Widespread genomic de novo DNA methylation occurs following CD8⁺ T cell activation and proliferation

Figures & data

Figure 1. Experimental design.

CD8+ T cells were isolated from the PBMC layer of the blood of eight healthy volunteers. T cells were stimulated using CD3 and CD28 antibodies for 24 h before being transferred to fresh wells. 200 µM GlyCl was added to the treatment wells, and the control wells received the volumetric equivalent of PBS. Samples were taken 24 h after treatment (48 h after initial stimulation) and 72 h after treatment (96 h after initial stimulation). Created with BioRender.com.

Figure 2. Unsupervised assessment of data variability.

(a) Multidimensional scaling of M-values, with the distances for leading log2FC in dimension 1 represented on the x-axis and the leading log2FC in dimension 2 are represented on the y-axis. Dots represent samples from the 48 h time point and triangles represent samples from the 96 h time point. Samples are coloured by participant. (b) Hierarchical clustering of β-values for all probes. The relative change in β-values is represented on the y-axis and individual samples are represented on the x-axis, samples are coloured according to participant.

Figure 3. Global DNA methylation (β-values) for all probes.

(a) Volcano plot with log₂ fold changes on the x-axis versus statistical significance on the y-axis -log₁₀P-value. Grey dots represent probes with an adj. P< .05. (b) Relative average methylation levels for each time point. Mean methylation levels for β-values are presented as percentages.

Figure 4. Scatterplot of probe significance ordered by genomic location for all probes across the array, calculated using M-Values.

All probes are ordered per chromosome position along the x-axis, and p-values as the – log10(p-values) are presented on the y-axis. Genome-wide significance was determined using the ‘Benjamini, Hochberg’ method within Limma and is approximately represented by the horizontal red line.

Figure 5. Box plots showing representative β-values at each time point.

β-values are represented on the y-axis and time on the x-axis. The CpG sites are marked with an orange dot in the gene scheme placed on top of each graph, with the TSS is marked by a red arrow: a) chr15: 58627950; b) chr7: 8132142; c) chr1: 101264831; d) chr12: 89760982; e) chr1: 30751002; f) chr17: 40310298.

Table 1. The top 20 most significant differentially methylated positions, ordered by significance*.

Probe ID	logFC	Adj p-val	Chromosome	Location	Gene	Genomic Feature**
cg06555928	1.61	5.31E–05	chr15	58627950	ADAM10	3’UTR
cg27060295	1.91	5.31E–05	chr7	8132142	ICA1	3’UTR
cg09190907	2.17	5.31E–05	chr1	101264831
cg21870144	2.28	5.31E–05	chr12	89760982
cg15459165	1.63	5.31E–05	chr1	30751002	LAPTM5	5’UTR
cg13762512	1.55	5.31E–05	chr17	40310298	RARA	5’UTR
cg01725626	1.41	5.31E–05	chr6	110687583	CDK1	Intronic
cg20732076	1.49	5.31E–05	chr6	42367492	TRERF1	5’UTR
cg07781212	1.61	5.31E–05	chr8	86523460	CPNE3	5’UTR
cg22608507	1.60	5.31E–05	chr12	122894760	VPS37B	Intronic
cg24362952	1.87	5.31E–05	chr1	108647018
cg05679475	1.57	5.31E–05	chr10	17428254	ST8SIA6	5’UTR
cg25060738	2.17	5.31E–05	chr6	107787354	SCML4	5’UTR
cg20641673	2.00	5.31E–05	chr19	16382871	EPS15L1	Intronic
cg27155939	1.52	5.31E–05	chr15	40054961	SRP14-DT	Intronic
cg24219822	2.10	5.31E–05	chr1	160839185	CD244	TSS200
cg20214179	1.85	5.31E–05	chr5	50702804	PARP8	5’UTR
cg17344223	1.44	5.41E–05	chr3	129576937	PLXND1	Intronic
cg14170423	1.30	5.41E–05	chr11	64746336	RASGRP2	TSS1500
cg08964365	1.23	5.41E–05	chr8	38368712	WHSC1L1	5’UTR

*The full dataset of differentially methylated CpGs is available in Supplementary File S1:Table S1.

**UTR = Untranslated Region, TSS = Transcription Start Site (distance in bp).

Figure 6. Topmost significant genomic regions displaying differential methylation, across adjacent probe loci.

Differentially methylated gene regions (DMRs) that displayed a significant change in methylation (y-axis represents β-values) across five or more CpGs (x-axis) between the 48 h (dark grey) and 96 h (light grey) time points. Gene structure is placed on top of each graph, with the TSS is marked by a red arrow. a) TRAJ44-TRAJ47 b) FOXP1 c) GPR114 d) TBC1D5, SATB1 e) TRAJ50 f) PRKAG2.

Table 2. Significant DMRs between 48 h and 96 h time points.

Chromosome	Start	End	Width (bp)	CpGs	FDR	Max β-values	Mean β-values	Overlapping genes
chr14	22960834	22964042	3209	13	0.006	−0.192	−0.122	AL132780.3
chr3	71353994	71355303	1310	8	0.009	−0.147	−0.102	FOXP1, AC097634.4
chr16	57576285	57577474	1190	6	0.002	0.230	0.137	ADGRG5
chr3	18459048	18460713	1666	5	0.008	−0.143	−0.109	SATB1-AS1
chr14	22957594	22958402	809	5	0.013	−0.177	−0.112	AL132780.3
chr7	151491913	151493647	1735	6	0.007	−0.348	−0.115	RHEB
chr13	114908155	114909333	1179	6	0.001	0.213	0.112	NA

*Annotated against hg38.

Table 3. GO pathway terms associated with each ontology type for the genes that demonstrated a significant DNA methylation change, and mapped to promoter regions.

Pathway	ontology	Description	DE	n	adj p value
GO:0001775	BP	cell activation	1070	188	1.02E–13
GO:0045321	BP	leukocyte activation	926	168	1.24E–13
GO:0046649	BP	lymphocyte activation	764	146	2.63E–13
GO:0051249	BP	regulation of lymphocyte activation	497	105	5.18E–12
GO:0050865	BP	regulation of cell activation	649	126	5.18E–12
GO:0002694	BP	regulation of leukocyte activation	592	116	2.07E–11
GO:0042110	BP	T cell activation	539	109	2.07E–11
GO:0007165	BP	signal transduction	5887	690	1.17E–10
GO:0046651	BP	lymphocyte proliferation	304	70	1.54E–10
GO:0032943	BP	mononuclear cell proliferation	311	71	1.61E–10
GO:0038023	MF	signaling receptor activity	1448	169	5.35E–05
GO:0060089	MF	molecular transducer activity	1448	169	5.35E–05
GO:0140375	MF	immune receptor activity	140	31	0.000132745
GO:0019955	MF	cytokine binding	139	32	0.00020508
GO:0005126	MF	cytokine receptor binding	270	45	0.000400818
GO:0004888	MF	transmembrane signalling receptor activity	1229	137	0.001570874
GO:0004896	MF	cytokine receptor activity	93	22	0.002898576
GO:0005102	MF	signaling receptor binding	1474	183	0.002964097
GO:0005125	MF	cytokine activity	237	34	0.016452127
GO:0019957	MF	C-C chemokine binding	24	8	0.018652189
GO:0009897	CC	external side of plasma membrane	375	71	4.67E–07
GO:0009986	CC	cell surface	873	135	2.20E–06
GO:0005887	CC	integral component of plasma membrane	1682	222	1.15E–05
GO:0031226	CC	intrinsic component of plasma membrane	1763	231	1.26E–05
GO:0098552	CC	side of membrane	593	96	2.55E–05
GO:0071944	CC	cell periphery	5958	656	4.11E–05
GO:0005886	CC	plasma membrane	5479	606	6.15E–05
GO:0016021	CC	integral component of membrane	5205	544	0.00088294
GO:0031224	CC	intrinsic component of membrane	5370	559	0.001237551
GO:0001772	CC	immunological synapse	45	13	0.048395896

n, number of genes in the pathway.

DE, number of genes that were differentially expressed.

Figure 7. Multidimensional scaling (MDS) plot of top 100 significant pathways.

Co-occurrence Association Score (CAS) is presented on the x- and y-axis. CAS is a measure used to quantify the frequency of co-occurrences of two GO terms in a single-gene annotation relative to random chance. Pathways contained within the left circle generally correspond with activation and proliferation, whereas pathways within the right circle generally correspond with cell signalling and immune response. The full list of significant pathways is available in (Supplementary file 1: Table S2).

Figure 8. T cell viability and growth.

Cell measures were conducted at 24 and 72 h post-treatment with GlyCl or PBS (Control). Circles represent control samples and triangles represent treatment samples, the colours represent data from the same donor (n = 8). Significant differences were determined with paired t-tests. ns, not significant (a) cellular viability after treatment (b) cell growth after treatment.

Figure 9. Analysis of overall DNA methylation change due to GlyCl treatment.

Volcano plots with log2 fold changes on the x-axis versus statistical significance on the y-axis -log10P-value. Black dashed lines approximately represent genome-wide significance probes with an adj. P < .05. a) 24 h post-treatment b) 72 h post-treatment.

Data availability statement

The authors confirm that the analysed data supporting the findings of this study are available within the article [and/or] its supplementary materials. Request for raw data files can be made available from the corresponding author AJS upon reasonable request; however, these represent private data and are not available for public use.