Identifying novel transcriptional regulatory elements of HLA-A alleles through the evaluation of the 5’ un-translated region sequences.

Abstract

Sub-Saharan Africa holds approximately half the population living with human immunodeficiency virus (HIV) in the world (~19.6 million), of which around 7.2 million cases are found in South Africa. Although antiretroviral therapy can suppress viral loads to below detectable levels in most cases, drug resistance is a growing problem. Therefore, identifying novel treatment strategies are warranted against HIV. The strongest human genetic associations with HIV disease have been found within the human leukocyte antigen (HLA) region. The expression levels of various HLA genes have been associated with HIV disease outcomes. Increased HLA-A mRNA expression results in poor HIV outcomes due to the inhibition of natural killer (NK) cells since high mRNA expression of HLA-A results in high protein expression of HLA-E which serves as an inhibitory receptor for NK cells. Identifying factors that regulate the expression of HLA-A has the potential to serve as an avenue for HIV drug target sites. DNA methylation has previously been identified as one of the factors responsible for HLA-A expression regulation. In this study, we aimed to identify additional regulatory mechanisms for the HLA-A gene. The identification of a putative CCCTC-binding factor (CTCF) binding site upstream of HLA-A suggested that CTCF may play a role in regulation of HLA-A. Sequence alignments about 2 kilobases (2KB) upstream of the transcriptional start site (TSS) were analysed for polymorphisms that associate with HLA-A expression. Six HLA-A promoter variants (rs9260084, rs9260086, rs9260092, rs9260101, rs9260116 and rs41560714) were observed to significantly associate with HLA-A mRNA expression. However, only one single nucleotide polymorphism (SNP), rs9260084 (-993G>A), was predicted to disrupt a CTCF binding site. Despite the predicted disrupted binding site, using a chromatin immunoprecipitation (ChIP) assay, we did not detect any difference in CTCF binding across the -993 G>A variants. Additional transcriptional regulators, Nuclear Factor 1 (NF1), Ras related protein (RAP1) and glucocorticoid receptor (GR), were predicted to have differential binding to -993G>A, -226G>A and -885C>G, respectively. The results provided here serve as a basis for further studies exploring the role HLA-A promoter variants have in regulating HLA-A expression. These variants may serve as potential target sites for future therapeutic intervention against HIV.