The Cis-regulatory Logic of the Mammalian Photoreceptor Transcriptional Network

PLOS ONE, Jul 2007

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.

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The Cis-regulatory Logic of the Mammalian Photoreceptor Transcriptional Network

Cepko CL et al (2007) The Cis-regulatory Logic of the Mammalian Photoreceptor Transcriptional Network. PLoS ONE 2(7): e643. doi:10.1371/journal.pone.0000643 The Cis -regulatory Logic of the Mammalian Photoreceptor Transcriptional Network Timothy H.-C. Hsiau 0 1 2 Claudiu Diaconu 0 1 2 Connie A. Myers 0 1 2 Jongwoo Lee 0 1 2 Constance L. Cepko cepko@genetics 0 1 2 Joseph C. Corbo 0 1 2 0 Funding: This work was supported by funds from NIH grant, K08EY014822, to JCC and by funds from the Foundation for Retina Research, Howard Hughes Medical Institute and an NIH grant , R01EY009676, to CLC 1 Academic Editor: Dave Raible, University of Washington , United States of America 2 1 Department of Pathology and Immunology, Washington University School of Medicine , St. Louis , Missouri, United States of America, 2 Howard Hughes Medical Institute, Department of Genetics, Harvard Medical School , Boston, Massachusetts , United States of America 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, photoreceptorspecific 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. - INTRODUCTION Transcriptional regulatory networks (TRNs) lie at the center of organismal development and physiology [1,2]. Transcription factors (TFs) within these networks control the spatiotemporal pattern and levels of expression of their target genes by binding to CREs, short (,300600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. Significant progress has been made in the computational identification of putative CREs in a variety of species [37]. One recent study demonstrated the effectiveness of using deep phylogenetic conservation of non-coding DNA to identify developmentally active CREs in the mouse [4]. However, given the importance of cis-regulatory change in evolution [8] and the relatively limited number of deeply conserved non-coding regions in the mouse (,3000 were identified in [4]), it is likely that there are many tissue-specific CREs which do not fall within such regions. Despite advances in the design of computational algorithms to identify CREs in mammalian genomes, the development of cheap, high-throughput assay systems for validating these computational predictions in vivo has lagged far behind. 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. On the other hand, 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. Photoreceptor cells are sensory neurons that elaborate a highly specialized, membrane-rich organelle, the outer segment, which is exquisitely sensitive to light. These cells are particularly susceptible to degeneration. There are currently over 180 mapped disease loci which cause blindness in humans (http://www.sph (...truncated)


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Timothy H.-C. Hsiau, Claudiu Diaconu, Connie A. Myers, Jongwoo Lee, Constance L. Cepko, Joseph C. Corbo. The Cis-regulatory Logic of the Mammalian Photoreceptor Transcriptional Network, PLOS ONE, 2007, 7, DOI: 10.1371/journal.pone.0000643