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. Author manuscript; available in PMC: 2020 Nov 24.
Published in final edited form as: Cell Stem Cell. 2020 Sep 28;27(5):765–783.e14. doi: 10.1016/j.stem.2020.09.001

Figure 4: Dissection of EC1.45 enhancer region uncovers a core role for the Coordinator motif and TWIST1 binding in developmental enhancer regulation.

Figure 4:

(A) TWIST1 ChIP-seq peaks (marked under track) at EC1.45 overlap p300 peaks Peak1 and Peak2 and minimally active sequences (min1 and min2).

(B) Luciferase assay for EC1.45 min1 and min2 tested separately and combined, along with Coordinator mutant sequences, schematic of constructs on left.

(C) Coordinator motif (upper, Prescott et al, 2015) compared to motif enriched at TWIST1 binding sites in hCNCCs (lower).

(D) Luciferase assay for heterologous enhancer sequence for human min1 plus vertebrate min2, schematic of constructs on left. A scatter plot depicts luciferase signal compared to the sum of Coordinator scores (ANOVA, p = 0.00035, right).

(E) TWIST1 is upregulated during hCNCC differentiation and reduced in chondrocytes (fragments per million, FPM).

(F) Schematic of plasmids, primers and probes for ChIP-ddPCR for wildtype (WT) and Coordinator mutant (4x mut) min1+min2 plasmids. F, forward; R, reverse.

(G) TWIST1 ChIP-ddPCR for P4 late hCNCCs transfected with plasmids in (F), normalized to input, and wildtype adjusted to 1. Two biological replicates are depicted.

See also Figure S5.