In recent years, the previously neglected 3’ untranslated region (UTR) has gained recognition as a critical region in the regulation of gene expression. Numerous studies have been conducted on disease-associated 3’ UTR variants to elucidate unidentified regulatory elements within the 3’ UTR. An example of an unresolved 3’ UTR variant is the G801A polymorphism (rs1801157) occurring within the 3’ UTR of the CXCL12β transcript isoform. The CXCL12 gene encodes a C-X-C motif chemokine 12 (CXCL12) that functions as a ligand for the chemokine (C-X-C motif) receptor 4 (CXCR4), which is involved in the entry of the human immunodeficiency virus (HIV) into host cells. Correspondingly, previous association studies have indicated a potential correlation between the A allele of the G801A polymorphism and increased resistance to the progression of acquired immunodeficiency syndrome (AIDS). This suggests that the G801A polymorphism may be linked to a regulatory element in the 3’ UTR, which modulates the expression of CXCL12β, and therefore plays an intermediary role in its reported association with HIV/AIDS.
In order to identify the regulatory elements that may be associated with the G801A polymorphism, a bioinformatic approach was adopted by utilising a webserver developed in the Brown Lab called ‘Scan For Motifs’. However, only a 26 base pair (bp) region of high conservation was identified at the site of the G801A polymorphism. Consequently, three variable lengths of the CXCL12β 3’ UTR, containing the highly conserved region with either alleles at the G801A polymorphism, were cloned downstream from the luciferase gene (luc+) in pGL3MS2site/basic cloning vectors to yield three sets of reporter gene constructs. The desired CXCL12β 3’ UTR inserts were prepared by either the polymerase chain reaction (PCR) amplification of said sequences from HepG2 genomic DNA (gDNA) or appropriate oligonucleotide design. Site-directed mutagenesis was implemented when applicable using PCR-based methods such as the ‘megaprimer’ method and the overlap extension method, to create a polymorphic counterpart for each reporter gene construct. Prepared reporter gene constructs and previously characterised control plasmids were transiently co-transfected with a renilla luciferase reporter gene construct (phRL-SV40) using Lipofectamine® 2000 into HeLa cells and HEK293T. The transfected cells were harvested after a 48-hour incubation period for subsequent luciferase assays in order to measure differences in gene expression with respect to the G801A polymorphism. Changes in gene expression in response to different experimental conditions were also analysed through the application of two independent stimuli: the replenishment of foetal bovine/calf serum (FBS/FCS) (10%) and the premature harvest of cells after 24 hours. The amount of DNA transfected was also standardised through the co-transfection of pUC18, a non-coding plasmid.
In contrast to initial expectations, different levels of gene expression were not observed between each pair of reporter gene constructs with allelic differences at the G801A polymorphism. In support of the experimental results, levels of gene expression in response to two different conditions behaved in agreement with initial expectations. Likewise, the standardisation of the amount of DNA transfected using pUC18 co-transfection only increased the gene expression associated with the larger plasmid relative to the other smaller DNA constructs. Despite the negative results obtained from a series of luciferase assays, the functional implications associated with the G801A polymorphism was not conclusively determined as absent given the conflicting indications reported in the wider literature. Further experimental analyses are required to confirm or reject the functional consequences of the G801A polymorphism. If there is a functional impact associated with allelic differences at the G801A polymorphism, the next step would be to elucidate the responsible regulatory element in the context of its associated diseases.
