Fig. 4: PHF14 serves as a CG-rich motif reader to recruit DNMT3B on SMAD7 gene.

a Schematic diagram of the truncated PHF14 protein constructions. Interactions between DNMT3B and various Flag-tagged truncated PHF14 constructions were evaluated by co-IP assays in 293FT cells without (b) or with DNase I treatment (c). d Wall-eye stereo view of the predicted interaction between the α-helix of PHF14 ePHD ___domain (right) and the major groove of DNA helix (left), in which key residues are shown as sticks, hydrogen bonds are shown as black dashes (upper panel), and electrostatic surface view of PHF14 ePHD ___domain bound to DNA helix (lower panel) was also shown. e Interactions between PHF14 ePHD ___domain or full-length PHF14 with unmethylated/methylated CG-rich oligonucleotides probes or AT-rich oligonucleotides probe were evaluated by EMSA using purified proteins. f, g Interactions between wild-type or mutated E430AK435A PHF14 ePHD ___domain or full-length PHF14 with unmethylated CG-rich oligonucleotides probes were evaluated by EMSA using purified proteins. Shifted DNA indicates protein-bound oligonucleotide probes, and free DNA indicates protein-free oligonucleotide probes. h SPR analysis measuring the affinity and kinetics of the interaction between PHF14 ePHD ___domain with CG-rich oligonucleotides probe. PHF14 ePHD ___domain was immobilized on a CM5 chip. i ChIP-qPCR assays validated the occupancy of Flag-tagged PHF14 on region 1 of SMAD7 gene locus in Calu3 and HCC827 cells with indicated treatments. j ChIP-qPCR assays validated the recruitment of DNMT3B on region 1 of SMAD7 gene locus in Calu3 and HCC827 cells with indicated treatments. k BSP validated the DNA methylation level of region 1 on SMAD7 gene locus in Calu3 and HCC827 cells with indicated treatments. l, m WB and qPCR analysis showed the effect of expressing wild-type or mutated PHF14 on the protein and mRNA levels of SMAD7 in Calu3 and HCC827 cells with indicated treatments. n Measurement of SBE-luciferase activity showed relative TGF-β signaling activates in Calu3 and HCC827 cells with indicated treatments. For aforementioned WB, qPCR, ChIP-qPCR, BSP and SBE-luciferase activity assays, cells were treated with TGF-β1 at final concentration of 5 ng/mL for 48 h before the indicated assays were performed (i–n). Error bars represent means ± SD derived from three independent experiments. Two-way ANOVA multiple comparison analysis was used for statistical analysis. **P < 0.01; ns, not significant, P > 0.05.