Figure 1.
Heatmap display of transcription factors that differential expressed (>1.5 fold) between Lin-CD34+ cell and Lin-CD34− cells.
Two replicas were shown for each cell type. Red color represents up-regulated genes and green color represents down-regulated genes. Genes mentioned in the text are labeled. Cd34 and Ly6a (Sca1) are cell surface markers. See “Methods” for the calculation of Z scores.
Figure 2.
Identification of transcription factor binding targets using ChIP–Sequencing.
(A) Tcf7 is bound by both itself and by RUNX1 (AML1). Peaks indicate ChIP-Sequencing signal. Input genomic DNA serves as the negative control. The “binding sites” tracks (black vertical bars) show the transcription factor binding loci determined using the PeakSeq program (normalized against genomic input DNA; q-value<0.001). Data are visualized in Integrated Genome Browser (B) Identification of evolutionarily conserved RUNX1 binding sites at Tcf7 promoter region using REGULATORY VISTA. The graph shows conserved and aligned AML1/RUNX1 transcription factor binding sites between mouse and human genomes using a matrix similarity score of 1 (the most stringent). Two versions of the AML1 binding sites were found (AML1 and AML_Q6). The *ECRs: Evolutionary Conserved Regions are indicated by deep red blocks. The degree of conservation (50%–100%) is indicated by the height of the peaks. Coding region is sown by blue and UTR is shown by yellow. (C) Runx1 promoter is bound by both TCF7 and itself.
Figure 3.
Binding site analyses suggest RUNX1 functions coordinately with TCF7 in regulating target genes.
(A) In the Venn diagram, the blue circle represents TCF7 target genes and red circle represents RUNX1 target genes. 3915 target genes (72% of all RUNX1 targets and 49% of all TCF7 target genes) are common to each other. The hypergeometric p-value of the intersection (compared to the entire UCSC known gene collection 24901 genes) is 6.72294e-56. (B) The graph represents the distribution of the distance of the RUNX1 peak to the TCF7 peaks. (C) The distribution of TCF7 binding motifs and RUNX1 motifs in binding regions of both factors are presented. Since TCF7 had two versions of the motifs, non-duplicate union of occurrence of both motifs was indicated in the table. The sequence of one (version) of the motif logos is shown in the table.
Figure 4.
Correlation of transcription factor binding targets with RNA–Seq differential gene expression data by GSEA.
(A). A statistically significant enrichment of TCF7 targets was shown among up-regulated genes in CD34+ cells (Nominal p-value = 0, FDR q-value = 0.02). Differential gene expression was ranked by fold change between Lin-CD34+ cells and Lin-CD34− cells (x-axis). The most up-regulated genes in CD34+ cells are shown on the left side (red), while the most up-regulated genes in CD34− cells were shown on the right side (blue). Black bars represent the positions of genes in the ranked list. Enrichment score (ES, Y-axis) reflects the degree to which TCF7 binding targets are overrepresented at the extremes of the ranked gene expression list. When the distribution is at random, the enrichment score is zero. Enrichment of TCF7 targets at the top of the ranked list results in a large positive deviation of the ES from zero. (B) A statistically significant enrichment of RUNX1 targets was shown among up-regulated genes in CD34+ cells (Nominal p-value = 0, FDR q-value = 0). Enrichment of RUNX1 targets at the top of the ranked list results in a large positive deviation of the ES from zero.
Figure 5.
Tcf7 shRNA gene knockdown experiments provide functional evidence.
(A) qRT-PCR validation of the effect of three Tcf7 shRNA knockdown constructs. Tcf7 shRNA constructs were shown in the figure using the last two digits of their product numbers: TRCN0000012678, TRCN0000012679, TRCN0000012680. (B) Western blot analysis in TCF7 knockdown cell lines shows TCF7 protein and the shorter isoforms of RUNX1 are absent. Scrambled shRNA serves as a negative control. The polyclonal RUNX1 antibody recognizes the three major isoforms of RUNX1 ranging from ∼25 kDa to ∼50 Kda. The anti actin antibody indicates equal loading. Tcf7 shRNA knockdown reduces cell proliferation in SCF (scrambled shRNA serves as a negative control). (C) The down-regulated genes in Tcf7 shRNA knockdown cells are significantly enriched among up-regulated genes in CD34+ cells (Nominal p-value = 0, FDR q-value = 0). (D) The up-regulated genes in Tcf7 shRNA knockdown cells are significantly enriched among up-regulated genes in CD34− cells (Nominal p-value = 0, FDR q-value = 0.01898734). See Figure 4 legend for GSEA plot details.
Figure 6.
TCF7, together with RUNX1, regulates a transcriptional regulatory network.
The network involved in HSC establishment and development (red nodes), cell growth control (blue nodes) and multipotency (orange nodes) was identified among up-regulated genes in CD34+ cells (>2 fold) and displayed by Ingenuity Pathway Analysis software (IPA). Gray lines are IPA annotated relations based on the literature. Pink lines indicate TCF7 or RUNX1 binding to gene targets that were identified by our ChIP-Seq experiments. The shades of green color of the nodes in the network indicate the level of up-regulation in CD34+ cells. Sox4, Mpo, Tal1 and Ppard were TCF7 binding targets that were added to the network manually because of their obvious interesting function in hematopoiesis and self-renewal. All other nodes were from default IPA analysis. Direct relations were indicated by solid line or arrows. Indirect relations were indicated by dotted line. Please see Ingenuity Pathway Analysis software (IPA https://analysis.ingenuity.com/) Online Help section for detailed definitions.