Fig. 1

Engineered p16 epimutation recapitulates key features of age-associated epigenetic silencing. A p16 DNA methylation profiles in cis-MEF following serial passaging (p). A schematic of the p16 promoter of the cis-element knock-in allele with the CpG maps is shown. B Kinetics of p16 promoter methylation and mRNA expression in ctr-MEF and cis-MEF. Methylation levels were averaged from CpGs from − 814 bp to − 589 bp relative to the TSS. Note that cis-MEF cells grew for more than 25 passages; in contrast, the controls (ctr-MEF) entered growth arrest at passage 8 (p8) via p16 up-regulation. The p16 gene expression is relative to β–actin. C UCSC Genome Browser tracks showing the DNA methylation status of p16, p19, and p15 in the INK4/ARF locus on chromosome 4. The chromosomal coordinates are annotated on the top. The WGBS tracks show DNA methylation profiling in ctr-MEF (p7) and cis-MEF (p10). The height of each bar represents the methylation level of an individual CpG between 0 and 1 (100%). Promoters are indicated by angled arrows, and the knock-in location is indicated by an asterisk. D Comparisons of p16 promoter methylation in cis-MEF before and after Ad-Cre mediated cis-element excision with increasing doses of DAC. The culture passage number of cis-MEF is at p23. E Side-by-side comparisons of p16 expression in cis-MEF under the same conditions as (D). For (D) and (E), data are shown as mean ± SEM with individual values from three independent experiments. P values were determined by a one-way ANOVA test. F dCas9-SunTagTET1-mediated targeted demethylation of p16 in cis-MEF. p16-gRNA indicates GFP-positive cells treated with a p16-specific gRNA that binds the promoter region from − 792 bp to − 769 bp relative to the TSS. Ctr indicates GFP-negative cells without targeted demethylation. Data are shown from three independent experiments. G Targeted promoter demethylation resulted in p16 gene reactivation. The culture passage number of cis-MEF is at p23. P values were determined by a two-tailed Student’s t-test