF, TFRC) van die geanaliseerde gene het ‘n genomiese area bevat wat meer as 75% konservering getoon het. Hierdie gekonserveerde area (GA) is 140 bp in lengte en is geïdentifiseer in elk van die promotors van die nege gene. Die GA was verder bloodgestel aan analises, met die hulp van spesifieke sgateware, wat gebruik maak van vergelykende algoritmes vir motief karakterisering. Vier cis-motiewe is identifiseer en kom voor in dieselfde volgorde en oriëntasie in elk van die gene. In silico voorspelling van moontlike transkripsie faktor bindingsplekke het getoon dat daar talle bindingsmotiewe van belang teenwoordig is en dié motiewe kan gekoppel word aan biologiese funksies in hierdie padweg, insluitend ‘n nuwe MTF-1 bindingsplek in vyf van die gene van belang. Die bioinformatiese analises is verder gevalideer om die relevansie van die resultate in ‘n in vitro sisteem ten volle te assesseer. Luciferase rapporteerder konstrukte is vir die nege gene ontwerp wat die volgende bevat: 1) die 2 kb promotor, 2) ‘n 1.86 kb promotor met die GA verwyder en 3) die 140 bp GA element. Die vlakke van uitdrukking van hierdie drie rapporteerder konstrukte was genormaliseer met ‘n dubbele-luciferase rapporteerder assay onder standaard kultuur kondisies en gesimuleerde ysteroorlading kondisies in twee verskillende soogdier sellyne. Resultate van die luciferase assays dui aan dat die GA promotor konstrukte statisties betekenisvolle variasie toon in vergelyking met die onbehandelde kontrole konstrukte. Verder, die GA blyk om transkipsionele regulatoriese effekte te medieer via ‘n yster-onafhanklike meganisme. Dit blyk duidelik dat die bioinformatiese voorspellings ook funksioneel getoon kon word en was dus relevant in dié studie en regverdig verdere ondersoek. Hierdie eksperimentele ontwerp verteenwoordig ‘n unieke en omvattende oorsig van nuwe transkripsionele beheer elemente wat voorkom in die yster metaboliese padweg. Die resultate van dié studie versterk die hipotese dat gene met soortgelyke promotor argitektuur en wat betrokke is in ‘n gemene padweg saam gereguleer kan word. Daarbenewens, die gekombineerde strategie wat in hierdie studie gebruik is het toepassings in alternatiewe metaboliese paaie, en kan dien as ‘n verfynde benadering vir die voorspelling en studie van die regulerende teikens in nie-koderende genomiese DNS.National Research Foundation (Thuthuka)Stellenbosch Universit

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