Fig. 3

Early pre-implantation alcohol exposure triggers DNA methylation alterations in developing embryonic forebrain. Genome-wide CpG methylation analyses of E10.5 control (n = 6) and EtOH-exposed (n = 16) forebrains. a Distribution of genomic tiles (100 bp) (all-tiles; n = 114 911) across ranges of CpG methylation levels in control and EtOH-exposed samples. ****p < 0.0001, ***p < 0.001, *p < 0.05; z-test proportion test. b Heatmap showing CpG methylation levels for the top 1% most variable tiles (n = 1 200) between control and EtOH-exposed forebrains. Gray lines in heatmap have no associated methylation values because of lack of sufficient sequencing coverage in sample. c Scatterplot representing the differentially methylated regions (DMRs) between control and EtOH-exposed forebrains (see "Methods" section for details). Red dots represent the tiles with a methylation increase of at least 10% in EtOH-exposed compared to control forebrains (n = 69); blue dots represent the tiles with a methylation decrease of at least 10% in EtOH-exposed compared to control forebrains (n = 1 440); grey dots represent the tiles with changes less than 10% in EtOH-exposed compared to control forebrains (n = 113 402). d Proportion of DMRs associated with the changes of CpG methylation levels between control and EtOH-exposed E10.5 forebrains. e Comparison of CpG methylation levels of specific DMRs-associated tiles in EtOH-exposed versus control forebrains. Blue bar graph represents the comparison for decreased-methylation DMRs (n = 1 440); red bar graph represents comparison for the increased-methylation DMRs (n = 69). f Functional enrichment analysis showing top 10 enriched pathways for decreased- and increased-methylation DMRs located in genic regions (n = 710 unique gene DMRs), based on Metascape analysis for pathways and p value. The size of the dot represents the number of DMR-associated genes in a pathway, and gene ratio represents the number of DMR-associated genes with regards to the number of genes in a pathway. g Examples of CpG methylation levels of individual samples for DMR-associated genes related to the top enriched pathways. Letters under gene name relate to the pathways in f). ǂ represents significant differences in CpG methylation levels of DMRs (e.g., ±  > 10% methylation difference, q < 0.01) between control and EtOH-exposed embryos (see "Methods " section for details)