Expression of the multiple sclerosis-associated MHC class II Allele HLA-DRB1*1501 is regulated by vitamin D

Abstract

Multiple sclerosis (MS) is a complex trait in which allelic variation in the MHC class II region exerts the single strongest effect on genetic risk. Epidemiological data in MS provide strong evidence that environmental factors act at a population level to influence the unusual geographical distribution of this disease. Growing evidence implicates sunlight or vitamin D as a key environmental factor in aetiology. We hypothesised that this environmental candidate might interact with inherited factors and sought responsive regulatory elements in the MHC class II region. Sequence analysis localised a single MHC vitamin D response element (VDRE) to the promoter region of HLA-DRB1. Sequencing of this promoter in greater than 1,000 chromosomes from HLA-DRB1 homozygotes showed absolute conservation of this putative VDRE on HLA-DRB1*15 haplotypes. In contrast, there was striking variation among non-MS-associated haplotypes. Electrophoretic mobility shift assays showed specific recruitment of vitamin D receptor to the VDRE in the HLA-DRB1*15 promoter, confirmed by chromatin immunoprecipitation experiments using lymphoblastoid cells homozygous for HLA-DRB1*15. Transient transfection using a luciferase reporter assay showed a functional role for this VDRE. B cells transiently transfected with the HLA-DRB1*15 gene promoter showed increased expression on stimulation with 1,25-dihydroxyvitamin D3 (P = 0.002) that was lost both on deletion of the VDRE or with the homologous "VDRE" sequence found in non-MS-associated HLA-DRB1 haplotypes. Flow cytometric analysis showed a specific increase in the cell surface expression of HLA-DRB1 upon addition of vitamin D only in HLA-DRB1*15 bearing lymphoblastoid cells. This study further implicates vitamin D as a strong environmental candidate in MS by demonstrating direct functional interaction with the major locus determining genetic susceptibility. These findings support a connection between the main epidemiological and genetic features of this disease with major practical implications for studies of disease mechanism and prevention.

Figure 1. HLA-DRB1 promoter.

Sequence shown is that for HLA-DRB1*15. Important regulatory elements (S, X and Y Boxes) are highlighted.

Figure 2. In vitro binding of VDR protein to the HLA-DRB1*15 VDRE.

Electrophoretic mobility shift assay showing binding of recombinant VDR and retinoic acid receptor beta (RXR) to radiolabelled oligoduplex probe corresponding to the VDRE in the proximal HLA-DRB1 promoter region for the HLA-DRB*15 haplotype. Two specific complexes are indicated, denoted I and II, together with a supershifted complex shown by an * symbol in the presence of antibody to VDR.

Figure 3. VDR is recruited to HLA-DRB1*15 VDRE in PGF cells.

Chromatin immunoprecipitation experiment using PGF cells either unstimulated (○) or after stimulation with 1,25-dihydroxyvitamin D3 (•). Input controls are shown (lanes 1 and 2), mock antibody immunoprecipitated controls (lanes 3 and 4) and VDR primary antibody immunoprecipitated DNA (lanes 5 and 6).

Figure 4. Reporter gene analysis of DRB1 promoter VDRE.

Raji B cells were transiently transfected with pGL3 luciferase constructs as indicated together with pRL_TK to normalise luciferase activity. Open bars indicate resting cells, grey shaded bars results following stimulation of transfected cells with 1,25-dihydroxyvitamin D3. Mean+/−SD of three independent transient transfection experiments are shown, each performed in quadruplicate.