Fig 2: PROSER1 regulates the chromatin association of TET1/2 to mediate UTX/H3K4me1–dependent enhancer activity.(A) Heatmaps centered on 18,017 reproducible PROSER1 binding sites obtained from three different PROSER1 antibodies in WT HEK293 cells. Occupancy of PROSER1, UTX, OGT, TET1, TET2, H3K4me1, H3K4me2, H3K4me3, and H3K27ac is displayed. (B) Venn diagram depicting the overlap between reproducible PROSER1, UTX, and TET2 peaks in WT HEK293 cells. (C) Heatmaps centered on 4,421 UTX/H3K4me1-down-regulated regions in PROSER1 KO compared with WT cells. Occupancy of PROSER1, UTX, OGT, TET1, TET2, H3K4me1, H3K4me2, H3K4me3, and H3K27ac is displayed. (D) Genome browser tracks depicting the ChIP-seq profiles of PROSER1, UTX, OGT, TET1, TET2, H3K4me1, H3K4me2, H3K4me3, and H3K27ac in WT and PROSER1 KO HEK293 cells at the DDB2 promoter (left) and SOX2 enhancer (right). (E) Genome browser tracks showing the RNA-seq profiles of the DDB2 and SOX2 genes in WT and PROSER1 KO HEK293 cells. Two replicates are displayed for each genotype. (F) Violin plots showing transcriptional down-regulation of the 400 genes associated with the 4,421 regions displaying lower enrichment of UTX and H3K4me1 in PROSER1 KO compared with WT cells. Expression values were standardized for each gene across the samples. The median interquartile range and the 1.5× interquartile range are shown for each violin plot.

Fig 3: Loss of PROSER1 function causes DNA hypermethylation encroachment at CpG islands.(A) Representative genome browser tracks depicting the ChIP-seq profiles of PROSER1, UTX, H3K4me1, TET1, TET2, and whole genome bisulfite sequencing results at CGIs with DNA hypermethylation encroachment in PROSER1 KO versus WT cells. Examples for 5' encroachment (MGA), bidirectional encroachment (IGFBP7), and 3' encroachment (HOXC6) in WT and PROSER1 KO cells are displayed. (B) Heat map showing the whole genome bisulfite sequencing results of 483 identified CGIs with DNA hypermethylation encroachment in PROSER1 KO compared with WT cells. Clustering was performed based on the pattern of DNA hypermethylation encroachment: 5' end encroachment (orange), bidirectional encroachment (gray), and 3' encroachment (green). The color scale shows the DNA methylation range difference between PROSER1 KO and WT cells. Missing values (Not-a-Number; NaN) are depicted in white. (C) Average per-bin DNA methylation level of the CGIs with DNA hypermethylation encroachment described in Fig 6B categorized into groups of 5' end encroachment (orange), bidirectional encroachment (gray), and 3' end encroachment (green).

Fig 4: PROSER1 co-localizes genome-wide with UTX, TET1, TET2, and OGT.(A, B, C, D) Heat maps centered on (A) 109,378 UTX peaks; (B) 69,815 TET1 peaks; (C) 16,982 TET2 peaks and (D) 23,386 OGT peaks in WT HEK293 cells. Occupancy of PROSER1, UTX, OGT, TET1, TET2, H3K4me1, H3K4me2, H3K4me3, and H3K27ac is displayed. (E) Genomic distribution of all possible 1 kb genomic regions (control) and identified reproducible PROSER1, UTX, TET1, TET2, and OGT peaks in WT HEK293 cells. TES, transcription end site. (F) Venn diagram showing the overlap between down-regulated UTX and down-regulated H3K4me1 peaks in PROSER1 KO versus WT HEK293 cells (both FC = 2). (G) Genomic distribution of all 4,421 UTX/H3K4me1 down-regulated peaks.

Fig 5: Phosphorylation at TET2 Y1902 results in decreased PTEN expression.A The levels of PTEN, AKT, and p-AKT were measured by immunoblotting in SMMC-7721 and HepG2 cells expressing FGFR3 (WT) or FGFR3∆7–9. B Western blot analysis for TET2, PTEN, AKT, and p-AKT in SMMC-7721FGFR3∆7–9 and HepG2FGFR3∆7–9 cell lines expressing TET2 (wild type and Y1902F mutant) or empty vector. C Western blot analysis for PTEN, AKT, and p-AKT in SMMC-7721FGFR3∆7–9 and HepG2FGFR3∆7–9 with indicated treatment. D CCK-8 cell proliferation assay for indicated cell lines. E The correlation of TET2 and PTEN from TCGA data analysis (R = 0.27, P < 0.001). F The mRNA levels of PTEN were measured by qRT-PCR in SMMC-7721FGFR3∆7–9 and HepG2FGFR3∆7–9 cell lines expressing TET2 (wild type and Y1902F mutant) or empty vector. G The 5-hmC content at the PTEN promoter in SMMC-7721FGFR3∆7–9 cell lines expressing TET2 (wild type and Y1902F mutant) or empty vector, **P < 0.01. H ChIP assay was performed to determine the interaction between TET2 and the PTEN promoter. **P < 0.01. I Inhibition of signaling downstream of PTEN decreases cell proliferation induced by FGFR3∆7–9. CCK-8 cell proliferation assay for cell treated with DMSO and LY294002.