Figure 4

D5E2 is a transcriptional enhancer that functions in NS/PCs. Candidate enhancer alignments were selected using information from the UCSC genome data base and by looking at p300-binding sites [52, 53] in the expected enhancer regions of introns and 55-65 Kb upstream. Each enhancer reporter gene used in the experiments is shown in (A). Nestin second intron-Tkp was used as a positive control. (B), (C) Candidate enhancer activities in ESCs and NS/PCs. Dissociated E14.5 cortex cells were cultured on a poly-D-Ornithine/fibronectin coated dish in an FGF-2/EGF mixed selective NS/PC conditioned medium. Enhancer reporter constructs were transfected into EB3 tg14 and NS/PCs, and the Luciferase activity was detected after 48 hours. The D2E and D5E2 sites enhanced the transcriptional activity in ESCs, and the D5E2 site enhanced it in NS/PCs [4.3-fold, 5.1-fold in ESCs, 2.2-fold, 15.2-fold in NS/PCs, respectively, compared with P1 (P1 = 1)], but D5E1 could not enhance the activity in either cell type. The data represent the mean ±SEM of three independent experiments. The data were subjected to non-repeated-measures ANOVA tests, and p values were calculated by Bonferroni multiple comparison tests. *p < 0.05: P1 to D2E-P1, D5E1-P1, or D5E2-P1, ns: not significant. FLU/RLU: firefly luciferase light unit/renilla luciferase light unit. (D), (E) The D2E and D5E2 enhancer activities were confirmed in EBs: 47 stable ES cell lines for P1, D2E-P1, and D5E2-P1were established and cultured in EB-formation conditions with or without RA. (D) Day 6 of neural-induced EB (+RA). Left panel shows bright field and right panel shows fluorescent images. Scale bar: 100 μm. (E) All cell lines were analysed for each condition on day 6. Average intensity is shown in comparison to RA-treated P1 (P1: +RA = 1, -RA = 0.33, D2E-P1: +RA = 3.47, -RA = 0.26, D5E2-P1: +RA = 16.7, -RA = 3.18.). Note that D5E2-P1 showed potent enhancer activity in the neural induced EBs.