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A. K. Kundzewicz, D. Skowronska-Krawczyk, C. Alliod, F. Chiodini, L. Matter-Sadzinski, J.-M. Matter; Linkage of Neuron Specification and bHLH Regulated Transcription by a "Chip-on-Chip" Strategy. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2940.
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© ARVO (1962-2015); The Authors (2016-present)
In the developing retina, the production of ganglion cells is dependent on the proneural protein ATH5, whose activity defines stages along the pathway converting progenitors into newborn neurons. Establishing the compendium of ATH5 transcriptional targets should help answer the question whether ATH5 is dedicated solely to the production of RGCs or whether it also promotes the development of other retinal subtypes.
Using genomic arrays, we have completed a global screen to locate the chromatin sites occupied by ATH5. The ChIP experiment was conducted using ATH5 antibodies on chromatin isolated at different stages of retina development. Immunoprecipitated DNA was hybridized to promoter arrays of ~500 non-coding sequences. We designed the chips ourselves, including numerous genes expected to be involved in (1) RGC specification/differentiation, (2) signaling and cell cycle pathways, (3) Notch/Delta and Shh pathways, (4) cytoskeleton and cell adhesion. A wide set of control genes, such as myogenic transcription factors and genes expressed in photoreceptors were also included. The sequences put on the arrays contain non-coding regions encompassing 2kb or 5kb upstream of initiation codons.
Data analysis of the ChIP-on-chip experiments has revealed several interesting properties of the ATH5 protein. Although less than 10% of the sequences on the array were found to bind ATH5, the majority of these putative targets are chromatin regions that control transcription of genes encoding a variety of key proteins involved in different metabolic and signaling pathways. The screen has identified promoter regions of genes encoding proteins important for cytoskeleton organization, cell adhesion, process extension, cytokinesis and axon formation. Analyzing the function and regulation of these genes should help us understand how ATH5 controls cell cycle exit and how it contributes to axon formation in newborn RGCs.
It appears that ATH5 may control or modulate multiple metabolic pathways in the retinal neuroepithelium in order to create the proper cellular background for neuron specification and differentiation.
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