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Timothy Joel Cherry, Milena Andzelm, David Harmin, Michael Greenberg; Regulation of photoreceptor development by competitive activation of cell type-specific enhancers. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):436.
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© ARVO (1962-2015); The Authors (2016-present)
Photoreceptor development is driven by a network of transcription factors (TFs), however the cis-regulatory elements (CREs) through which these factors regulate gene expression are not fully understood. We sought to comprehensively identify active CREs in developing photoreceptors and to dissect the combinatorial mechanisms by which TFs regulate photoreceptor gene expression. Such mechanisms may provide a roadmap for reprogramming of somatic cells to replace photoreceptors lost to disease.
We performed ChIP-Seq for markers of active chromatin and total RNA-Seq to assess enhancer RNA (eRNA) and gene expression in the developing mouse retina. These datasets were compared to DNAse I hypersensitivity (DHS) data (ENCODE) to identify candidate active CREs. Motif analysis was performed to identify TF binding motifs enriched at active CREs. ChIP-Seq analysis was then performed to determine binding of corresponding TF proteins. ChIP-Seq of H3K27Ac and total RNA-Seq was performed in TF KO retinas to determine the contribution of these factors to CRE activity and gene expression. ChIP-Seq of MEF2D in Crx KO retinas was performed to determine the requirement of CRX for competitive recruitment of MEF2D to photoreceptor specific CREs.
Our analysis identified approximately 20,000 DHS sites with high levels of both H3K27Ac and eRNA expression in the p11 mouse retina. These sites were enriched for DNA sequences corresponding to CRX, RORA/B and MEF2 consensus binding sites and demonstrated CRX and MEF2D protein occupancy. A subset of these of these sites demonstrated a significant reduction in H3K27Ac and eRNAs in CRX KO and Mef2d KO retinas respectively. 330 MEF2D binding sites were reduced >2x in CRX KO retinas, while 227 sites demonstrated an increase in MEF2D binding. Many of the latter sites demonstrated an increase in eRNAs and proximal gene expression.
These studies identify candidate active CREs in the developing retina and suggest mechanisms by which TFs regulate gene expression in photoreceptors. TFs bind to distal enhancers to regulate their activity and demonstrate a functional interdependence whereby MEF2D requires CRX for binding to and activation of certain photoreceptor enhancers. This suggests that an active competition for MEF2D recruitment to photoreceptor enhancers may be required for normal photoreceptor development.
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