Fig. 5: Increased CEBPA p30 binding to the Gata2 distal hematopoietic enhancer drives expression of Gata2 via TET2.

a Gata2 mRNA expression in mouse Cebpa^p30/p30 leukemic granulocyte/monocyte progenitors (GMPs) vs normal GMPs (samples from 4 Cebpa^+/+ and 2 Cebpa^p30/p30 mice) and, b CEBPA binding to the Gata2 distal hematopoietic enhancer (G2DHE; −77 kb) region (samples from 2 mice per group), data from Jakobsen et al.¹¹. Data are presented as mean ± SEM. Differential analysis was performed with DESeq2 (P < 0.05). c Schematic genomic view of the Gata2 distal hematopoietic enhancer (G2DHE), including normalized chromatin immunoprecipitation sequencing (ChIP-seq) signal of CEBPA (data from Heyes et al.¹²), TET2 and H3K27Ac (data from Heyes et al.¹²), as well as assay for transposase-accessible chromatin using sequencing (ATAC-seq) in Cebpa^p30/p30 cells without (light blue) and with (green) mutation in Tet2. d Gata2 mRNA levels in response to targeting of the G2DHE by CRISPR-Cas9 in Cebpa^p30/p30 cells in vitro using indicated sgRNAs and e the averaged change in Gata2 mRNA levels of the 12 deletions (averaged data from 2 separate experiments). Data are presented as median ± range and analyzed by two-tailed Wilcoxon signed-rank test. f Experimental setup for evaluating the effects of Cebpa knockout on Gata2 V2 mRNA expression and DNA methylation of the CpG island at the promoter of Gata2 V2 in MLL-fusion driven AML (iMLL-AF9). The illustration was created with BioRender.com. g Gata2 V2 mRNA expression (leukemic cell lines generated from 2 separate mice were assayed on 2 separate days in 2–3 technical replicates each). Data are presented as mean ± SEM and the individual cell lines are indicated by circles or squares. Data were log-transformed and analyzed by two-tailed unpaired t-test. h DNA methylation of the Gata2 V2 promoter CpG-island (2 separate leukemic cell lines). Data are presented as median±range and the individual cell lines are indicated by circles or squares. i Experimental setup for evaluating the effects of Cebpa knockdown on TET2 binding to the G2DHE in Cebpa^p30/p30 cells with inducible expression of shRNA targeting Cebpa and control (Renilla), respectively. The illustration was created with BioRender.com. j TET2 binding to the G2DHE assessed by ChIP-qPCR (3 replicates per condition). Data are presented as mean±SEM and analyzed by two-tailed unpaired t-test. k Frequency of GATA2 and/or TET2 mutations (GATA2^MUT and TET2^MUT, respectively) in CEBPA high expressing (CEBPA^HIGH; 45 cases) vs. CEBPA low expressing (CEBPA^LOW; 61 cases) AML cases, data from Beat AML cohort¹. The distributions of GATA2^WTTET2^WT vs. GATA2^MUT and/or TET2^MUT cases were analyzed by Wilson/Brown binominal test. Source data are provided as a Source Data file.