For analyzing differentiation of hESCs in Fig. 4h, we used a second set of DMRs. We used a pairwise comparison strategy between ESCs and three in vitro derived cell types representative of the three germ layers (mesoderm, endoderm, ectoderm) and performed DMR calling as previously described 52 . Only DMRs losing more than 30% methylation compared to the ESC state at a significance level of p ≤ 0.01 were retained. Subsequently, we computed weighted methylation levels for all three DMR sets across HUES64, mesoderm, endoderm and ectoderm as well as three consecutive stages of in vitro derived neural progenitors (please see companion52 paper for details on the cell types). Finally, we plotted the corresponding distribution using the R function vioplot in the vioplot package. In order to identify potential regulators associated with the loss of DNA methylation at these regions, we determined binding sites of a compendium of transcription factors profiled in distinct cell lines and types (see Ziller, 2013 #45 for details) that overlapped with each set of hypomethylated DMRs. Next, we determined a potential enrichment over a random genomic background by randomly sampling 100 equally sized sets of genomic regions, respecting the chromosomal and size distribution of the different DMR sets and determined their overlap with the same transcription factor binding site compendium to estimate a null distribution. Only transcription factors that showed fewer binding sites across the control regions in 99 of the cases were considered for further analysis. Next, we computed the average enrichment over background for each TF with respect to the 100 sets of random control regions for each germ layer DMR and report this enrichment level in Fig. 4h right, where we capped the relative enrichment at 12.