Abstract
In vertebrate retinal progenitor cells, the proneural factor Atoh7 exhibits a dynamic tissue and cellular expression pattern. Although the resulting Atoh7 retinal lineage contains all seven major cell types, only retinal ganglion cells require Atoh7 for proper differentiation. Such specificity necessitates complex regulation of Atoh7 transcription during retina development. The Notch signaling pathway is an evolutionarily conserved suppressor of proneural bHLH factor expression. Previous in vivo mouse genetic studies established the cell autonomous suppression of Atoh7 transcription by Notch1, Rbpj and Hes1. Here we identify four CSL binding sites within the Atoh7 proximal regulatory region and demonstrate Rbpj protein interaction at these sequences by in vitro electromobility shift, calorimetry and luciferase assays and, in vivo via colocalization and chromatin immunoprecipitation. We found that Rbpj simultaneously represses Atoh7 transcription using both Notch-dependent and –independent pathways.
Highlights
During vertebrate embryonic development, multipotent retinal progenitor cells (RPCs) undergo a prolonged period of differentiation during which six neuronal and one glial (Müller) cell type are produced and assembled into a highly laminated tissue
We discovered that simultaneous loss of all four binding sites reduced Atoh[7] transcription, suggesting a distinct mode of Rbpj regulation, consistent with locus priming for efficient transcriptional regulation by other factors[33]
In the adult frog retina, this conserved noncoding elements (CNEs) is critical for maintaining Atoh7/Ath[5] expression in the ciliary marginal zone[8]
Summary
Multipotent retinal progenitor cells (RPCs) undergo a prolonged period of differentiation during which six neuronal (retinal ganglion, cone and rod photoreceptor, amacrine, horizontal and bipolar) and one glial (Müller) cell type are produced and assembled into a highly laminated tissue. Multiple signaling pathways and transcription factors, including proneural basic helix-loop-helix (bHLH) transcription factors, regulate retinal cell fate, as reviewed in[1] Among these factors, Atoh[7] (Atonal homolog 7, Math[5], Ath5) is dynamically expressed by subsets of embryonic RPCs and required for retinal ganglion cell (RGC) formation[2,3,4,5,6]. Recombination Signal Binding Protein for immunoglobulin kappa J region) in human/mouse, Su(H) (Suppressor of Hairless) in Drosophila, Lag-1 (lin[12] and glp-1 phenotype) in C. elegans) and the Maml (Mastermind-like) co-activating factor This protein complex activates downstream target gene transcription by binding to a variety of CSL sites within noncoding DNA13–16. The mechanism(s) underlying Notch pathway regulation of Atoh[7] remain unresolved
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