Supplementary Figure 5: An extended Otx binding site (motif 1) is required for SBE1 activity.
From: Cis-regulatory architecture of a brain signaling center predates the origin of chordates
(a) Sequence logo based on the position weight matrix (PWM) of motif 1 (Otx) from the seven most specific SBE1-like enhancers (top). Comparison of the PWMs for a 10-nt sequence encompassing the consensus Otx binding site, GATTA, derived from 53 SBE1-like enhancers (middle) and 200 random genomic sequences matched for GC content and length (bottom). Adenine nucleotides preferentially flank the GATTA core sequence of the Otx binding site in SBE1-like enhancers as compared to random genomic sequence (**P < 0.01, ***P < 0.001, Fisher’s exact test). (b) Results of luciferase reporter assays in COS-1 cells cotransfected with increasing doses of Otx2 and wild-type (WT) or mutant versions of SBE1-luciferase reporter constructs. The mutations include deletion of motif 1 (blue), point mutations within the GATTA consensus sequence (magenta) and point mutations immediately adjacent to the GATTA binding site in motif 1 (green). (c) ChIP-qPCR performed with chromatin isolated from COS-1 cells that were cotransfected with FLAG-Otx2 and wild-type or mutant versions of SBE1-luciferase constructs as described in b. qPCR results represent an average of three biological replicates (*P < 0.05, **P < 0.01, two-sided Student’s t test). Error bars in all graphs represent s.d.m.
