Fig 2: Identification of ZEB1‐binding DNA regions. (A) ZEB1‐binding signals at the ESRP2 and HBB gene loci. The y‐axis scales are not equal as they were adjusted to show the binding signals clearly for each cell line. The kb sizes denote the ranges shown in the panels. The bars below the binding signals indicate the significant binding regions (q < 0.05). (B) De novo motif prediction was performed from the 500‐bp sequences flanking the summit position of each ZEB1‐binding region using the Gibbs Motif Sampler provided by CisGenome. Default parameters were used except for the mean motif length (8), maximum motif length (15), and K (15). The identified E‐box‐like motif was compared to known motifs using the Tomtom motif comparison tool in MEME suite version 4.11.2. The alignment of the calculated motif with a top‐ranked motif (MA0103.2) that was deposited as a ZEB1‐binding site is shown. Y‐axis scale: bits. (C) Motif centrality analysis was performed using the motif calculated in (B) and CentriMo of the MEME suite. The x‐axis indicates the relative position of the best site from the peak summit of each ZEB1‐binding region. (D) A heatmap representation of the ZEB1‐binding regions in the genomes of the indicated cell lines. The y‐axis indicates the union of the ZEB1‐binding regions that were obtained from five cell lines as sorted by the signal intensities of MDA‐231‐D and HepG2. X‐axis: the position of each binding region relative to the peak summits of the ZEB1‐binding regions shown on the y‐axis. (E) Frequencies of the ZEB1‐bound genes showing expression that was up‐ or downregulated more than 1.5‐ or 2‐fold by siRNA targeting ZEB1 and ZEB2 (siZEB1‐1 and siZEB2‐1) in the absence of TGF‐β. Of the 15 175 ZEB1‐bound genes (ZEB1‐binding regions within 50 kb from the transcription start site, TSS), the genes encoding small RNA or showing expression that was < 10 FPKM (fragments per kilobase of exon per million mapped reads) were excluded from the evaluation. In panels (E) and (F), MDA‐231‐D cells were transfected with siRNA and treated with or without 1 ng·mL−1 of TGF‐β for 24 h. RNA‐seq was then performed. siNC, control siRNA. (F) A scatter plot showing the relationship between the effect of ZEB1/2 siRNA (siZEB1‐1 and siZEB2‐1) and TGF‐β as determined by RNA‐seq. ZEB1‐bound, TGF‐β‐regulated genes showing expression that was induced or suppressed ≥ 2‐fold were selected (n = 5975). siZEB1/2‐1, siZEB1‐1 + siZEB2‐1. Each dot represents a single gene.

Fig 3: The effect of TGF‐β signaling on the expression of ZEB1‐regulated secretory proteins. (A) The expression of ZEB1 and ZEB2 in MDA‐231‐D cells as determined by RNA‐seq. Error bars, C.I. (B) qRT‐PCR analysis of IL6 and IL8 expression in the presence or absence of TGF‐β (1 ng·mL−1, 48 h) and a TGF‐β inhibitor (LY364947: 1 μm, 48 h) in MDA‐231‐D cells transfected with ZEB1/2 siRNA. Data represent the means of two biological replicates. Error bars, S.D. *P < 0.05; n.s., not significant. siZEB1/2‐1, siZEB1‐1 + siZEB2‐1; siZEB1/2‐2, siZEB1‐2 + siZEB2‐2. (C) A heatmap showing the effect of exogenous ZEB1 expression versus control vector infection as determined by antibody array analysis of the conditioned medium obtained from a 48‐h culture of serum‐starved HCC1954‐Luc cells. The list of proteins is the same as in Fig. 2E, and it is arranged in the order of induction by ZEB1 in HCC1954‐Luc cells. HCC‐ZEB1, ZEB1‐expressing HCC1954‐Luc cells; HCC‐GFP, GFP‐expressing HCC1954‐Luc cells. n.d., not detected using the same criteria as in Fig. 2E. (D) qRT‐PCR analysis to determine the effects of TGF‐β on IL6 expression in HCC1954‐Luc cells that were overexpressing ZEB1. RNA was obtained from the cells 48 h after TGF‐β stimulation. Data are shown as the means of two biological replicates. Error bars, S.D. *P < 0.05.

Fig 4: ZEB1 regulates the expression of inflammatory response genes. (A–C) GSEA was performed using the RNA‐seq data in Fig. 1E and F. ZEB1‐bound genes were selected for evaluation. (A) Gene sets in MDA‐231‐D cells that were differentially expressed after treatment with ZEB1/2 siRNA (siZEB1‐1 and siZEB2‐1). The most enriched MSigDB hallmark signatures that had normalized enrichment scores (NES) > 1.5 or < −1.5 are listed. (B) Enrichment plot of the HALLMARK_INFLAMMATORY_RESPONSE gene set. ZEB1‐bound genes (vertical black bars in the bottom panel) were enriched toward the left; ZEB1 and ZEB2 functioned as transcriptional activators. siZEB1/2‐1, siZEB1‐1 + siZEB2‐1. (C) Venn diagram showing the overlap of genes that were downregulated by ZEB1/2‐1 siRNA in the presence and absence of TGF‐β. Genes with maximum FPKM values ≥ 10 in the four samples were selected for further evaluation, and those with fold changes (FPKM (siZEB1/2) + 0.01)/(FPKM(siNC) + 0.01) ≤ 0.5 were defined as downregulated genes. (D) Visualization of the ZEB1 ChIP‐seq data tracks at the IL6,IL8, and CSF2 gene loci. Data are shown as in Fig. 1A. (E) Antibody array analysis of proteins in conditioned medium obtained from serum‐starved MDA‐231‐D cells. ZEB1‐bound genes and genes regulated by ZEB1/2 siRNA according to ChIP‐seq and RNA‐seq analysis are shown in a heat map in the order of downregulation by siRNA (left panel). Conditioned medium was collected from MDA‐231‐D cells treated with ZEB1/2‐1 siRNA and analyzed using an antibody array. The effects of ZEB1/2 siRNA on cytokine secretion are shown in the right panel. Genes were selected according to the following criteria: Maximal expression values (FPKM) were ≥ 10, and there was a fold change ≥ 1.5 in response to ZEB1/2 siRNA. Note that the amount of IL‐6 protein in this cell line was very high and could not be quantitatively evaluated in this array. n.d.: not detected. (F) The effect of ZEB1/2 siRNA on the secretion of IL‐6 and IL‐8 by MDA‐231‐D cells as determined by ELISA. Error bars indicate the S.D. *P < 0.05. (G) The effect of ZEB1/2 siRNA on the secretion of IL‐6 and IL‐8 by Hs578T cells as determined by ELISA. Error bars indicate the S.D. *P < 0.05. In (F) and (G), siZEB1/2‐1, siZEB1‐1 + siZEB2‐1; siZEB1/2‐2, siZEB1‐2 + siZEB2‐2.

Fig 5: Requirement of Notch signaling and hematopoietic cytokines for hematopoietic enhancer activation(A) Heatmap and boxplots showing the effects of Notch signaling inhibition by DAPT and of the absence of hematopoietic cytokines on the H3K27ac ChIP-seq signal at the hematopoietic enhancers defined in Figure 1C. DAPT was added from day 4 at a concentration of 10 μM, and hematopoietic cytokines (SCF, TPO, FLT3L and FP6) were removed the same day. ns, p > 0.05; ∗∗∗∗P ≦ 0.0001 (repeated-measures one-way ANOVA followed by Dunnett’s multiple comparisons test).(B) Flow cytometry of control and DAPT-treated live cells on day 10 for identifying HPCs by the expression of CD34 and CD45. DAPT was added from days 4–10 at a concentration of 10 μM. Representative (left) and summary data (right) are shown. ∗P ≦ 0.05 (paired t-test).(C) Percentage of hematopoietic enhancers occupied by Notch1 on day 7. Hematopoietic enhancers are divided into 3 clusters, as shown in Figure 1C.(D) Fold-change of the indicated gene expressions in day 7 CD34+ cells by DAPT treatment. Genes were selected from the transcription factors whose binding motifs were enriched at cluster 2 enhancers (see Figure 2A). ns, FDR >0.05; ∗∗FDR ≦ 0.01; ∗∗∗∗FDR ≦ 0.0001 (calculated by DESeq2).(E) Binding of the indicated transcription factors to hematopoietic enhancers on day 7. The ChIP-seq signal distribution at enhancer regions (5′ to 3′ ends) +/− 1 kB (top) and the proportions of hematopoietic enhancers bound by the transcription factors (bottom) are shown. Hematopoietic enhancers were grouped according to the timing of their activation, as shown in Figure 1C.(F) Expression of the indicated genes during hematopoietic cell differentiation. Genes were selected from candidate regulators of cluster 1 and 2 hematopoietic enhancers, as shown in Figure 2A.(G) Expression patterns of ZEB2 and MEIS1 during in vitro hematopoietic cell differentiation. The averages and standard deviations are shown.(H) Occupancy of the genomic loci of the genes shown in Figure 3E by the five indicated regulators. The average of two independent experiments (A, C, D, G), representative and summary of four independent experiments (B), individual data of two or three independent experiments (F), and representative data from one or two experiments (E, H) are shown.