Loss of hemogen increases the recruitment of corepressors and decreases enhancer/promoter looping in the context of the LDB1 complex in human K562 cells. (A) Volcano plot showing differential gene expression between hemogen-KO and WT K562 cells. Dashed vertical lines represent log₂ fold change (−0.6 and +0.6). Dashed horizontal line represents adjusted P value < .05. Light blue dots highlight erythroid genes that were significantly downregulated upon hemogen KO (n = 3 biological replicates). (B) Lfc-lfc plot comparing HEMGN-KO and CBFA2T3 (ETO2)-KO dysregulated genes in K562 cells. Red squares represent genes that were positively regulated by HEMGN and negatively regulated by ETO2. Genes of interest are labeled. ChIP-qPCR for hemogen (C), ETO2 (D), and CHD4 (E) occupancy in the β-globin locus in hemogen-KO and WT K562 cells. ChIP-qPCR for GATA1 (F) and LDB1 (G) occupancy in the β-globin locus in hemogen-KO and WT K562 cells. (H) Coimmunoprecipitation (IP) and western blot analysis of interaction between hemogen (hem) and LDB1, GATA1 or BRG1. (I) Chromosome conformation capture (3C) interactions between the β-globin LCR (anchor) and genes in hemogen-WT and -KO K562 cells. Error bars represent standard deviation. N = 3 biological replicates. *P < .05, **P <.01, 2-tailed Student t test. IgG, immunoglobulin G.