DNA methylation - discovery
We identified DNA methylation at 1173 CpG sites which clearly differentiated specific immune cell populations using publicly available data from whole blood [16]; hierarchical clustering and principal components analyses provide a visual presentation and highlight that these markers cluster the cell populations in a biologically meaningful way (Figure 1). Pathway analyses of the genes to which these 1173 CpG sites mapped strongly supported their discriminatory nature, and, as expected, enrichment for immune cell biological function was observed: enrichment for CD56 (> 79 genes), CD4 (> 68 genes), CD8 (> 34 genes), CD14 (> 69 genes) and CD19 (> 194 genes) was observed. Furthermore, these results suggest that discriminatory CpG marker loci may map to genes with a hitherto unrecognised role in immune cell discrimination and/or function.
This robust differentiation between cell types was explained by non-overlapping sets of CpGs specific for each cell population: CD8+ (n=70); CD4+ (n=96); CD19+ (n=347); CD56+ (n=112); CD14+ (n=126); Gran (n=128); Neu (n=128), and Eos (n=166). The majority of these sites were relatively hypo-methylated in the cell type of discrimination and hyper-methylated in all other cell populations analysed: CD8+ (46/70), CD4+ (71/96) CD19+ (344/347), CD56+ (111/112), CD14+ (126/126), Gran (94/128), Eos (165/166) with Neu being the exception (33/128). Interestingly, the majority of CpG marker sites identified (~95% of CpGs) mapped to annotated gene loci, with many in regions involved in regulating mRNA expression (e.g. promoters). For each cell type marker the proportion of CpG sites mapping to annotated loci was: CD8+ (62/70), CD4+ (78/96) CD19+ (255/347), CD56+ (99/112), CD14+ (82/126), Gran (102/128), Eos (136/166) and NEU (108/128). For individual marker information including annotation see https://github.com/sirselim/immunecell_methylation_paper_data [DOI: 10.5281/zenodo.336694].
The largest DNA methylation difference observed was 87% between CD19+ cells against all others. This 87% difference was observed in two genes, WIPI2 and CARS2; while WIPI2 has a reported role in the immune system [25], no such function has been reported for CARS2 to our knowledge to date, and neither has been previously considered as a CD19+ discriminatory marker. Ranked by the largest change in methylation the top five CpG sites mapping to annotated loci for each cell type were:
CD19+: 87% (WIPI2, CARS2), 83% (RERE), 82% (LOC100129637), 80% (POU2F2)
CD4+: 69% (CD40LG), 56% (PUM1), 54% (DCP2, BAG3), 48% (SF1)
CD8+: 67% (CD8A), 64% (CD8A), 51% (CD8B), 49% (CD8B, CD8A)
CD56+: 63% (SLC15A4), 52% (RASA3), 48% (MAD1L1), 45% (KLRB1/CD161),
43% (KLRB1/CD161)
CD14+: 79% (PARK2), 70% (CENPA, PARK2), 69% (KIAA0146, FAR1)
EOS: 73% (FAM65B), 72% (KIAA0317, APLP2), 70% (MEF2A, CCDC88A)
GRAN: 60% (VPS53, PCYOX1), 59% (ARG1), 58% (CSGALNACT1),
56% (SH3PXD28)
NEU: 14% (CUL9), 12% (LASP1), 7% (GFl1), 6% (LRFN1, NFAT5)
DNA methylation - validation
In order to validate our initial observations from the in silica experiment we selected 11 differentially methylated loci for analysis in 12 independent samples (n=6 female, n=6 male). These DNA methylation sites included 2 discriminatory loci mapping to annotated genes, including the loci with the maximum methylation difference, for each of CD4+ (CD40LG, DCP2) , CD8+ (CD8A, CD248) , CD19 (POU2F2, WIPI2) , CD56+ (KLRB1, SLC15A5), CD14+ (FAR1, PARK2) , with CD19 included as an additional positive control. We observed a strong agreement with the expected discriminatory patterns of DNA methylation for all loci examined (Figure 2).
RNA expression
Given the role that DNA methylation plays in regulation of gene expression we also explored the mRNA levels of the 11 loci from our DNA methylation validation experiment. We investigated gene expression by QRTPCR in the 12 independent samples. A clear differentiation between immune cells types at the gene expression level was observed for PARK2, POU2F2, DCP2, CD248, CD8A, SLC15A4, CD4A0LG and CD19 but not for FAR1, WIPI2, KLRB1 (Figure 2) .