DNA methylation and gene expression changes in mouse mammary tissue during successive lactations: part I – the impact of inflammation

Figures & data

Figure 1. Gene expression (∆∆Ct) in three comparisons: inflammation during 1^st lactation (L1-I, n = 5; L1-NI, n = 7) (orange), inflammation during 2^nd lactation after inflammation in 1^st (L2-I (L1-I), n = 7; L2-NI (L1-I), n = 5) (green), and inflammation during 2^nd lactation with no prior inflammation (L2-I (L1-NI), n = 5; L2-NI (L1-NI), n = 7) (purple). The genes listed are differentially expressed (p < 0.05) in at least one comparison (colour-coded circles). Each column represents one individual. Lowest gene expression is represented in blue (highest ∆∆Ct values), highest – in red (lowest ∆∆Ct values), no data available – in black.

Table 1. Library characterization, mapping efficiency on the murine genome (GRCm38), coverage and average methylation in RRBS libraries.

	L1-I	L1-NI	L2-I (L1-NI)	L2-NI (L1-NI)	L2-I (L1-I)	L2-NI (L1-I)
	(n = 5)	(n = 7)	(n = 5)	(n = 7)	(n = 7)	(n = 5)
Number of read pairs (million)	32.3 (±6.8)	41 (±7.5)	36 (±9.6)	33 (±6.7)	35.2 (±6.4)	35.6 (±2.4)
Average quality score (Phred score)	35.7 (±0.2)	35.8 (±0.1)	35.7 (±0.1)	35.7 (±0.2)	35.7 (±0.1)	35.7 (±0.1)
Uniquely mapped reads (%)	68 (±1)	68.3 (±1)	68.7 (±0.6)	68.1 (±0.9)	68.2 (±0.7)	68.5 (±0.4)
Ambiguous reads (%)	17.2 (±0.5)	16.2 (±0.9)	17.1 (±0.4)	17.4 (±0.4)	17.4 (±0.3)	17.4 (±0.2)
Unmapped reads (%)	14.8 (±1.3)	15.5 (±1.5)	14.1 (±0.3)	14.6 (±1.1)	14.4 (±0.9)	14.1 (±0.6)
Bisulfite conversion rate (%)	99.8 (±0.1)	99.8 (±0.1)	99.9 (±0)	99.8 (±0.1)	99.9 (±0.1)	99.9 (±0.1)
Number of covered CpGs (million)	2.05 (±0.3)	2.14 (±0.07)	1.98 (±0.1)	1.92 (±0.03)	1.97 (±0.07)	1.97 (±0.06)
Average cover per CpG	67.2 (±15.2)	84.5 (±16.2)	74.6 (±15.6)	69.8 (±13)	72.2 (±11.2)	74.8 (±4.2)
Percentage of CpGs10	67.7 (±7.1)	67.9 (±1.9)	72.1 (±2)	73.3 (±1.9)	72.2 (±2.2)	72.2 (±2.2)
Average DNA methylation at CpGs10%)	37.9 (±1)	44.3 (±1.3)	38.2 (±0.8)	38.5 (±0.8)	38.6 (±0.5)	44.1 (±0.6)
Percentage of CPGs10 hypermethylated (DNA methylation > 80%)	31.6 (±0.9)	31.5 (±1.4)	31.6 (±0.7)	31.5 (±0.8)	31.9 (±0.5)	31.9 (±0.3)
Percentage of intermediate CpGs10 (DNA methylation in [20%; 80%])	14.1 (±0.6)	14.8 (±0.3)	14.8 (±0.4)	15.4 (±0.4)	15.1 (±0.3)	14.9 (±0.1)
Percentage of CpGs10 hypomethylated (DNA methylation < 20%)	56.3 (±4.7)	53.7 (±1.5)	53.5 (±1)	53 (±0.8)	53 (±0.6)	53.3 (±0.3)

Figure 2. Hierarchical clustering and principal component analysis of samples from 1^st lactation, with (orange) or without inflammation (black).

Figure 3. Violin plots of the overall distribution of methylation levels in all three comparisons: inflammation during 1^st lactation (L1-I, n = 5; L1-NI, n = 7) (orange), inflammation during 2^nd lactation after inflammation in 1^st (L2-I (L1-I), n = 7; L2-NI (L1-I), n = 5) (green), inflammation during 2^nd lactation with no prior inflammation (L2-I (L1-NI), n = 5; L2-NI (L1-NI), n = 7) (purple). The abscissa represents the different conditions in each comparison, the ordinate represents the level of methylation of the DMCs in that condition, and each violin represents the density of the point at that methylation level.

Figure 4. Venn diagrams of the total number of differentially methylated cytosines (DMCs) as well as shared DMCs in all three comparisons: inflammation during 1^st lactation (L1-I, n = 5; L1-NI, n = 7) (orange), inflammation during 2^nd lactation after inflammation in 1^st (L2-I (L1-I), n = 7; L2-NI (L1-I), n = 5) (green), inflammation during 2^nd lactation with no prior inflammation (L2-I (L1-NI), n = 5; L2-NI (L1-NI), n = 7) (purple) (A) and of the total number of differentially methylated regions (DMRs) as well as shared DMRs in all three comparisons (B).

Figure 5. Pie charts showing the distribution of DMCs for all three comparisons: inflammation during 1^st lactation (L1-I, n = 5; L1-NI, n = 7) (orange), inflammation during 2^nd lactation after inflammation in 1^st (L2-I (L1-I), n = 7; L2-NI (L1-I), n = 5) (green), inflammation during 2^nd lactation with no prior inflammation (L2-I (L1-NI), n = 5; L2-NI (L1-NI), n = 7) (purple). Distribution is shown according to gene regions (A), CpG density (B), and repeats (C). All three comparisons are discussed in comparison to the background (control comprised of all CpGs found after RRBS analysis).

Table 2. GO (gene ontology) terms (MF: molecular function; BP: biological process) and signalling pathways enriched by DAVID analysis for all three comparisons: inflammation during 1^st lactation (L1-I, n = 5; L1-NI, n = 7) (orange), inflammation during second lactation after inflammation in 1^st (L2-I (L1-I), n = 7; L2-NI (L1-I), n = 5) (green), inflammation during second lactation with no prior inflammation (L2-I (L1-NI), n = 5; L2-NI (L1-NI), n = 7) (purple).

	L1-I vs. L1-NI		L2-I (L1-I) vs. L2-NI (L1-I)		L2-I (L1-NI) vs. L2-NI (L1-NI)
	p value	Number of unique genes targeted by DMCs	p value	Number of unique genes targeted by DMCs	p value	Number of unique genes targeted by DMCs
GO: MF
DNA binding	2.1×10^−3	32	4.5×10^−2	21	0.17	23
Transcription factor activity	7.0×10^−3	21	0.12	9	0.2	10
Transcriptional repressor activity	3.1×10^−2	9	1.6×10^−3	10	0.068	8
Methyltransferase activity	0.6	3	1.0	0	0.31	4
GO: BP
Negative regulation of transcription	6.2×10^−3	20	1.2×10^−2	15	2.3×10^−2	17
Positive regulation of transcription	0.078	20	0.15	14	0.3	10
Methylation	0.61	3	0.12	13	0.56	3
Canonical Wnt signalling	0.32	3	1.0	0	0.087	4
Cell-cell adhesion	4.2×10^−2	3	0.26	7	0.6	3
Signaling pathways
Cell junction organization	3.2×10^−2	4	1.0	0	1.0	0
Wnt signalling pathway	0.1	5	0.55	3	0.24	4
PI3K-Akt signalling pathway	0.69	4	1.0	0	0.66	4
IL-6 signalling pathway	1.0	0	4.5×10^−2	4	1.0	0

Data availability statement

RRBS fastq files have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB57878 https://www.ebi.ac.uk/ena/data/view/PRJEB57878.