genes and tested their ability to regulate reporter gene expression in the intact retina in a MEF2D-dependent manner (Figure 2.10C,D). All 5 tested enhancers were sufficient to drive reporter gene expression in photoreceptors (data not shown; Figure 2.10C). Additionally, the activity of 4/5 of these reporters was significantly reduced in the presence of MEF2D shRNA or when the MRE was mutated, demonstrating that direct MEF2D binding to these elements is required to drive gene expression (Figure 2.10C,D). Together these results show that MEF2D regulates target gene expression by selectively activating only a subset of the sites to which it is bound. This reveals an additional level of control in how MEF2D regulates gene expression in photoreceptors, and helps explain why the number of MEF2D-bound enhancers greatly outnumbers the number of MEF2D target genes.
CRX determines the selective activation of MEF2D-bound retinal enhancers
To determine the mechanism of selective MEF2D enhancer activation we considered the possibility that CRX could serve as a co-activator at MEF2D-bound enhancers, at sites where
CRX. To test this hypothesis, we first asked if CRX binding correlated with enhancer activity at MEF2D-bound sites. Genome-wide, the subset of MEF2D-bound enhancers co-bound by CRX was significantly more active than the subset of MEF2D-bound elements where CRX does not bind, even when the amount of MEF2D binding was similar (Figure 2.11A). This strongly suggested that the presence of CRX together with MEF2D might be required for the maximal activation of MEF2D-bound enhancers.
To determine if CRX co-binding was required for activation of MEF2D-bound regulatory elements, we performed RNA-Seq in WT and Crx KO retinae at p11 and quantified the levels of eRNAs at MEF2D-bound enhancers (Figure 2.11B, C). In addition, we performed ChIP for H3K27Ac at select CRX-bound enhancers in WT and Crx KO retinae to confirm that eRNAs and H3K27Ac correlated in reflecting loss of activity (Figure 2.11D). As expected we found that CRX was required for the majority of eRNA expression at active sites where MEF2D binding is dependent upon CRX (Figure 2.11E). CRX was also required for eRNA expression at an additional 38% of MEF2D-bound enhancers that are active in WT retinae, but do not require CRX for binding. These sites included enhancers of clinically relevant MEF2D target genes such as Pcdh15, Guca1a and Guca1b. These results indicate that CRX is required for the selective activation of MEF2D-bound regulatory elements not only by recruiting MEF2D, but also by directly activating these promoters and enhancers.
MEF2D and CRX coordinate gene expression through enhancer co-binding and co- activation
Figure 2.11. CRX determines the selective activation of MEF2D-bound enhancers
(A) Three sets of aggregate plots centered on summits of MEF2D-bound regions that are either co-bound by CRX (left) or do not have a CRX peak (right). Top, aggregate plots of H3K27Ac ChIP-Seq signal in MEF2D WT retinae. Middle, aggregate plots of MEF2D ChIP signal (purple) or CRX ChIP signal (blue), demonstrating differential peak size of CRX and normalization of data analysis to MEF2D peak size. Bottom, aggregate plots of RNA-seq reads (coding reads removed) for forward (dark blue) and reverse (light blue) strands in Mef2d WT retinae.
(B) MEF2D and CRX ChIP-Seq tracks at Tnfaip3 example genomic locus. RNA-seq data from CRX WT (dark blue) and KO (yellow) retinae is also shown. Arrow denotes transcriptional start site (TSS). A light gray vertical bar highlights the identified MEF2D peak.
(C) eRNA read density calculated from RNA-Seq in CRX WT and KO retinae +/- 1 kb from the center of all MEF2D-bound enhancers where RNA-seq reads met minimum criteria for eRNAs (see methods). N=2 retinae per genotype. Gray line indicates unity.
(D) Right, eRNA read density for 2 CRX-bound enhancers, 1 co-bound by MEF2D (fscn2) and one not (rho), in CRX WT and KO retinae. Left, H3K27Ac ChIP-qPCR results from CRX WT and KO retinae for same 2 CRX-bound enhancers. Error bars represent S.E.M.
Figure 2.11. (Continued) CRX determines the selective activation of MEF2D-bound enhancers
(E) Aggregate plots are shown centered on MEF2D enhancers whose MEF2D binding levels are unchanged or decreased in CRX KO retinae. Top, aggregate plots of RNA-seq reads (coding reads removed) for forward (dark blue) and reverse (light blue) strands in Crx WT retinae as well as forward (red) and reverse (pink) strands in Crx KO retinae. Bottom, aggregate plots of MEF2D ChIP-Seq signal in CRX WT and KO retinae for same groups of enhancers.
WT Input
WT H3K27Ac ChIP Signal
ChIP-fragment depth (per bp per peak)
ChIP-fragment depth (per bp per peak)
0 1 2 3 4 -2800 -2000 -1200 -400 400 1200 2000 2800 0 1 2 3 4 -2800 -2000 -1200 -400 400 1200 2000 2800 0 10 20 30 40 50 60 -2800 -2000 -1200 -400 400 1200 2000 2800 CRX ChIP signal MEF2D ChIP Signal
0 10 20 30 40 50 60 -2800 -2000 -1200 -400 400 1200 2000 2800 0 0.02 0.04 0.06 -2800 -2000 -1200 -400 400 1200 2000 2800 0 0.02 0.04 0.06 -2800 -2000 -1200 -400 400 1200 2000 2800 WT RNA-Seq (+) reads WT RNA-Seq (-) reads
RNA-seq read density (per bp per peak)
B 62 kb Tnfaip3 locus Conservation ChIP-Seq MEF2D CRX Crx KO Crx WT RNA-Seq
Crx WT RNA-Seq (+) reads Crx WT RNA-Seq (-) reads Crx KO RNA-Seq (+) reads Crx KO RNA-Seq (-) reads
E
Figure 2.11. (Continued) CRX determines the selective activation of MEF2D- bound enhancers 0 0.02 0.04 0.06 -2800 -2000 -1200 -400 400 1200 2000 2800 0 10 20 30 40 50 60 -2800 -2000 -1200 -400 400 1200 2000 2800 0 0.1 0.2 0.3 Crx WT Crx KO
RNA-seq read density
fscn2 0 0.1 0.2 0.3 0.4 Crx WT Crx KO
RNA-seq read density
rho 0 0.02 0.04 0.06 Crx WT Crx KO Fraction binding normallized to input fscn2 0 0.01 0.02 0.03 0.04 Crx WT Crx KO Fraction binding normallized to input
rho
H3K27Ac from ChIP-qPCR
eRNA read density from RNA-seq 0 10 20 30 40 50 60 -2800 -2000 -1200 -400 400 1200 2000 2800 C 0 0.02 0.04 0.06 -2800 -2000 -1200 -400 400 1200 2000 2800 0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100 eRNA read density , CRX KO
eRNA read density, CRX WT
D
eRNAs at MEF2D-bound enhancers
ChIP-fragment depth (per bp per peak)
RNA-seq read density (per bp per peak)
MEF2D enhancer peak size unchanged in CRX KO
MEF2D enhancer peak size decreased in CRX KO
MEF2D ChIP Signal in CRX WT MEF2D ChIP Signal in CRX KO
Ultimately, the interactions of TFs at enhancers and promoters are read out through target gene expression. Given the interactions of MEF2D and CRX at critical retinal enhancers it is expected that MEF2D and CRX would share specific target genes. Furthermore, the shared loss of function phenotype for these two factors predicts that target genes shared between these TFs would be critical for photoreceptor development and function. To identify shared MEF2D and CRX target genes we compared the previously described RNA-Seq results from WT, Mef2d KO and Crx KO retinae at p11 and found that ~51% of MEF2D direct target genes are also highly regulated by CRX (36/71 genes) (Figure 2.12A,B). These shared target genes included Sag,
Guca1a, Guca1b and Fscn2, genes that together are essential for photoreceptor development and
function. We found that ~92% of shared target genes have MEF2D and CRX co-bound at nearby enhancers or promoters, demonstrating that CRX and MEF2D are largely working at the same regulatory elements to direct expression of these genes. Of these regulatory sites, ~31% require CRX for MEF2D binding and activation (Figure 2.12C). The majority of remaining sites do not have significant changes in MEF2D binding but lose activity in the Crx KO (Figure