Figure 4.

Chromosome conformation capture (3C) showed that KLF4 facilitated 3D chromatin loop formation. A. Hypothesized 3D structure of the BLK gene locus activation showing KLF4 binds to the 5ʹUTR and 3ʹUTR fragments of the BLK gene through a chromatin loop to activate gene expression. Primers were designed to match the 5ʹUTR and 3ʹUTR KLF4 binding sites, respectively. A PCR fragment with the size of 298 bp was predicted based on our model, even though these two binding sites are ~37 kb apart. B. 3C was performed under four conditions: U87 KLF4 WT +/- Dox and U87 KLF4 R458R +/- Dox. Cross-linked genomic DNA was fragmented by digesting with HindIII followed by ligation of adjacent fragments. Same amount of template was used for PCR to amplify the putative linked site. PCR fragment with the predicted size of 298 bp was enriched in KLF4 expressing cells. C. Quantification of the enrichment of the PCR product in U87 KLF4 WT and KLF4 R458A cells indicated a 9.2-fold increase in KLF4 WT expressing cells. D, E. Sanger sequencing confirmed that the PCR fragment 100% matched our predicted linker sequencing as shown in D. F. 5-Aza treatment in U87 KLF4 WT expressing cells prevented loop formation, and no enrichment of PCR product was detected. G. Luciferase assay of the binding activity of KLF4 to the 3ʹUTR region of the BLK locus showing ~4-fold increase in luciferase activity upon Dox treatment. For panel B, C, F, and G, independent experiments were repeated at least three times. ***: P <0.001.