Fig. 3

Active enhancer sub-region e6-1-a interacts with Pgam5 promoter after TBI A Relative enhancer RNA expression of putative enhancers sub-regions in injured cortical neurons on DIV9, normalized to un-injured groups. B Relative enhancer RNA expression of e6-1 sub-regions in injured cortical neurons on DIV9. The enhancer RNA expression is assessed by PCR. Grey dashed lines indicate fold change equal to 1. Data are presented as mean ± SEM (n = 5). * p < 0.05, Student’s t-test. C Schematic flow chart of 3C assays. The interaction between e6-1-a region and Pgam5 promoter was assessed by 3C assays. Red arrows indicate primers for predicted ligation product. Black arrows indicate primers for loading control. EcoRI: Restriction site of EcoRI. TSS, transcription start site. D Electrophoresis of predicted PCR products of 3C assays. The length of PCR products of loading control and predicted ligation product are 457 bp and 293 bp, respectively. Arrow indicates the band of predicted ligation product of 3C assays. E Quantification of relative intensity of predicted ligation product in (D), normalized to loading control. Data are presented as mean ± SEM (n = 7). * p < 0.05, Student’s t-test. F Constructs of pPgam5-GFP and e6-1-a-pPGAM5-GFP. pPgam5: Pgam5 promoter. G Neuro2a cells were transiently transfected with pPgam5-GFP or e6-1-a-pPgam5-GFP constructs, together with pmCherry-expressing plasmid, for 24 h. Fluorescence images were taken using Carl Zeiss Observer Z1 microscope. Scale bar, 100 μm. H Schematic model of genomic architecture transformation of enhancer-promoter looping of Pgam5 after TBI