Redundant roles of Zfp281 and Zfp148 in γ-globin gene expression in K562 cells. (A) Western blot showing knockout (KO) of Zfp148 in K562 cells by CRISPR/Cas9. (B) Western blot showing knockdown of Zfp281 protein levels in K562 cells using lentiviral transduction of shRNA constructs c#1 and c#2. A lentiviral shRNA against luciferase (shLuc) is shown as a control. (C) Western blot showing Zfp148 and Zfp281 protein levels in Zfp148 KO cells or control cells transduced with shZfp281c#1 or shLuc. (D) Representative BioChIP-seq signals at the γ-globin locus in hemin-induced K562 cells expressing Bir A alone or Bir A and ^BioZfp148 or ^BioZfp281. Peak calls for H3K27ac, H3K27me3, H3K4me1, H3K4me2, H3K4me3, and H3K9ac from ENCODE³⁵  are indicated. (E) Quantitative RT-PCR analysis of γ-globin mRNA transcripts from cells in panel C. The levels were normalized to GAPDH mRNA transcript levels and are shown relative to the Zfp148 WT and shLuc control. HBG, hemoglobin subunit gamma.