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Fig. 4 | Clinical Epigenetics

Fig. 4

From: Aberrant DNA hydroxymethylation reshapes transcription factor binding in myeloid neoplasms

Fig. 4

5hmC enrichment reshapes CEBP-α binding in human leukemia cells. A Histogram and Heatmap representation of 5hmC enrichment within the C/EBP-α binding sites in MOLM13 leukemia cells. B Genome-browser views of 5hmC (red), C/EBP-α (blue) and BRD4 (green) peaks in MOLM13 cells at the indicated regions. BRD4, but not C/EBP-α, was enriched at 5hmC-enriched regions. C Histogram and Heatmap representation of 5hmC enrichment within the BRD4 binding sites in MOLM13 cells. BRD4 ChIP-seq data were obtained from GSM1557123. A total of 54,411 BRD4 peaks were identified. D Dotblot analysis of global 5hmC (top) and 5mC (bottom) levels in MOLM13 cells treated with or without 250 µM Vitamin C (ViC) at the indicated time points. Methylene blue (Methyl Blue) staining was used on the same blot as the loading control. ViC treatment led to 5hmC increase, but had minor effects on 5mC levels in MOLM13 cells. E Immunofluorescent staining of 5hmC (red) and C/EBPα (green) in MOLM13 cells before and after Vitamin C (ViC) treatment for 72 h. DAPI was used for nuclear staining. ViC treatment resulted in a significant increase of 5hmC, but had minor effects on the fluorescent signal of C/EBPα. Scale bar: 5 µm. F Histogram and Heatmap representation of C/EBP-α enrichment within the newly emerged 5hmC peaks following ViC treatment in MOLM13 cells compared with the untreated group. C/EBP-α enrichment was plotted within the new 5hmC peaks gained in ViC-treated MOLM13 cells. C/EBP-α enrichment was significantly reduced in regions showing ViC-induced 5hmC increase. G Immunoblot (left) and statistical quantification (right) of C/EBP-α protein expression in MOLM13 cells treated without and with 250 µM ViC at the indicated time points. Anti-tubulin was used as the loading control. n = 3 biological replicates. H Motif analysis of DHMRs identified in MOLM13 cells before and after ViC (250 µM) treatment for 72 h. I Representative histogram and statistical analysis of flow cytometry analysis on CD11b expression in MOLM13 cells treated with or without ViC (250 µM) for 72 h. MFI: mean fluorescent intensity. Data were shown as mean ± S.D; n = 3. P = 0.0012, by two-tailed Student’s t-test

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