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. 2015 Dec 8;5(6):1155–1170. doi: 10.1016/j.stemcr.2015.11.003

Figure 5.

Figure 5

TBX3 Depletion Leads to Complex Compensational Processes

(A) Molecular circuitry of pluripotency markers (inner layer) and TBX3 target genes (outer layer) generated by the STRING database. The color of a node reflects the change in gene expression level between wild-type (WT) and TBX3-null (KO) ESCs. Black edge shows an evident interaction (confidence score > 0.7) between two connected genes from the STRING database. The gray edge connects TBX3 with its target genes identified by ChIP-seq data (GSE19219; Han et al., 2010a).

(B) Venn diagram of upregulated genes in TBX3-null ESCs and Tbx3 knockdown ESCs (GSE26520), compared to respective wild-type controls, shows 20 candidate genes.

(C) Heatmap of gene expression level of these candidate genes. Light green, below median expression; dark green, above median.

(D) The expression level of Dppa3 in TBX3+/+and TBX3−/− iPSCs, wild-type (+/+), and TBX3-null (ven/ven) ESCs.

(E) Clustering of single-cell Dppa3 expression (n = 26) suggests the reciprocal expression between Tbx3 and Dppa3 (hypergeometric test; p = 0.023). Dark green, group of low Dppa3 expression; light green, group of high Dppa3 expression. TBX3-low-sorted single cell is shown (GFP-L no. X). TBX3-high-sorted single cell is shown (GFP-H no. X).

(F) TBX3 occupancy (GSE19219) and H3K4me3/H3K27me3 bivalent modifications (GSE31039) of DPPA3 promoter region in ESCs.

(G) Dppa3 expression in WT or TBX3−/− iPSCs upon transduction with scramble control virus or various combinations of different shRNAs targeting Dppa3. Two independent experiments performed with triplicate technical replicates are shown.

(H) Colony formation of TBX3−/− iPSC lines with shRNAs against Dppa3 upon clonal density seeding. Top, representative AP-stained plates; bottom, quantification of three independent experiments performed with triplicate technical replicates. Black bar, AP positive; grey bar, partially AP positive; light gray bar, AP negative.

(I) qPCR of pluripotency markers in Dppa3 knockdown and scramble control TBX3−/− iPSC lines (three independent experiments performed with duplicate technical replicates).

(J) Comparisons of differentially regulated genes between TBX3-high and -low ESC and between TBX3 WT (+/+) and TBX3-null (ven/ven) ESCs.

(K and L) The GO terms of DNA methylation (GO0006306) show no bias toward TBX3-high or -low ESCs (K) but were significantly enriched in TBX3-null ESCs (L).