p19INK4D is directly regulated by AML-1 (RUNX1). (A) Schematic representation of the p19INK4D human promoter region. The arrowhead represents the putative transduction start site. Black bars with diamonds designate genomic positions of the amplicons used in ChIP analysis (UCSC genomic localization http://genome.ucsc.edu/). plucF2 and plucF3 represent schematically the length of the promoter regions cloned in the pluc-mcs reporter. (B) Luciferase assay performed by transient transfection of HEL cells with the indicated micrograms of MPI vector containing AML1c and of pEF6/V5-His-TOPO vector containing CBFβ. Luciferase levels are shown as fold change relative to cells transfected with reporter construct alone. The total amount of transfected DNA was kept constant by transfection with an empty MPI vector. The histograms show 1 representative experiment of 2, each in triplicate. Error bars represent the standard deviation of triplicate. (C) Chromatin immunoprecipitation assay performed in CD34+-derived MKs (day 10 in culture) with primer sets (p19INK4DA, B, and C) directed toward AML-1–binding site predicted in silico, as well as control primer sets. Control rabbit IgG and 2 anti–AML-1 antibodies were used for immunoprecipitation. The histograms indicate relative occupancy of the AML-1–binding site in the p19INK4D promoter by AML-1. The figure illustrates 1 representative experiment using 2 different anti–AML-1 antibodies. Error bars represent the standard deviation of experiments performed on the same cell extracts with 2 different anti–AML-1 antibodies in duplicate. A similar result was obtained in a second independent experiment. (D,E) CD34+ cells were transduced at days 1 and 2 of culture with a control lentivirus and the lentivirus encoding either shRNA AML-1 or control shRNA. GFP+ cells were analyzed at day 9. (D) p19INK4D and AML-1 mRNAs were measured by QRT-PCR in GFP-positive cells. Error bars are standard deviations of the mean of 3 repeated experiments. (E) p19INK4D and AML-1 protein level was analyzed by Western blot. HDAC-1 was used as internal control of a quantity. The data illustrate 1 of 2 representative experiments that produced similar results.
