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. Author manuscript; available in PMC: 2020 Jan 1.
Published in final edited form as: Nat Methods. 2019 Jun 24;16(7):633–639. doi: 10.1038/s41592-019-0436-5

Figure 3. LADL redirects and reinforces a long-range interaction between a stretch enhancer and a new target gene upon blue light illumination.

Figure 3.

(a) Heatmap of long-range interactions around an ~800 kb genomic region encompassing the Klf4 and Zfp462 genes. SE, the Klf4 stretch enhancer. E1, E2, E3, E4, the Zfp462 enhancers. Mouse embryonic stem cells were co-transfected with LADL (Anchor + Bridge + Target) plasmids and then exposed to 24 hours blue light illumination. Box 1, the target de novo engineered loop between the pluripotency-specific Klf4 stretch enhancer and the Zfp462 promoter. Box 2, Klf4 interaction with its upstream, pluripotency-specific stretch enhancer (SE). Additional controls are shown in Supplementary Figure 6. Zoomed-in heatmaps of (c) Box 1 or (f) Box 2. Top, relative interaction frequency 5C signal. Bottom, distance-corrected interaction score 5C signal. (d, e, g) Classic 4C looping efficiency plots from the viewpoint of (d) the Zfp462 promoter targeted gRNA, (e) the stretch enhancer targeted gRNA, or (g) the Klf4 promoter. Additional negative controls and replicates are shown in Supplementary Figures 7–10. (h) Model of looping interaction reconfiguration in response to LADL and blue light illumination.