DNA methylation and gene expression changes in mouse mammary tissue during successive lactations: part II – the impact of lactation rank

Figures & data

Table 1. The total number of DMCs and DMRs and the per cent upmethylation found in all three comparisons: 1^st lactation (n = 7) vs. 2^nd lactation (n = 7) (blue), 1^st lactation (n = 5) vs. 2^nd lactation with inflammation (n = 5) (green), and 1^st lactation (n = 5) vs. 2^nd lactation with inflammation and previous inflammation history (n = 7) (red).

Condition	Total DMCs	Upmethylated DMCs in 2^nd lactation (%)	Total DMRs	Upmethylated DMCs in 2^nd lactation (%)
L1-NI vs. L2-NI (L1-NI)	1981	82.7%	97	92.9%
L1-I vs. L2-I (L1-NI)	964	23.9%	55	18.2%
L1-I vs. L2-I (L1-I)	2590	66.6%	153	77.8%

Figure 1. Violin plots of the overall distribution of methylation levels in all three comparisons: 1^st lactation (n = 7) vs. 2^nd lactation (n = 7) (blue), 1^st (n = 5) vs. 2^nd lactation with inflammation (n = 5) (green), and 1^st (n = 5) vs. 2^nd lactation with inflammation and previous inflammation history (n = 7) (red). 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 2. Pie charts showing the distribution of DMCs for all three comparisons: 1^st lactation (n = 7) vs. 2^nd lactation (n = 7) (blue), 1^st (n = 5) vs. 2^nd lactation with inflammation (n = 5) (green), and 1^st (n = 5) vs. 2^nd lactation with inflammation and previous inflammation history (n = 7) (red). 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. Molecular function and biological processes GO (gene ontology) terms (MF: molecular function; BP: biological process) as well as signalling pathways enriched by DAVID analysis for all three comparisons: 1^st lactation (n = 7) vs. 2^nd lactation (n = 7) (blue), 1^st lactation (n = 5) vs. 2^nd lactation with inflammation (n = 5) (green), and 1^st lactation (n = 5) vs. 2^nd lactation with inflammation and previous inflammation history (n = 7) (red).

	L1-NI vs. L2-NI (L1-NI)		L1-I vs. L2-I (L1-NI)		L1-I vs. L2-I (L1-I)
	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	3.3×10^−3	90	0.21	26	1.7×10^−2	93
Transcription factor activity	5.0×10^−3	39	0.14	16	3.1×10^−4	47
GO: BP
Multicellular organism development	8.2×10^−8	79	1.8×10^−2	29	1.6×10^−5	76
Positive regulation of transcription	1.2×10^−2	64	0.17	5	7.9×10^−3	70
Negative regulation of transcription	1.7×10^−3	55	1.0	0	1.0	0
Canonical Wnt signalling	2.3×10^−3	12	0.27	4	0.074	9
Cell–cell signalling	0.53	4	0.16	4	3.2×10^−2	8
Lipid transport	0.71	6	1.0	0	2.9×10^−2	13
Signalling pathways
Wnt signalling pathway	2.5×10^−2	13	2.9×10^−2	8	1.3×10^−2	15
cAMP signalling pathway	4.7×10^−3	17	0.53	5	3.2×10^−3	19
PPAR signalling pathway	0.13	6	0.48	4	1.0	0
Regulation of pluripotency of stem cells	0.078	10	3.0×10^−3	9	3.6×10^−2	12
Regulation of lipid metabolism by PPARα	1.7×10^−2	4	1.0	0	1.0	0

Figure 3. Gene expression (∆∆Ct) in three comparisons: 1^st (n = 7) vs. 2^nd lactations in physiological conditions (n = 7) (blue), 1^st (n = 5) vs. 2^nd lactations with inflammation (n = 5) (green), and 1^st (n = 5) vs. 2^nd lactations with inflammation and previous inflammation history (n = 7) (red). The expression of 60 genes was analysed. The genes listed in this figure are differentially expressed (p < 0.05) in at least one comparison (colour-coded rectangles). 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.

Figure 4. Venn diagrams of the total number of differentially methylated cytosines (DMCs) as well as shared DMCs in three comparisons: 1^st lactation (n = 7) vs. 2^nd lactation (n = 7) (blue), 1^st (n = 5) vs. 2^nd lactation with inflammation (n = 5) (green), and 1^st (n = 5) vs. 2^nd lactation with inflammation and previous inflammation history (n = 7) (red) (a), as well as the total number of differentially methylated regions (DMRs) and shared DMRs in all three comparisons (b).

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.