American Society of Hematology

Figure 5

$Figure 5. Dnmt1 directly binds to the Gata1 locus and promotes Gata1 gene inactivation in A6 cells. (A) Dnmt1 ChIP assay with A6 and MEL cells. The PCR amplifications of the c-Kit, Gata2, and first intron of Actb (Actb int1) genomic regions were used as negative controls. The data are normalized to the level of DNA methylation at the Actb promoter locus. (B) MeDIP assay with A6 and MEL cells. The data are normalized to the level of DNA methylation at the Actb promoter locus. (C) Gata1 and Gata2 mRNA levels analyzed by qRT-PCR in whole bone marrow cells (WBM), the bone marrow LSK fraction, the A6 cells, and the bone marrow ProEB and MEL cells. The results of the qRT-PCR are normalized to glyceraldehydes-3-phosphate dehydrogenase level. (D) Gata1 and Gata2 mRNA levels in the A6 cells determined by qRT-PCR 96 hours after treatment with PBS and 0.05 or 0.1 μM of 5-Aza-dC. (E) Relative mRNA level of Dnmt1, Gata1, and Gata2 in the A6 cells transfected with shRNA targeting Dnmt1 (sh-1 or sh-2) or control shRNA. Results of the qRT-PCR are normalized to GAPDH level. (F) Effect of Dnmt1 knockdown on DNA methylation at the Gata1 dbG region. Bisulfite sequencing is performed using A6 cells transfected with shRNA targeting Dnmt1 (sh-1 or sh-2) or control shRNA. Both methylated (filled circles) and unmethylated (open circles) CpG motifs are depicted. The DNA methylation ratio of the each locus is denoted by the percentage. Data are presented as the mean ± SD with P values from the Student unpaired t test; ***P < .001; **P < .01; *P < .05; n.s., not significant).$

Dnmt1 directly binds to the Gata1 locus and promotes Gata1 gene inactivation in A6 cells. (A) Dnmt1 ChIP assay with A6 and MEL cells. The PCR amplifications of the c-Kit, Gata2, and first intron of Actb (Actb int1) genomic regions were used as negative controls. The data are normalized to the level of DNA methylation at the Actb promoter locus. (B) MeDIP assay with A6 and MEL cells. The data are normalized to the level of DNA methylation at the Actb promoter locus. (C) Gata1 and Gata2 mRNA levels analyzed by qRT-PCR in whole bone marrow cells (WBM), the bone marrow LSK fraction, the A6 cells, and the bone marrow ProEB and MEL cells. The results of the qRT-PCR are normalized to glyceraldehydes-3-phosphate dehydrogenase level. (D) Gata1 and Gata2 mRNA levels in the A6 cells determined by qRT-PCR 96 hours after treatment with PBS and 0.05 or 0.1 μM of 5-Aza-dC. (E) Relative mRNA level of Dnmt1, Gata1, and Gata2 in the A6 cells transfected with shRNA targeting Dnmt1 (sh-1 or sh-2) or control shRNA. Results of the qRT-PCR are normalized to GAPDH level. (F) Effect of Dnmt1 knockdown on DNA methylation at the Gata1 dbG region. Bisulfite sequencing is performed using A6 cells transfected with shRNA targeting Dnmt1 (sh-1 or sh-2) or control shRNA. Both methylated (filled circles) and unmethylated (open circles) CpG motifs are depicted. The DNA methylation ratio of the each locus is denoted by the percentage. Data are presented as the mean ± SD with P values from the Student unpaired t test; ***P < .001; **P < .01; *P < .05; n.s., not significant).

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