NR4A3 is a target gene of RUNX1 in hematopoietic progenitors. (A) Effect of RUNX1 knockdown on NR4A3 expression. Q-PCR analysis of CD34⁺GFP⁺ cells transduced with the lentivirus expressing shRUNX1_1, shRUNX1_2, or a shSCR (scramble control sequence). The mRNA level of RUNX1 and NR4A3 was normalized to HPRT mRNA level. The histograms show 1 representative experiment of 2, each in triplicate. Error bars represent ± SD of triplicate. (B) Schematic representation of the NR4A3 human promoter region. The arrowhead represents the putative transcription start site. 101,622,464_101,623,969 designates Chr9 genomic positions of the region cloned in luciferase analysis (UCSC hg18 assembly, http://genome.ucsc.edu/). Two-side black arrows designate genomic positions of the amplicons used in ChIP analysis. NR4A3-Luc and NR4A3-MutR1-Luc designate luciferase reporter vector with the region of NR4A3 promoter with or without the mutated R1 RUNX1 binding site in italic bold. (C) Luciferase levels are shown as fold increase relative to cells transfected with promoter construct alone. Error bars represent ± SD of 2 experiments, each in triplicate. *P < .05. (D) ChIP assay was performed on CD34⁺ cells and shows the fold difference in chromatin immunoprecipitated between anti-RUNX1 and irrelevant IgG after normalization to input chromatin (means ± SD, n = 2). Amplified NR4A3 promoter regions R1, R2, and R3 illustrated in panel B were tested. A nearby region without RUNX1 binding site (C1) was amplified and used as a negative control. (E) Luciferase levels are shown as the fold increase relative to cells transfected with normal promoter construct (NR4A3-Luc) alone (on left) and with mutated promoter construct (NR4A3-MutR1-Luc) alone (on right). Error bars represent ± SD of 2 experiments, each in triplicate. *P < .05.