DNA hypomethylation at the Gata1 locus leads to GATA2-mediated aberrant Gata1 gene activation. (A) The in vitro methylated 659-bp minigene fragment ligated to the GFP expression cassette. Methylated and unmethylated vectors were electroporated into A6 cells along with the tdTomato expression vector. (B) GFP expression in the tdTomato-positive cells analyzed by flow cytometry. The data are presented as the mean ± SD from 3 independent experiments. (C) The GATA2 ChIP assay with the c-Kit−positive bone marrow progenitors. A representative dataset from the experiments, which were repeated 3 times, is shown. (D) The relative GFP intensity in the LSK CD34-negative (LT-HSC) fraction of the G1B-GFP (n = 5), MG-GFP (n = 7), MG-GFP::Gata2^+/− (n = 9) and MG-GFP::Gata2^fGN/fGN (n = 3) mice. The data are presented as the mean ± SD. The statistical significance is indicated (**P < .01; *P < .05; n.s., not significant; Student unpaired t test). (E) A GFP histogram of the LSK CD34-negative fraction. Gray line, G1B-GFP (line 392); black line, MG-GFP (line 20); dotted line, MG-GFP::Gata2^fGN/fGN (line 20). (F) Dnmt1 recruited to G1MDR sequences confers Gata1 gene inactivation by maintaining the CpG-methylation of the Gata1 gene enhancer sequences in the HSCs (upper diagram). The DNA hypomethylation in the MG-GFP transgenic allele increases GATA2-binding, thereby promoting abundant GFP expression in the HSCs (lower diagram). (G) A proposed model for the initiation of erythropoiesis through proper temporary-specific Gata1 gene regulation elicited by the GdC-region. In HSCs, the Gata1 gene is protected from GATA2-mediated Gata1 gene activation through DNA methylation, which in turn maintains the prematurity of the HSCs (upper diagram). When DNA methylation decreases, GATA2 transactivates the Gata1 gene expression to initiate erythropoiesis in the MEPs (lower diagram).