Figure 6. CRX determines the selective activation of MEF2D-bound retinal enhancers.

(A) Aggregate plots of H3K27Ac ChIP-seq signal (top) and eRNA expression (bottom) at active (N=660) or inactive (N=584) MEF2D-bound enhancers (reads per bp per peak). Plots are centered on summits of MEF2D-bound regions that are >1kb away from the nearest gene TSS. (B) Cumulative distribution of WT average exon density (from RNA-Seq data, n=2) for genes nearest active enhancers (red) versus genes nearest inactive enhancers (black) (KS test; p=9.73e-17). (C) The Pcdh15 genomic locus in WT and Mef2d KO retinas with ChIP-seq tracks for MEF2D and H3K27Ac and RNA-Seq tracks. The upstream MEF2D-bound enhancer is highlighted in gray. (D) Top, aggregate plots of H3K27Ac ChIP-seq signal at enhancers bound by MEF2D alone (left) versus those bound by MEF2D and CRX (right) in WT retinas (Student’s t test; p=4.79e-65). Middle, aggregate plots of eRNAs at enhancers bound by MEF2D alone (left) versus those bound by MEF2D and CRX (right) in WT retinas. Bottom, aggregate plots of MEF2D ChIP signal (purple) or CRX ChIP signal (blue) for same regions, demonstrating normalization of data analysis to MEF2D peak size and differential peak size of CRX. (E) Average eRNA expression at individual MEF2D-bound enhancers in WT versus Crx KO retinas. N=2 for each genotype. Read density was calculated for the 400bp window centered on the summit of each MEF2D-bound region. Gray line indicates unity. Expression of eRNAs where Crx KO/WT expression was equal or less than 0.5 is indicated with red points. (F) Expression of eRNAs at a putative enhancer bound by MEF2D and CRX upstream of the Tnfaip3 genomic locus in WT, Mef2d KO, and Crx KO retinas with RNA-seq tracks and ChIP-seq tracks for MEF2D and CRX in WT retinas. eRNA expression and MEF2D and CRX binding in the upstream enhancer is highlighted in gray.