Impact of targeted DNA methylation induction on NRF1 binding. (A) Genome Browser display of the targeted NRF1 binding site in the TMEM206 promoter. Sets of experiments include (from top to bottom): bsPCR-seq (mCG/CG), NRF1-ChIP-bs-seq (mCG/CG), NRF1-ChIP-bs-seq coverage, and NRF1 ChIP-seq-coverage. The NRF1 core binding site is highlighted in shaded green. (B) Quantitation of mCG/CG in the NRF1 core binding site (green shaded region) and adjacent to core binding site, comparing targeted bsPCR-seq (gray circles) to ChIP-bs-seq (cyan circles for αGCN4-DNMT3A and yellow circles for αGCN4-mCherry) at the TMEM206 promoter (NRF1 ChIP-bs samples combined n = 3 for coverage; bsPCR-seq, n = 3; error bars, SD; statistic: Fisher's exact test). (C) Quantitation of the TMEM206 promoter NRF1 ChIP-seq peak counts (TMM normalized) in samples treated with αGCN4-D3A (orange) compared to αGCN4-mCherry (gray) (mCherry n = 2; D3A n = 3; statistic edgeR, Benjamini-Hochberg multiple test corrected P-values). (D) qRT-PCR analysis of TMEM206 expression (normalized to geometric mean of the housekeeping genes RPS18, GAPDH, and HSPC3) comparing dC9Sun-D3A and dC9Sun-mCherry targeted to the NRF1 binding site in the TMEM206 promoter. Cells tested are HEK293T and HeLa cells, respectively (biological replicates n = 6; reference GFP-Puro transfected, puromycin-treated HeLa or HEK293T cells n = 4; statistic: Wilcoxon/Mann-Whitney U test, one-tailed). (E) Comparison of average mCG/CG in the CTCF and NRF1 core binding sites, respectively, by binning them into intervals of no (≥0 and ≤0.05), low (>0.05 and ≤0.1), intermediate (>0.1 and ≤0.5), or high (>0.5) levels of DNA methylation.
