|M.Sc Student||Avigad Ornit|
|Subject||The Effect of Human NRAMP1 Promoter Polymorphism on|
|Department||Department of Biotechnology and Food Engineering||Supervisor||Professor Ben-Zion Levi|
|Full Thesis text|
Nramp1 is exclusively expressed in monocyte/macrophage cells with a unique role in innate resistance to intraphagosomal pathogens. In mice, we have shown in previous studies that the restricted expression of Nramp1 is regulated by a myeloid cell-specific transcription factor termed IRF-8. Furthermore, the induction of Nramp1 expression in activated macrophages is accompanied by a promoter shift from a repression state elicited by c-Myc to an activation state elicited by the induction of IRF-8 in activated macrophages. This transition from repression to activation is facilitated by a competitive protein-protein interaction with the transcription factor Miz-1. IRF-8 interacts with Miz-1 and these two factors in conjunction with a third partner, PU.1, which is also hematopoietic specific, synergistically activate Nramp1. In humans, the promoter region of NRAMP1 possesses a polymorphism within a possible enhancer element containing a Z-DNA forming dinucleotide repeat. Other studies demonstrated that, in the absence of exogenous stimuli, allele 1 is a poor promoter of NRAMP1 expression, while allele 3 drives high expression. Moreover, allele 3 is activated significantly in response to IFN-γ and LPS, whereas allele 1 responses mildly to these stimuli.
Here we demonstrate for the first time using luciferase reporter gene assay that Miz-1, IRF-8 and PU.1 can activate the human allele 3 promoter to a level that is high from allele 1. This laid the molecular basis of the altered Nramp1 expression regulation driven by the different alleles, indicating that the Z-DNA forming repeat polymorphism affects the binding capacity of transcription factors and their synergistic interactions. Using 5' progressive deletions of the human NRAMP1 allele 3 promoter combined with luciferase reporter gene assay we identified regions important for Miz-1, IRF-8 and PU.1 binding and a suppressor element of Miz-1. Site directed mutagenesis revealed a PU.1 or IRF-8 binding site.
To investigate the effect of human Nramp1 promoter polymorphism on innate resistance to intraphagosomal pathogens we have introduced to Raw264.7 cells (null for Nramp1) the functional murine Nramp1 under the regulation of human allele 1 or 3 promoters. This was done by classic retroviral vectors and self inactivating vectors. However, both were found to be unsuitable because of strong constitutive expression of exogenic Nramp1 derived by the 5'LTR viral promoter. Stable plasmid transfection system demonstrated high basal expression level driven by allele 3 promoter compared to allele 1. This suggested that the Z-DNA forming repeat polymorphism may affect Miz-1/c-Myc interactions, resulted in altered Nramp1 inhibition by c-Myc.