Fig. 5: EGR1 is recruited to the MIF promoter in trTET2-mutant CMML monocytes.

a Quantitative ChIP-PCR analyses of MIF promoter (R3) were performed in sorted peripheral blood monocytes collected from three healthy donors (controls, circles) and 6 CMML patients, including 3 TET2^WT (squares) and 3 trTET2^MUT (triangles) cases. EGR1-associated DNA was immunoprecipitated. Results are expressed as fold enrichment relative to DNA immunoprecipitated with control immunoglobin G (IgG). Data are mean +/− SEM of each sample run in triplicate. Dunnett’s multiple comparison test using control monocytes as control, ns, non-significant, **P < 0.01. b siRNA-mediated downregulation of EGR1 in trTET2^MUT monocytes isolated from CMML3549 (purple) and CMML3565 (blue) with 2 and 3 sets of EGR1-siRNA respectively. RT-qPCR analysis of EGR1 and MIF mRNA expression normalized to RPL32. Data are mean +/− SEM of each sample run in triplicate. Unpaired Students-t test using control monocytes transfected with SCR as control, **P < 0.01. c Quantitative ChIP-PCR analyses of MIF promoter performed in 2 controls, one TET2^WT CMML and 2 trTET2^MUT CMML using antibodies against HDAC1 and HDAC2, focusing on R4 of MIF gene. Data are mean +/− SEM of each sample run in duplicate or triplicate. Dunnett’s multiple comparison test using control monocytes as control, ns non-significant, *P < 0.05, **P < 0.01. d 293T cells were transfected using Lipofectamine 2000 with empty vectors or pcDNA3-EGR1 and pcDNA3-HA-TET2. Immunoblot analysis of EGR1 and HA expression in transfected 293T cell. Actin, loading control. e Co-immunoprecipitation experiments in 293T-transfected cells. An anti-HA (HA 16B12, Covance), an anti-EGR1 (303–390 A, Bethyl Laboratories) or a control IgG were used for IP, followed by immunoblotting with anti-EGR1, anti-HA, and HDAC1 (#39531, Active Motif).