Detection of differential DNA methylation in repetitive DNA of mice and humans perinatally exposed to bisphenol A

Figures & data

Figure 1. Flowchart for Experimental and Analysis Pipeline. Both humans and mice underwent MethylPlex enrichment prior to sequencing. Reads were Repeat Masked and used to identify group level differences, and aligned to reference genomes to identify individual differences in repeat DNA methylation. Asterisks denote significance, *P < 0.05, **P < 0.01.

Figure 2. Percent CpG Enrichment by Exposure. Both human and mouse samples exhibited increased CpG content after MethylPlex enrichment. (A) The human low BPA group was slightly but significantly lower than the medium group (P = 0.01), and the high group (P < 0.05). (B) The mouse samples exhibited no significant differences in CpG enrichment by exposure group.

Figure 3. Percent Reads by Repeat Class and Exposure. (A) A subset of classes in human was hypomethylated in the medium BPA group. (B) No classes were differentially methylated by exposure group in mice. Asterisks denote significance, *P < 0.05, **P < 0.01.

Figure 4. Percent Reads by Repeat Family and Exposure. (A) A subset of repeat families was hypomethylated in the medium BPA group. (B) No families were differentially methylated by exposure group in mice. Asterisks denote significance, *P < 0.05, **P < 0.01.

Table 1. Repeat subfamilies differentially methylated by exposure.

Human	Element Subfamily	Condition	Condition	Condition	Direction of Methylation	Adjusted P-value (Tukey)	Element Age (MYA)
	AluSx3	Low	Medium		↓	2.23E-05	44
	AluSq	Low	Medium		↑	5.70E-09	44
	AluSq		Medium	High	↓	0.0003	44
	AluSx	Low	Medium		↓	5.68E-09	37
	AluSx	Low		High	↑	3.27E-07	37
	AluSx		Medium	High	↑	5.68E-09	37
	AluSg	Low	Medium		↑	5.68E-09	31
	AluSg	Low		High	↑	0.0072	31
	AluSg		Medium	High	↓	5.71E-09	31
	AluY	Low	Medium		↑	0.0026	24
	AluY	Low		High	↑	0.0110	24
	AluY		Medium	High	↑	5.68E-09	24
	AluYc	Low	Medium		↓	5.68E-09	4 to 16
	AluYc	Low		High	↓	5.68E-09	4 to 16
	AluYc		Medium	High	↑	5.68E-09	4 to 16
Mouse	GSAT_MM	UG	control		↑	1.26E-12
	GSAT_MM	MG	control		↑	1.26E-12

Figure 5. Individual Transposon Detection Pipeline. Both mouse and human reads were filtered for reads containing partial unique and partial repetitive sequence. The unique sequence was mapped to the reference genome and read count differences corresponding to DNA methylation differences were tested for significance with EdgeR.

Figure 6. Individual Repeats Differentially Methylated by Exposure. (A) Number of human transposon insertions exhibiting significantly different read counts across BPA exposure groups. (B) Mouse transposon insertions exhibiting differential read counts across BPA treatment groups.

Table 2. Genomic distribution counts of differentially methylated transposons.

	Human			Mouse
Annotation Category	Med vs. High	Med vs. Low	Low vs. High	UG vs. Control	UG vs. MG	MG vs. Control
1to5kb	156^**↑	3	0	0	0	0
3UTRs	16	0	0	0	0	1
5UTRs	249	4	0	0	2	2
Exons	19	0	0	0	0	1
Intergenic	549^**↓	17	0	1	4	7
Introns	897^**↑	14	0	1	5	6
Promoters	28	0	0	0	0	0

* P < 0.05,

** P < 0.01, ↑ or ↓ indicates an enrichment or depletion, respectively, relative to the human genome.