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. Author manuscript; available in PMC: 2022 Oct 20.
Published in final edited form as: Nat Struct Mol Biol. 2022 Sep 12;29(9):891–897. doi: 10.1038/s41594-022-00821-8

Figure 1. Synthetic Shh activation.

Figure 1

(a) Hi-C heatmap of the Shh TAD from wild type mESCs at 16kb resolution. Data are from ref 33 and were created using HiGlass. Genes, positions of TALE target sequences and the CTCF ChIP-seq track – including CTCF motif orientation - are shown below. Genome coordinates: mm9 assembly of the mouse genome. (b) Schematic of TALE-VP64 constructs used to target the Shh promoter (tShh-VP64), SBE6 (tSBE6-VP64) SBE2 (tSBE2-VP64) or ZRS (tZRS-VP64) enhancers. NLS: nuclear localisation sequence; 2A: self-cleaving 2A peptide. Repeat variable diresidue (RVD) code is displayed below using the one letter amino acid abbreviations. Equivalent TALE-Δ constructs lack the Vp64 module (c-e) Representative images of nuclei from mESCs transfected with (c) tShh-VP64, (d) tSBE2-VP64 and (e) tZRS-VP64 showing RNA FISH signals for Shh (white) and Lmbr1 (red). Scale bars = 5 μm. (f) Timecourse of TALE transfection and auxin treatment is shown above. The percent of Shh (left) and Lmbr1 (right) expressing alleles in wild-type mESCs transfected with TALE-Vp64 or TALE-Δ constructs assayed by RNA FISH in the absence or presence of auxin. The data were compared using a two-sided Fisher’s Exact Test, n = numer of alleles scored, ns – not significant (p>0.05). Source Data Fig1. The biological replicate for these data, and the full statistical evaluation of all comparisons for Shh are in Extended Data Fig. 1c. and d.