Decitabine-dependent downregulation of NFE2 occurs selectively in the presence of JAK2^V617F. (A) Effect of decitabine treatment on NFE2 expression and total H3K9me2 levels in K562 cells. K562 cells were treated with 400 ng/μL decitabine or DMSO. Lysates were interrogated by western blot. (B) Effect of decitabine treatment on H3K9me2 levels in the NFE2 locus in K562 cells. Cells were treated as in panel A. Cell lysates were chromatin immunoprecipitated with an antibody against H3K9me2 or an IgG control. Chromatin immunoprecipitated DNA was amplified with primers covering the NFE2 locus as illustrated in Figure 3C. One representative result of 3 independent experiments is shown. Bottom panel; densitometric analysis. Graphs represent mean ± SEM. (C-D) NFE2 expression after decitabine treatment in JAK2^V617F-positive and JAK2^wt cell lines. Whole-cell extracts of the indicated cell lines were treated as noted and analyzed as described in panel A. (C) JAK2^V617F-positive; (D) JAK2wt. (E) Effect of decitabine treatment on SHP1, phosphorylated JAK2 (p-JAK2), JAK2, and HP1α expression. HEL cells were treated as in panel A. Total cell lysates were interrogated by western blotting. One representative result of 3 independent experiments is shown. (F) Effect of JAK2 inhibitor treatment on NFE2 and JMJD1C expression. HEL cells were treated with the indicated concentrations of the JAK2 inhibitor TG101348 (fedratinib)²⁶  for 48 hours as indicated. Whole-cell lysates were analyzed by western blotting for JMJD1C, NFE2, PU.1, and actin. One representative result of at least 2 independent experiments is shown. (G) H3Y41P ChIPseq density profiles at the NFE2 gene locus in HEL cell ± JAK2 inhibition. Visualization of H3K4me3, H3Y41P before and after inhibition of JAK2 by TG101209 and IgG control. ChIPseq data from Dawson et al.²⁷  (H) Effect of Jmjd1c knockdown on cytokine-independent cell growth. Baf/3-Jak2^V617F cells expressing Jak2^V617F were transduced with a lentivirus encoding an shRNA against Jmjd1c or a scrambled control. Western blot analysis showed successful knockdown of JMJD1C (left, top). The cells were cultured without (left) or with (right) IL3. Cells were counted every 24 hours. Three independent experiments were performed in triplicates. Data points represent mean ± SEM. ***P < .001; **P < .01, *P < .05 by 2-way ANOVA.