Altered transcription profile in ST-HSCs upon dnTCF4 expression. (A) Volcano plot showing the differentially up- and downregulated genes in dnTCF4 ST-HSCs compared with WT ST-HSCs (n = 3 WT samples and n = 3 dnTCF4 samples, all isolated from 12-week-old mice). Numbers indicate the number of differentially expressed genes. (B) Transcription factor perturbations followed by expression analysis via online Enrichr tool. The list of downregulated genes in dnTCF4 ST-HSCs is enriched for genes that were deregulated in murine models with deletion (KO [knockout]) or overexpression (OE) of certain transcription factors. Gene Expression Omnibus accession numbers are indicated on the right. The x-axis indicates q values. (C) Quantitative RT-PCR in WT and dnTCF4 (dn) c-Kit⁺ cells. The y-axis represents Csf3r expression relative to the percentage of Gapdh. Data represent mean ± SD; n = 6 WT and n = 5 dnTCF4 samples. Two-tailed Student t test was used to assess statistical significance, **P < .01. (D) Flow cytometric analysis of G-CSF-R expression on the surface of WT and dnTCF4 LKS (left) and c-Kit⁺ cells (right). (E) Quantification of panel D. The y-axes indicate mean fluorescence intensity (MFI) for G-CSF-R levels. Data represent mean ± SD; n = 5 WT and n = 6 dnTCF4 samples. Two-tailed Student t test was used to assess statistical significance (**P < .01 and ***P < .001). (F) TCF4 binding profiles in the CSF3R gene locus in HEK293T, HepG2, MCF-7, PANC-1, and Hela S3 cells. The enhancer histone modification profiles (H3K4me2, H3K27ac, and H3K4me1) in the CSF3R gene locus in Hela S3 and HepG2 cells are also illustrated. Data were obtained from the ENCODE database. The arrow indicates direction of transcription; blue lines (bottom) depict positions of TCF/LEF consensus binding sequence GCTTTG; and boxes R1, R2, and CR describe the position of regions analyzed in the following ChIP-qPCR experiment. (G) ChIP-qPCR assay showing LEF1 binding to the TSS (R1), putative enhancer −3.3 kb from TSS (R2), and negative control region CR (−6.1 kb) of human CSF3R gene locus in cross-linked chromatin from K562 cells. The panel is a representative of 3 independent experiments, data represent mean ± SD.