Fig. 4

Epigenetic characteristics of differentially methylated region over a predicted enhancer near the DLL1 gene and validation by pyrosequencing. a UCSC Genome Browser screenshot showing the differentially methylated HMD region (circled in blue-purple). Methylation data for the 23 typical MARBLES placentas and 24 autism MARBLES placentas were combined to produce composite methylation tracks with each vertical bar showing percent methylation at individual CpG sites. H3K27ac and H3K4me1 tracks are from ENCODE. ChromHMM tracks are from the Roadmap Epigenomics Consortium [28]. HMD highly methylated domain, CGI CpG island, H1Bmp4T H1 human embryonic stem cells differentiated into trophoblasts, IMR90 human fetal lung fibroblasts, TSS transcription start site, TF transcription factor. b Long-range interactions in the DLL1 locus revealed by “Juicebox” visualization tool of human HUMEC HiC data (HUMEC in situ combined, observed, red; expected, blue) [29]. Red arrow designates DLL1 promoter; orange arrow, putative DLL1 enhancer; yellow circles highlight evidence for observed 3D interactions over expected for these two loci. c Three CpG sites corresponding to the H3K27ac/H3K4me1 peaks were assayed by pyrosequencing on the same samples analyzed by MethylC-seq (region shown by gray bar in a within circled HMD). Average MethylC-seq % methylation for the three CpG sites is plotted compared to the pyrosequencing values. ASD and TD pyrosequencing values from three replicates per sample were tested for significance by t test. *p < 0.05. d An “Other Developmental Concerns” (ODC) group that was neither TD nor ASD was compared to TD and ASD by pyrosequencing as in c. No significant differences were observed between ODC and ASD or ODC and TD, although the values were between the two groups, as expected. N = 24 ASD, 23 TD, 23 ODC. e DLL1 pyrosequencing analysis of methylation levels by placental region. Results shown are from six different control placentas and samples obtained from five different regions as labeled