Autoimmune vitiligo is associated with gain-of-function by a transcriptional regulator that elevates expression of HLA-A*02:01 in vivo

Abstract

HLA-A is a class I major histocompatibility complex receptor that presents peptide antigens on the surface of most cells. Vitiligo, an autoimmune disease in which skin melanocytes are destroyed by cognate T cells, is associated with variation in the HLA-A gene; specifically HLA-A*02:01, which presents multiple vitiligo melanocyte autoantigens. Refined genetic mapping localizes vitiligo risk in the HLA-A region to an SNP haplotype ∼20-kb downstream, spanning an ENCODE element with many characteristics of a transcriptional enhancer. Convergent CTCF insulator sites flanking the HLA-A gene promoter and the predicted transcriptional regulator, with apparent interaction between these sites, suggests this element regulates the HLA-A promoter. Peripheral blood mononuclear cells from healthy subjects homozygous for the high-risk haplotype expressed 39% more HLA-A RNA than cells from subjects carrying nonhigh-risk haplotypes (P = 0.0048). Similarly, RNAseq analysis of 1,000 Genomes Project data showed more HLA-A mRNA expressed in subjects homozygous for the high-risk allele of lead SNP rs60131261 than subjects homozygous for the low-risk allele (P = 0.006). Reporter plasmid transfection and genomic run-on sequence analyses confirm that the HLA-A transcriptional regulator contains multiple bidirectional promoters, with greatest activity on the high-risk haplotype, although it does not behave as a classic enhancer. Vitiligo risk associated with the MHC class I region thus derives from combined quantitative and qualitative phenomena: a SNP haplotype in a transcriptional regulator that induces gain-of-function, elevating expression of HLA-A RNA in vivo, in strong linkage disequilibrium with an HLA-A allele that confers *02:01 specificity.

Keywords: HLA; autoimmune disease; enhancer; transcription; vitiligo.

Fig. 1.

Vitiligo association in the HLA-A region of human chromosome 6p. Nucleotide positions, HLA-A transcriptional orientation, and the 22 SNPs that define the vitiligo high-risk haplotype are shown. Layered H3K27Ac, H3K4Me1, and H3K4Me3 marks, hidden Markov model chromatin state segmentation (ChromHMM), DNase I hypersensitive site cluster (DNase I Clusters), and transcription factor chromatin immunoprecipitation sequencing (Txn Factor ChIP-seq) data are from ENCODE (14). For layered H3K27Ac, H3K4Me1, H3K4Me3 marks, data are shown for the seven cell lines studied by ENCODE. For ChromHMM, red indicates active promoters, orange indicates strong enhancers, and blue indicates an insulator. Data shown are for GM12878 lymphoblastoid cells. For DNase clusters, darkness indicates relative signal strength in 125 cell types from ENCODE (V3). For Txn factor ChIP-seq, darkness indicates relative signal strength of aggregate binding of 161 transcription factors, and green bars indicate ENCODE Factorbook (15) canonical motifs for specific transcription factors.

Fig. 2.

HLA-A RNA in subjects homozygous for the high-risk and nonhigh-risk HLA-A region haplotypes. HLA-A RNA was measured in peripheral blood RNA from subjects homozygous for the high-risk MHC class I haplotype (nos. 1–3) or nonhigh-risk haplotypes (nos. 4–10) using two different qPCR assays (Table S2), and was normalized to 18S rRNA. Black bars, primer set 1; gray bars, primer set 2; each shows the mean of triplicate assays.

Fig. 3.

Normalized HLA-A mRNA expression data from the 1,000 Genomes Project subjects classified by genotype of lead HLA-A region SNP rs60131261. RNAseq mRNA profiles for 358 EUR subjects of the 1,000 Genomes Project were obtained along with their genotypes for rs60131261 and subjected to ANOVA. RPKM, reads per kilobase of transcript per million mapped reads. The gray box denotes the first through third quartile and the horizontal line in the box denotes the median. Black squares indicate means. Short horizontal lines denote 99% confidence limits. Crosses denote outliers.

Fig. 4.

Hi-C analysis of the HLA-A region of chromosome 6p. In situ Hi-C data for the HLA-A region of chromosome 6p of GM12878 lymphoblastoid cells (21) were analyzed by X-Y comparison using Juicebox (www.aidenlab.org/juicebox/). RefSeq genes, CTCF binding sites and orientation, DNase I hypersensitive sites, and H3K27ac, H3K4me1, and H3K4me3 marks are indicated. The box denotes the segment from HLA-A through the predicted downstream transcriptional regulatory element. HLA-A is the only protein coding gene in the region; HLA-H, HCG4B, HCG9, and ZNRD-AS1 are all nonprotein-coding RNAs.

Fig. 5.

GRO-seq data in the HLA-A region of chromosome 6p. Histogram of reads from HCT116 GRO-seq data shows transcription of both the HLA-A gene and a region 20 kb downstream of the gene coincident with the predicted downstream transcriptional regulatory element. Blue are reads on forward strand and red are reads on reverse strand. Reads from two 1-h replicates were summed from cells treated with control DMSO alone (Gene Expression Omnibus GSE53964). File is a Bedgraph with reads mapped and normalized to millions (22). DNase I clusters track (GM12878) is from ENCODE.

Fig. 6.

Transient transfection assay of promoter activities in the HLA-A downstream transcriptional regulatory element. Luciferase reporter constructs containing segments of the HLA-A downstream regulatory element inserted immediately upstream of the luc2 gene. (A) Full-length high-risk haplotypes (HR1, HR2) from subject 1 (orange and yellow) or full-length nonhigh-risk haplotypes (NHR1, NHR2) from subject 10. (B) Subfragments of the high-risk HR1 haplotype. (C) Subfragments of the high-risk HR2 haplotype. Arrowheads denote forward (F) and reverse (R) orientations relative to genomic orientation in chromosome 6. Relative light units denote fold-change of transcriptional activity relative to the pGL4.10 backbone plasmid. SEMs are indicated.

Fig. S1.

Transient transfection assay of enhancer activities in the HLA-A downstream regulatory region. (A) Luciferase reporter constructs containing the full-length HLA-A downstream regulatory region inserted immediately upstream of a minimal promoter (green) driving transcription of the luc2 gene. HR1, HR2, high-risk haplotypes from subject 1 (orange and yellow); NHR1, NHR2, nonhigh-risk haplotypes from subject 10 (blue and pale green). Arrowheads denote forward (F) and reverse (R) orientations relative to genomic orientation in chromosome 6. Relative light units denote fold-change of transcriptional activity relative to the pGL4.23 backbone plasmid. SEMs are indicated. (B) Subfragments of the high-risk HR1 haplotype. (C) Subfragments of the high-risk HR2 haplotype. (D) Full-length haplotypes inserted downstream of the promoterless luc2 reporter gene. Relative light units denote fold-change of transcriptional activity relative to the pGL4.10 backbone plasmid. (E) Full-length haplotypes inserted downstream of the minimal promoter:luc2 reporter gene. Relative light units denote fold-change of transcriptional activity relative to the pGL4.23 backbone plasmid.