Fig. 4: M⁺N⁺ and M^-N⁺ progenitor-derived β cells have DMRs.

a The flow of DNA methylation assays. b Boxplot distribution of methylated cytosine fraction in eYFP⁺ or eYFP^- β-cell subtypes. Center line, median; box limits, upper and lower quartiles; whiskers, upper and lower limit range; points, outliers. c The proportional distribution of DMRs in gene promoter, intron, exon, intergenic regions, etc. d DMRs near the Arx locus. Two methylation tracks (eYFP^- and eYFP⁺ β cells) are shown. The two DMRs have higher (red bar) or lower (orange bars) methylation levels in eYFP⁺ cells. The number following “X:” indicates the number of CpG dinucleotides within the DMR. A few predicted motifs specific to the two DMRs are labeled. e Predicted DNA motifs enriched in the DMRs between P2 β-cell subtypes using HOMER. The p-values were determined by a two-sided hypergeometric test and displayed. f, g Pathways/terms that are enriched in the DMR-associated genes (analyzed using DAVID), with either lower- (f) or higher levels of (g) methylation. The p-values, from one-sided Fisher’s exact tests, were adjusted using the Benjamini-Hochenberg procedure for multiple comparisons. h P2 DEGs (down- or up-regulated) associated with P2 DMRs (with lower or higher methylation in eYFP⁺ cells). P-values were from a two-sided Fisher exact test. i A DMR in the first intron of Syt7 with a lower methylation in eYFP+ β cells (orange bar). Two motifs associated with this DMR are labeled (Tcf4 and Mafb). j–l The effects of Syt7 DMR manipulation. j An U6 promoter was used to express a guide RNAto bring dCas9-DNMT3a (reported by eGFP expression) to methylate DNA close by¹⁸. k The effects of co-expressing a Syt7 gRNA anddCas9-DNMT3a (green) on Syt7 (red) levels in MIN6 cells. Three experiments were done with similar findings. Scale bar, 20 μm. l Real-time RT-PCR assays of three samples (mean + SEM, 3 RNA samples isolated from different plates), with p-values from unpaired t-test, two-sided type-two errors.