G1MDR sequences between G1HE and dbG exert a repressive function in A6 cells. (A) A schematic diagram depicting the assay system used to test the silencer activity of the 3.2-kb sequence between G1HE and dbG (G1MDR). Gata2-GFP, G1MDR-GFP, G1MDR5′-GFP, G1MDR3′-GFP, or G1MDR3′E2Fmut-GFP construct was introduced into A6 cells along with the tdTomato-expression reporter vector (red fluorescence reporter) by electroporation. Resulting GFP expression in the tdTomato-positive cells was analyzed by flow cytometry 24 hours after electroporation. Autoregulatory GATA-binding sites located 77 kb (light gray), 2.8 kb (dark gray), and 1.8 kb (black) upstream of the Gata2 gene are linked in the reporter constructs. IS depicts the hematopoietic cell-specific Gata2 promoter. (B) Genomic sequence conservation among mammalian species around G1MDR is derived from UCSC genome browser (http://genome.ucsc.edu/). The horizontal arrow denotes G1MDR. The dotted rectangles indicate the G1MDR5′ and G1MDR3′ regions. Conserved E2F binding sequences in the G1MDR3′ region is predicted in TRANSFAC database (https://portal.biobase-international.com). (C) Relative mean GFP intensity in the A6 cells introduced with Gata2-GFP, G1MDR-GFP, G1MDR5′-GFP, G1MDR3′-GFP, and G1MDR3′E2Fmut-GFP. Data are presented as the mean ± SD from 3 independent experiments with P values from a Student unpaired t test. (D) GFP histogram of the A6 cells introduced with the Gata2-GFP, G1MDR-GFP, G1MDR3′-GFP, and G1MDR3′E2Fmut-GFP. The mean fluorescence intensities (MFI) of GFP are analyzed by flow cytometry.