MYL9 and MYH9 are direct target genes of RUNX1 in MKs. (A) Schematic representation of MYL9 and MYH9 promoters. Gray numbers indicate genomic location in UCSC database (GRCh37/hg19). Gray box indicate RUNX1 putative binding sites (MYL9_1, MYL9_2, MYH9_1, MYH9_2) and black lines show MYL9_A, MYH9_A and MYH9_B amplicon fragments studied in ChIP experiment, respectively localized in position: chr20:35169598-35169802, chr22:36784345-36784527, chr22:36785559-36785780. TSS indicates transcription start site and ATG translational start site. (B) Quantitative ChIP analysis was performed in MKs. Primers encompassing (MYL9_A, MYH9_A,_B) or not (control) RUNX1 site were used to amplify input genomic DNA and the DNA precipitated by antibodies against either normal IgG or RUNX1. A genomic region without RUNX1 binding site was used as negative control sequence (control). Values are normalized to input genomic DNA. Values obtained for fold enrichment using MYH10_A and _B were statistically significant as indicated. Data are representative of 3 independent experiments performed in duplicate (n = 3, P < .001, error bars represent SD of duplicate). (C) MYL9-promoter luciferase assay with mutated RUNX1 binding sites (plucMYL9mut1, plucMYL9mut2, plucMYL9mut1/2) or with WT promoter (plucMYL9) in HEL cells. Luciferase assay was performed by transient HEL cell cotransfection with 500 ng of MPI vector containing RUNX1 WT and pEF6/V5-His-TOPO vector con-aining CBFβ. Luciferase levels are shown as fold change relative to cells transfected with the promoter construct (plucMYL9, plucMYL9mut1, plucMYL9mut2) or plucMYL9mut1/2 alone. The total amount of transfected DNA was kept constant by transfection with an empty vector. (C) Illustrates 1 representative experiment (n = 3, P < .01, error bars represent SD of triplicate). (D) MYH9-promoter luciferase assay with (plucMYH9mut1) or without mutated RUNX1 binding site (plucMYH9) in HEL cells. (D) Illustrates 1 representative experiment (n = 3, P < .001, error bars represent SD of triplicate).