Abstract
The photoreceptor cells of the retina are subject to a greater number of genetic diseases than any other cell type in the human body. The majority of more than 120 cloned human blindness genes are highly expressed in photoreceptors. In order to establish an integrative framework in which to understand these diseases, we have undertaken an experimental and computational analysis of the network controlled by the mammalian photoreceptor transcription factors, Crx, Nrl, and Nr2e3. Using microarray and in situ hybridization datasets we have produced a model of this network which contains over 600 genes, including numerous retinal disease loci as well as previously uncharacterized photoreceptor transcription factors. To elucidate the connectivity of this network, we devised a computational algorithm to identify the photoreceptor-specific cis-regulatory elements (CREs) mediating the interactions between these transcription factors and their target genes. In vivo validation of our computational predictions resulted in the discovery of 19 novel photoreceptor-specific CREs near retinal disease genes. Examination of these CREs permitted the definition of a simple cis-regulatory grammar rule associated with high-level expression. To test the generality of this rule, we used an expanded form of it as a selection filter to evolve photoreceptor CREs from random DNA sequences in silico. When fused to fluorescent reporters, these evolved CREs drove strong, photoreceptor-specific expression in vivo. This study represents the first systematic identification and in vivo validation of CREs in a mammalian neuronal cell type and lays the groundwork for a systems biology of photoreceptor transcriptional regulation.
Highlights
Transcriptional regulatory networks (TRNs) lie at the center of organismal development and physiology [1,2]
This study demonstrates the feasibility of a high throughput, in vivo, non-transgenic approach to mammalian cis-regulatory elements (CREs) analysis which can be applied to a wide range of different tissues
Using stringent criteria to define up- and downregulation, a total of 628 genes were identified as dysregulated in at least one of the three mutants (Fig. 1A; Tables S1, S2, S3, S4, S5 and S6). 179 genes were downregulated in Crx-/(compared to 140 in Nrl-/- and 12 in Nr2e3-/-) whereas 93 genes were upregulated
Summary
Transcriptional regulatory networks (TRNs) lie at the center of organismal development and physiology [1,2]. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs [4]. This technique is time-consuming, costly and subject to insertion site effects. Rapid assays for mammalian CRE function have been developed in tissue culture systems, but it is not clear how such results translate into the in vivo behavior of CREs. We aim to demonstrate in this paper that rapid, inexpensive, high throughput analysis of mammalian CREs can be achieved by exploiting electroporation to introduce CRE-reporter fusion constructs either into living tissue in vivo or in ex vivo explant culture. This approach retains many of the desirable features of in vivo transgenic approaches to CRE analysis but is much more rapid and inexpensive
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