Fig 2: GATA1 Is a Potentially Interacted Protein with FAM122A and the Gene Regulation Profiling upon FAM122A Knockout(A and B) The potential interacted proteins with FAM122A were analyzed by in vitro binding assay and MS analysis. Schematics showed the process of the experiments (A). Coomassie brilliant blue-stained gel of proteins bound to GST and GST-FAM122A are shown, followed by identification with tandem mass spectrometry (B). The indicated bands referred to GST-FAM122A, GATA1, and the known FAM122A interacting proteins, PP2A-Aa, -Ba, and -Ca (left panel). The indicated proteins were analyzed by scaffold 4 proteome software and their information is presented (right panel). Peptides and coverage indicate the numbers of unique peptides and the percentage of sequence coverage for identified proteins.(C and D) FAM122A KO and NC K562 cells were subjected to RNA sequencing and bioinformatics analysis. Differentially expressed genes were analyzed by gene ontology (C), and the most upregulated genes in FAM122A KO K562 cells were enriched as indicated in molecular functions, cellular components and biological processes. Heatmap presents the enriched candidate and upregulated genes involved in erythroid maturation and function as indicated with red words (D).(E) The representative candidate genes regulated by FAM122A were confirmed by qPCR (n = 3). Data indicate means with bar as SD in an independent experiment.

Fig 3: FAM122A Inhibits the Transcriptional Activity of GATA1 by Reducing Its Association with the Targeting DNA(A) EMSA assay was performed to analyze the effect of FAM122A on GATA-1 binding to target DNA sequences by purified proteins expressed in E. coli. Specificity of the shift was confirmed by the complete competition with wild-type competitors but not the mutant one (upper panel). The protein expressions were examined by western blot (bottom panel) (n = 3).(B) 293T cells were co-transfected with the increasing concentrations of GFP-FAM122A with or without Flag-GATA-1, together with pro-ALAS2-pGL3-basic (ALAS2-pro)/pro-PRG2-pGL3-basic (PRG2-pro) and pRL-SV40 plasmids (as an internal reference plasmid), and the luciferase activities were measured after transfection for 24 h. The efficiency of protein expression was confirmed by western blot (bottom panels) (n = 3).(C) ChIP assay was performed to analyze GATA1 binding to the promoter and enhancer regions of PBGD, AHSP, and AQP1 genes in FAM122A knockout (upper panels) or overexpression (bottom panels) K562 cells treated with 50 µM hemin for 48 h. Input, 1% of chromatin lysate subjected to immunoprecipitation; IgG, normal IgG as a negative control (n = 3).

Fig 4: FAM122A Interacts Directly with GATA1 by Its C-Terminal Zinc Finger Domain(A–D) FAM122A directly interacted with GATA1. 293T cells were transiently co-transfected with GFP-FAM122A, with or without Flag-GATA1 plasmids, and co-immunoprecipitation (coIP) was performed with anti-Flag (left panel) or anti-GFP (right panel) antibodies, respectively. Western blots were used to examine the proteins in immunoprecipitates (n = 3) (A). Interaction of endogenous FAM122A and GATA1 are shown by IP with anti-FAM122A (upper panel) or GATA1 (bottom panel) antibody in K562 cells, followed by immunoblotting with the indicated antibodies (n = 3) (B). Representative images of colocalization of endogenous FAM122A (red) and GATA-1 (green) proteins are shown in K562 (upper panels) and CD34+ cells treated with EPO (lower panels) (n = 3) (C). Direct interaction of FAM122A and GATA1 was analyzed by GST pull-down assay by in vitro expressed GST-FAM122A and His-GATA1 proteins (n = 3) (D).(E) Structure diagram of full-length and truncated GATA1 plasmids used in (F) and (G) is shown. GATA1-expressing plasmids were sequentially truncated to produce the mGATA1 (84-413), mGATA1 (233-413), mGATA1 (286-413), mGATA1 (84-254), or mGATA1 (?C-ZF), which specifically deleted the C-terminal zinc finger domain for subsequent analysis of the critical domains for GATA1 and FAM122A interaction.(F and G) 293T cells were transiently co-transfected with Flag-FAM122A and GFP-tagged different truncated/mutated GATA-1 plasmids, followed by coIP assay with anti-Flag antibody (n = 3).