Fig. 1: JMJD2C constitutes a novel NFE2 target gene.
A Top: NFE2 ChIP-seq at the JMJD2C locus [7]. Bottom: ChIP PCR of HEL cells using an antibody against NFE2 or an IgG control, as depicted. PCR primers flank the predicted binding site at +260 bp or negative control sites at +4.6 kb of the JMJD2C locus and the myogenin locus. B Jmjd2c mRNA expression in BM of hNFE2tg mice (n = 12) or littermate controls (n = 9) determined by RT-qPCR. Expression is normalized to B2m expression. C Jmjd2c mRNA levels in CB3 cells following infection with pLeGO-iG-NFE2-wt or an empty control vector. GFP positive cells were sorted and Jmjd2c expression measured as described in panel 1B. D Illustration of the JMJD2C reporter gene construct. A circle indicates the predicted NFE2 binding site +260 bp relative to the transcription start site (TSS). E Cotransfection of HEK-293T cells with the JMJD2C luciferase reporter gene construct and different combinations of expression plasmids encoding NFE2 and MafG cDNAs, as depicted. F Disruption of the potential NFE2 binding site by site directed mutagenesis (crossed circle). Altered bases are shown in bold. E, F Data were normalized to the wt JMJD2C reporter gene construct cotransfected with MafG alone, which was set as one. G Exemplary western blots depicting JMJD2C expression in lysates from PB granulocytes. GAPDH was used as a loading control. H Densitometric analysis of JMJD2C western blots, n = 26 MPN patients and n = 9 healthy controls. I JMJD2C protein expression across different MPN subtypes (ET = 5, PV = 12, PMF = 9). J JMJD2C protein expression according to mutational status (JAK2V617F = 19, CALR = 7). C, E, F Data are represented as mean of at least three independent experiments. B, C, E, F, H–J *p < 0.05, **p < 0.01 by Student’s t test.
