Fig. 3: KLF5 overexpression partially rescues K5aSE-KO phenotypes in ESCs.

a Showing rankings for K5aSE in ESC super-enhancers. b Hi-C (GSE96107) and H3K27ac ChIP-seq data (generated in this study) indicating that both K5aSE and Klf5 are located within the same TAD in ESCs. Red dashed box marking K5aSE region. Hi-C data are analyzed and presented using online tools: 3D Genome Browser (https://3dgenome.fsm.northwestern.edu/)¹³³. c Volcano plot based on RNA-seq revealing Klf5 to be the most significant DEGs in K5aSE-KO relative to WT ESCs. Genes showing at least a twofold change and p < 0.05 were considered differentially expressed. The data showed down-regulated expression of 388 genes and up-regulated expression of 255 genes in K5aSE-KO ESCs, respectively. d RT-qPCR analysis showing relative expression of Klf5 mRNA in V-WT, V-K5aSE-KO and KLF5-K5aSE-KO ESCs. Data shown as mean ± SD, n = 3 independent replicates; two-sided unpaired t tests. e Western blot showing KLF5 protein expression in indicated ESCs (V-WT, V-K5aSE-KO and KLF5-K5aSE-KO, n = 1). KLF5 expression was normalized according to GADPH expression using ImageJ. f AP-staining of indicated ESCs (V-WT, V-K5aSE-KO and KLF5-K5aSE-KO). Scale bar: 200 μm. g Analysis of clone size in indicated ESCs (V-WT, V-K5aSE-KO and KLF5-K5aSE-KO). In V-WT, n = 34 clones; in V-K5aSE-KO, n = 96 clones; in KLF5-K5aSE-KO, n = 204 clones; two-sided unpaired t tests. The clone size is quantified using ImageJ. h Partial rescue of K5aSE-KO ESC proliferation by KLF5 overexpression. Data shown as mean ± SD, n = 3 independent replicates; two-sided unpaired t-tests. i Heatmap showing DEGs in V-K5aSE-KO compared to V-WT ESCs (FC ≥ 2, p < 0.05). j Heatmap showing rescue of DEGs shown in (i) by KLF5 overexpression (KLF5-K5aSE-KO compared to V-K5aSE-KO, FC ≥ 1, p < 0.10). k Statistical analysis of genes significantly regulated by KLF5 but aberrantly expressed in K5aSE-KO ESCs. Rescued Up-regulated genes, n = 160 genes; rescued Down-regulated genes, n = 165 genes; two-sided paired t tests. l IGV screenshots show KLF5 (GSM1208218) and H3K4me3 (GSM1871952) binding at loci of indicated genes (examples include Wnt3a, Klf10, Nek2, Sp5, Fgfbp1 and Uqcrc1) rescued by KLF5. Black arrows, direction of transcription. The relative RNA expression levels of these genes were obtained from RNA sequencing (n = 2 independent replicates). m, The schematic shows that K5aSE may regulate ESCs identity through the transcriptional regulatory network of its adjacent transcription factor KLF5. Source data are provided as a Source Data file.