Differential transcriptional and epigenomic activity of mutant R307C/H and GATA1 WT. (A) Schematic of experiment. Indicated expression vectors were introduced to murine G1E cells via lentiviral integration followed by in vitro culture. (B) Frequency of Ter119⁺ cells at indicated day postinfection (pi) as assessed by flow cytometry. (C) Volcano plot of differentially expressed genes as determined by RNA-seq comparing G1E cells expressing mutant and GATA1 WT at day 3 pi. Selected genes are highlighted. (D) Heatmap showing results of k-means clustering of differentially expressed genes in G1E cells expressing indicated GATA1 constructs. Results of 2 replicates for each construct were pooled for this analysis. Color bar: z-scored expression. (E) Mean expression (log₂ counts per million [cpm]) of genes per cluster from panel D across the process of human erythroid differentiation, including hematopoietic stem cells (HSCs), multipotent progenitor cells (MPPs), common myeloid progenitor (CMPs), megakaryocyte erythroid progenitor cells (MEPs), myeloid progenitor cells (MyPs), colony-forming unit erythroid cells (CFU-Es), proerythroblasts (ProE1 and ProE2), basophilic erythroblasts (BasoEs), polychromatic erythroblasts (PolyEs), orthochromatic erythroblasts (OrthoEs), and OrthoEs further enriched with reticulocytes (Orth/Ret). (F) Gene ontology (GO) terms for cluster K4 from panel D that identify key pathways associated with impaired gene induction by G1E cells expressing R307C/H compared with GATA1 WT. (G) Differences in chromatin accessibility as determined by ATAC-seq in G1E cells expressing indicated GATA1 constructs compared with GATA1 chromatin occupancy at respective sites in G1E-ER4 cells at indicated hours after differentiation induction. (H) Meta gene pileup analysis of chromatin accessibility patterns of genes in clusters K1 to K4 as determined in panel D. The position relative to the transcription start site (TSS) is shown. (I) Cross-species analysis of differential expression patterns of genes differentially regulated in the primary patient cell rescue data (Figure 2) and the G1E complementation experiments. The dot plot shows the log₂ fold change (FC) for each gene comparing GATA1 WT relative to R307C/H mutant transduced cells for all genes that were differentially expressed in both species (adjusted P < .1; n = 226). Two replicates for GATA1 WT and all 4 mutant replicates were pooled for this analysis. BP, biological process; MF, molecular function.