Fig. 2

Validation and extension of the results for DS-DM in the NLGN2, MZF1 and STK19 genes by bis-seq. a CpG hypermethylation is localized to the promoter region of the NLGN2 gene in DS neurons. The bis-seq shows that the DM affects multiple CpGs in the promoter region. As shown by the DM track (Δ Methyl; mean difference in AVG_Beta in DS compared with control samples, showing all peaks with a p value < 0.001) aligned to ENCODE and Zhu et al. [100] data (GSE17312 and GSM733758, respectively) covering 50 kb of this gene-rich region, strong signals for DM are localized to the downstream edge of the NLGN2 promoter (marked by the activating H3K4me3 histone modification in H9-derived neurons) and one downstream region, with weaker signals at several other locations. The asterisk indicates the position of the bis-seq amplicon. b Validation of CpG hypermethylation at the 3′ end of the MZF1 gene in DNA from purified neuronal and glial cell nuclei. The bis-seq amplicon (asterisk) spans a CGI at the 3′ end of the gene that appears to be the promoter for an antisense transcript, LOC100131691, which our RT-PCR results show is expressed in fetal brain but not in adult FC (data not shown). Strong DM is restricted to this region, which carries the H3K27m3 poised chromatin mark in human embryonic stem cells (hESC). c Validation of CpG hypermethylation in the promoter region of the STK19 gene in DNA from neurons and whole FC. The bis-seq amplicon (asterisk) covers the promoter region, which is marked by H3K4m3 in H9-derived neuronal cells. This promoter region also gives rise to an opposite-strand transcript (DOM3Z). As is true for the other examples above, the strong DM is tightly localized to this single region