Zhx2 directly represses Zeb2 transcription during NK cell development. (A and B) RNA-seq with purified splenic NK cells from Zhx2^+/+ and Zhx2^Δ/Δ mice. A shows a volcano plot (log₂ [fold change] versus −log₁₀ [P value]). Fold change determinant is Log₂MEANFPKM (Zhx2^Δ/Δ/Zhx2^+/+). B shows the results of GSEA of DP NK (CD27⁺CD11b⁺) or CD27^low NK (CD27⁻CD11b⁺) represented genes. RNA-seq data of DP and CD27^low NK are adopted from Gene Expression Omnibus accession no. GSE45842. NES, normalized enrichment score.(C) Venn diagram showing shared genes in RNA-seq and ATAC-seq of Zhx2^+/+/ Zhx2^Δ/Δ NK cells and reported NK cell maturation–related genes (Gene Expression Omnibus accession no. GSE45842). A subset of genes for different profiles is highlighted. (D) Real-time RT-qPCR showing Zeb2 expression level in Zhx2^+/+ and Zhx2^Δ/Δ NK cells (representative of three independent experiments). (E) Luciferase reporter gene assays were performed in HEK293T cells cotransfected with ZHX2-encoding (ZHX2-OE [overexpression]) or empty (mock) vector and ZEB2 promoter reporter plasmid containing −2,000 to −100 nt of Zeb2 gene (n = 6, representative of three independent experiments). (F) ChIP assay of ZHX2 binding to the ZEB2 promoter in human NK92 cells. PCR primers were designed across the region around −1,438 to −1,276 nt shown in H as a red dashed line to amplify the ZHX2-binding region. Primers in the β-actin exon region were used as a negative control (NC). qPCR analyzed the quantities of fold change (representative of at least three independent experiments). (G) GSEA of Zhx2 mRNA expression in RNA-seq data from Zhx2^+/+ and Zhx2^Δ/Δ NK cells and reported Zeb2-repressed gene signatures (Gene Expression Omnibus accession no. GSE72162). (H) Integrative Genomics Viewer (IGV) screenshot showing Zeb2 locus in Zhx2^+/+ and Zhx2^Δ/Δ NK cells (from ATAC-seq with Zhx2^+/+ and Zhx2^Δ/Δ NK cells). TSS, one open peak in Zhx2^Δ/Δ NK cells, and the primer region for ChIP-qPCR (red dashed line) are shown. (I and J) Adoptive transfer assay. Splenic NK cells (5 × 10⁶) from Zhx2^+/+ or Zhx2^Δ/Δ mice pretreated with mock lentivirus (shNC)/Zeb2 interference lentivirus (shZeb2) were transferred into C57BL/6 recipients. Graph shows the percentage of recipient splenic CD27⁻CD11b⁺ (CD27^low) NK cells (I) and CD107a expression (J; representative of two independent experiments). Dots represent data from individual mice, and error bars represent SEM per group in one experiment. Data were analyzed using Student’s t test (two-tailed paired t test). **, P < 0.01; ***, P < 0.001.