Human-specific epigenetic variation in the immunological Leukotriene B4 Receptor (LTB4R/BLT1) implicated in common inflammatory diseases

Abstract

Background: Common human diseases are caused by the complex interplay of genetic susceptibility as well as environmental factors. Due to the environment's influence on the epigenome, and therefore genome function, as well as conversely the genome's facilitative effect on the epigenome, analysis of this level of regulation may increase our knowledge of disease pathogenesis.

Methods: In order to identify human-specific epigenetic influences, we have performed a novel genome-wide DNA methylation analysis comparing human, chimpanzee and rhesus macaque.

Results: We have identified that the immunological Leukotriene B4 receptor (LTB4R, BLT1 receptor) is the most epigenetically divergent human gene in peripheral blood in comparison with other primates. This difference is due to the co-ordinated active state of human-specific hypomethylation in the promoter and human-specific increased gene body methylation. This gene is significant in innate immunity and the LTB4/LTB4R pathway is involved in the pathogenesis of the spectrum of human inflammatory diseases. This finding was confirmed by additional neutrophil-only DNA methylome and lymphoblastoid H3K4me3 chromatin comparative data. Additionally we show through functional analysis that this receptor has increased expression and a higher response to the LTB4 ligand in human versus rhesus macaque peripheral blood mononuclear cells. Genome-wide we also find human species-specific differentially methylated regions (human s-DMRs) are more prevalent in CpG island shores than within the islands themselves, and within the latter are associated with the CTCF motif.

Conclusions: This result further emphasises the exclusive nature of the human immunological system, its divergent adaptation even from very closely related primates, and the power of comparative epigenomics to identify and understand human uniqueness.

Figure 1

Genome-wide view of human s-DMRs. Pooled DNA from uncultured whole blood cell samples including both sexes (all 60% male) were analyzed for each species. Methylated fragments via MeDIP-seq were aligned to the appropriate species’ genome; then peaks were called within these, using two peak-calling algorithms (MACS v1.4.1 [47] and BayesPeak v1.8 [48]). Triangulation reciprocal LiftOver [49] comparison then identified human s-DMRs. Outer ring, human chromosomes; blue ring, hypermethylated human s-DMRs (15,858); yellow ring, hypomethylated human s-DMRs (22,758); green ring, CNV density; inner ring, gene density.

Figure 2

Proportional genomic annotation coverage of s-DMRs compared with HapMap B-lymphocyte EBV GM12878 ChromHMM data[68]. (A) Hypomethylated s-DMRs; (B) hypermethylated s-DMRs. Various genomic annotations were significantly enriched, as defined by this segmentation analysis, calculated via the Genomic Hyperbrowser [54] (P-value overlap MC * < 0.05, ** < 0.005).

Figure 3

Subcategorization of repeat element increase in hypermethylated s-DMRs via Epiexplorer in comparison with a reshuffled control set[53](medium overlap ≥10%). Increased hyper s-DMRs within the SINE group are identified, which comprises predominately Alus. LINE, long interspersed nucleotide element; LTR, long terminal repeat; RC, rolling circle; snRNA, short nuclear RNA; srpRNA, signal recognition particle RNA.

Figure 4

Human s-DMRs in CpG islands versus CpG island shores. Number of hypo- and hypermethylated s-DMRs for island (77 and 45) and shore (821 and 431), respectively, is corrected for proportion of genome size (genomic space for islands = 23.8 Mb and for shores = 89.5 Mb). Directly comparing island and shore s-DMRs with regard to possible locations of hypomethylated s-DMRs (all co-locating chimpanzee and rhesus macaque peaks in these regions), χ²P = 2.90 × 10^-29, and hypermethylated s-DMRs (from all location of human peaks in these regions), χ²P = 1.93 × 10^-8; combined χ²P = 1.80 × 10^-32. Therefore, human-specific peaks are more likely than non-human-specific peaks to reside within CpGi shores.

Figure 5

Sequence divergence between human and chimpanzee within peak regions. There was no significant difference (Wilcoxon P > 0.05 for both) between s-DMRs in islands and all chimpanzee and rhesus macaque (C&M) combined peaks or all human peaks in islands.

Figure 6

Change in transcription factor motif binding prediction within s-DMRs between primates calculated via TRAP [[55]] with TRANSFAC motifs[56]. (A,B) Difference in binding prediction (total corrected Benjamini-Hochberg -log₁₀P-value) between human and chimpanzee (y-axis) and human and rhesus macaque (x-axis) for each motif within the total set of hypomethylated (A) and hypermethylated (B) CpGi s-DMRs. Known TFBSs with MDR effects are highlighted in color (SP1 in red, CTCF in blue, RFX motif family in green). Both CTCF motifs show a consistent increase in the hypomethylated s-DMRs, as well as a consistent decrease in the hypermethylated DMRs, with respect to human. The MeCP2 motif is identified as a strongly increased outlier in the hypermethylated s-DMRs (orange).

Figure 7

Comparative DNA methylation of LTB4R visualized in the UCSC browser. Human hypomethylated s-DMRs (yellow) are shown in the promoter CpGi (CpG: 99) of LTB4R (major isoform LTB4R-001 outlined in red). Methylation scale is in reads per millions (RPM) for each species from MeDIP-seq (human, light blue; chimpanzee, orange; rhesus macaque, olive green). As well as reduced promoter methylation, larger gene-body methylation, which is related to higher expression [69], was also seen in human compared with the other species over the sole exon (approximately 1.29-fold stronger peak MAC P-value over gene body CpGi (CpG:76)). In this complex locus the promoter of the major isoform of LTB4R (highlighted with a red rectangle) also co-locates with the gene body of the low-specificity receptor LTB4R2 and CIDEB. LTB4R has strong expression in all blood subtypes, particularly the myeloid lineage, including monocytes (Additional file 6).

Figure 8

Results of HpaII digestion of LTB4R followed by sequencing for promoter and gene body CpGs in human (Hs) and chimpanzee (Pt) (Martin et al.[19]via GEO). Methylation within the (A) promoter (Prom = CpG:99 in Figure 7) and (B) gene body (Body = CpG:76 in Figure 7) CpG islands of LTB4R from reverse scores (1 - p(U)) of all included HpaII MethylSeq sites analyzed by MetMap6 in purified neutrophils. These data replicate the significant difference identified in MeDIP peripheral blood. Four human and four chimpanzee samples had average methylation of 18.4% and 70.3% in the promoter (Prom), and 75.9% and 62.0% in the exonic gene body (Body) orthologous CpGis (Wilcoxon P < 0.05 for both).

Figure 9

Human-specific H3K4me3 enrichment in the LTB4R promoter from ChIP-seq data derived from B cells (lymphoblastoid cell line; data from Cain et al. [[21]] via GEO). These data identified significant activating peaks in all three human samples, but the signal was not strong enough for any peaks to be called in all three chimpanzee and all three rhesus macaque samples analyzed.

Figure 10

Data from the MARMAL-AID Human 450k Methylation array repository[94]for the LTB4R promoter and gene body CpG islands. These data are derived from 1,665 whole blood samples from healthy and non-cancer disease subjects from multiple experiments. This also showed a consistent low average level of human LTB4R promoter methylation (mean 29.4%, standard deviation 7.4%) and high average LTB4R gene body methylation (mean 87.9%, standard deviation 2.3%). Human LTB4R methylation whole blood 1,665 samples.

Figure 11

Expression and signaling of LTB4R (BLT1) in PBMCs isolated from human and rhesus macaque peripheral blood. (A) Real-time RT-PCR analysis of LTB4R mRNA in isolated human and rhesus macaque PBMCs normalized to the reference gene (18s); results from seven different human donors and from pooled rhesus macaque PBMCs run as four separate experiments; mean ± standard error of the mean. (B) Human and rhesus macaque PBMCs were stimulated with indicated concentrations of LTB₄ and intracellular calcium mobilizations were recorded. Results are expressed as the ratio of stimulated over basal (S/B) peak calcium fluxes obtained from three different human donors and pooled rhesus macaque PBMCs run as four experiments analyzed simultaneously, mean ± standard error of the mean. (C) Human PBMCs were pre-incubated for 10 minutes with different concentrations of BLT1 inhibitor LY22398 and calcium mobilization was analyzed in response to LTB₄ (300 nmol/L). Data are presented as mean ± standard error of the mean percentages of maximum response to LTB₄ (N = 3).