Prdm16s activates myeloid gene regulatory networks in MEPs. (A) A volcano plot showing genes with log₂ fold change >0.5 (shaded in yellow; 2135 genes) or < −0.5 (shaded in blue; 1252 genes) with adjusted P < .05 that are differentially expressed in L-MEPs compared with normal MEPs. Two ETS factors, Spi1 and Elf1, are indicated. (B) Pairwise GSEA assessing the enrichment of L-MEP gene signature in different hematopoietic populations revealed that normal MEPs, erythroid progenitors, HSCs, and macrophages are enriched in L-MEP genes, as shown by the positive cumulative enrichment scores (ESs). (C) GSEA showed that L-MEPs were enriched for HSC signature, myeloid development genes, and PU.1 target genes compared with normal MEPs. (D) Gene ontology (GO) analysis of L-MEPs compared with normal MEPs. (E) A heatmap showing the z scores of the differentially expressed genes in L-MEPs compared with normal MEPs. (F) Immunoblotting to detect PU.1, C/EBPα, Elf1, and FLAG-tagged Prdm16s from normal bone marrow (BM) cells (C1-3) and Prdm16s-induced AML cells (P1-3; n = 3). (G) Several Spi1 target genes were highly expressed in L-MEPs compared with normal MEPs (n = 3). (H) Megakaryocytic/erythroid regulators Gata1, Fog1, and Klf1 were highly expressed in MEPs but were downregulated in L-MEPs, as determined by quantitative polymerase chain reaction analysis. Expression levels are relative to those in control BM cells (n = 4). All data represent mean ± standard deviation. *P < .05, **P < .01, ***P < .001 by Student t test. B, B cell; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; Ery, erythrocyte; FDR, false discovery rate; FPKM, fragments per kilobase million; Gran, granulocyte; LT, long term; Mf, macrophage; Mono, monocyte; NES, normalized enrichment score; NK, natural killer cell; ST, short term; WT, wild type.