Fig. 4

A Three differentially methylated regions (DMRs) spanning exon 4 to exon 6 of ITGB7 were selected for targeted induction of DNA methylation, on the basis of their epigenetic regulatory roles in gene expression. B Targeted DNA methylation was aimed by designing a fusion protein of deactivated cas9-endonuclease (dCas9) and catalytic domain of DNA-methyltransferase 3A (DNMT3A-CD), and ITGB7-DMR targeting single guide RNAs (sgRNAs). C MM.1S cell line [t(14;16) translocation] was co-transduced with dCas9-DNMT3ACD and a combination of sgRNAs in the presence and absence of doxycycline for induction. Cells transduced with individual constructs or dual constructs were sorted with flow cytometry, based on the tagged fluorophore. D–F We performed region-specific pyrosequencing to determine changes (p < 0.01 or p < 0.001) in DNA methylation per CpG site at DMR-1, -2 and -3 before and after induction of dCas9-DNMT3ACD plus sgRNAs. G The impact of targeted changes in DNA methylation on ITGB7 expression was determined with qPCR