Figure 3.
Regulation of NFE2 expression by JMJD1C via a positive feedback loop entailing H3K9 demethylation. (A) JMJD1C expression in PV, ET, and PMF patients and healthy controls (HC). Peripheral blood granulocytes from n = 10 PV patients, n = 4 ET patients (3 JAK2V617F, 1 CALR-mutated), n = 6 PMF (2 JAK2V617F, 4 CALR-mutated) patients, and n = 19 healthy controls were analyzed by western blotting and densitometry (below). Representative blots are shown. Patient characteristics are given in supplemental Table 6. The Kruskal-Wallis showed that groups did not originate from the same distribution (P < .0001). Groups were therefore compared by using the Wilcoxon signed-rank test. *P < .05; **P < .001. (B) Global H3K9me3, H3K9me2, H3K9me1 levels in PV patients and healthy controls. Peripheral blood granulocytes from n = 10 PV patients and n = 9 healthy controls were analyzed by western blotting. One representative blot of 3 and the densitometric analysis of n = 10 and n = 9, respectively, are shown. ***P < .001; *P < .05. (C) JMJD1C binding in the NFE2 locus in PV patients and healthy controls. Peripheral blood granulocytes of n = 4 PV patients and n = 3 healthy controls were chromatin immunoprecipitated with an antibody against JMJD1C or an IgG control. Top: chromatin immunoprecipitated DNA was amplified with primers covering the -1A and -1F promoter as well as the −5-kb region in the NFE2 locus, illustrated in the schematic representation. Bottom: densitometric analysis of ChIP results. Graphs represent mean ± SEM. *P < .05 by the Student t test. (D) H3K9me2 levels in the NFE2 locus in PV patients and healthy controls. Peripheral blood granulocytes of n = 6 PV patients and n = 7 healthy controls were chromatin immunoprecipitated with an antibody against H3K9me2 or an IgG control. Chromatin immunoprecipitated DNA was amplified as in panel C. One representative PV patient and healthy control each are depicted. Bottom: densitometric analysis. Graphs represent mean ± SEM. ***P < .001; *P < .05 by the Student t test. (E) HP1α binding in the NFE2 locus in PV patients and healthy controls. Peripheral blood granulocytes of n = 4 PV patients and n = 3 healthy controls were chromatin immunoprecipitated with an antibody against HP1α or an IgG control. Chromatin immunoprecipitated DNA was amplified as in panel C. Right, Densitometric analysis. Graphs represent mean ± SEM. *P < .05 by the Student t test. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.