Limits...
The cis-regulatory logic of the mammalian photoreceptor transcriptional network.

Hsiau TH, Diaconu C, Myers CA, Lee J, Cepko CL, Corbo JC - PLoS ONE (2007)

Bottom Line: Examination of these CREs permitted the definition of a simple cis-regulatory grammar rule associated with high-level expression.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, United States of America.

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.

Show MeSH

Related in: MedlinePlus

In silico evolution of functional photoreceptor cis-regulatory elements.A, Flatmount and cross-sectional images of mouse Rho-CRE fused to DsRed co-electroporated with CAG-eGFP. All constructs were electroporated at P0, cultured as explants, and harvested at P8. The column labeled ‘CRE architecture’ shows the distribution of Crx (top half) and Nrl (bottom half) sites with the indicated ‘affinity’ as described in the legend at the top of the figure. The orientation of a given site is indicated by the direction of the triangle representing that site. The score on the y-axis is the log odds score for that site which reflects its closeness to consensus. Size bar = 500 µm for flatmount images and 100 µm for cross-sections B-D, Images of retinas electroporated with the indicated synthetic CREs. For example, ‘Syn1-G70’ indicates a sequence corresponding to the genome of the ‘fittest’ organism of generation 70 in evolutionary run 1. The CREs in B-D derive from three separate evolutionary runs. E, Graph of first 700 generations of evolutionary run 3. The four ‘organisms’ from this run whose genomes were tested for CRE activity are circled and images of their expression patterns are given in Fig. S4. F, Graph of expression strength of four synthetic CREs from evolutionary run 3.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC1916400&req=5

pone-0000643-g005: In silico evolution of functional photoreceptor cis-regulatory elements.A, Flatmount and cross-sectional images of mouse Rho-CRE fused to DsRed co-electroporated with CAG-eGFP. All constructs were electroporated at P0, cultured as explants, and harvested at P8. The column labeled ‘CRE architecture’ shows the distribution of Crx (top half) and Nrl (bottom half) sites with the indicated ‘affinity’ as described in the legend at the top of the figure. The orientation of a given site is indicated by the direction of the triangle representing that site. The score on the y-axis is the log odds score for that site which reflects its closeness to consensus. Size bar = 500 µm for flatmount images and 100 µm for cross-sections B-D, Images of retinas electroporated with the indicated synthetic CREs. For example, ‘Syn1-G70’ indicates a sequence corresponding to the genome of the ‘fittest’ organism of generation 70 in evolutionary run 1. The CREs in B-D derive from three separate evolutionary runs. E, Graph of first 700 generations of evolutionary run 3. The four ‘organisms’ from this run whose genomes were tested for CRE activity are circled and images of their expression patterns are given in Fig. S4. F, Graph of expression strength of four synthetic CREs from evolutionary run 3.

Mentions: Comparative analysis of the CREs identified in this study demonstrated a correlation between a closely linked pair of Crx and Nrl sites and strong expression in photoreceptors. In order to test the generality of this association, we created a genetic algorithm to evolve photoreceptor CREs from random DNA sequences in silico using a selection filter based on clustering and affinity of Crx and Nrl binding sites (see MATERIALS AND METHODS for details). The purpose of this experiment was to produce sequences that retain the essential features of photoreceptor-specific CREs (i.e., closely clustered Crx and Nrl binding sites), while randomizing all intervening sequences. We found that in less than 100 generations it was possible to evolve CREs with features highly reminiscent of naturally occurring photoreceptor-specific CREs, including some with a strong resemblance to the well characterized Rho-CRE (compare Fig. 5A with B-D; Table S10). In order to test whether these in silico evolved CREs could drive photoreceptor-specific transcription in vivo, we selected three ‘organisms’ from three independent evolutionary runs which had a distribution of Crx and Nrl binding sites resembling that of Rho-CRE and which contained the cis-regulatory motif described above. None of these evolved sequences had significant linear sequence homology with each other or with Rho-CRE. We then synthesized these three 400 bp sequences and cloned them upstream of a minimal basal promoter driving DsRed. This basal promoter alone does not drive any expression in photoreceptors (Fig. S2). When electroporated into explanted P0 retinas, these synthetic CREs drove photoreceptor-specific expression after several days in culture. All three synthetic CREs were scored as ‘strong’ with syn1-G70 and syn2-G65 being somewhat stronger than syn3-G55. These results strongly suggest that closely clustered Crx and Nrl sites are a critical determinant of strong photoreceptor-specific expression and that there is significant flexibility in the architecture of photoreceptor-specific CREs.


The cis-regulatory logic of the mammalian photoreceptor transcriptional network.

Hsiau TH, Diaconu C, Myers CA, Lee J, Cepko CL, Corbo JC - PLoS ONE (2007)

In silico evolution of functional photoreceptor cis-regulatory elements.A, Flatmount and cross-sectional images of mouse Rho-CRE fused to DsRed co-electroporated with CAG-eGFP. All constructs were electroporated at P0, cultured as explants, and harvested at P8. The column labeled ‘CRE architecture’ shows the distribution of Crx (top half) and Nrl (bottom half) sites with the indicated ‘affinity’ as described in the legend at the top of the figure. The orientation of a given site is indicated by the direction of the triangle representing that site. The score on the y-axis is the log odds score for that site which reflects its closeness to consensus. Size bar = 500 µm for flatmount images and 100 µm for cross-sections B-D, Images of retinas electroporated with the indicated synthetic CREs. For example, ‘Syn1-G70’ indicates a sequence corresponding to the genome of the ‘fittest’ organism of generation 70 in evolutionary run 1. The CREs in B-D derive from three separate evolutionary runs. E, Graph of first 700 generations of evolutionary run 3. The four ‘organisms’ from this run whose genomes were tested for CRE activity are circled and images of their expression patterns are given in Fig. S4. F, Graph of expression strength of four synthetic CREs from evolutionary run 3.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1916400&req=5

pone-0000643-g005: In silico evolution of functional photoreceptor cis-regulatory elements.A, Flatmount and cross-sectional images of mouse Rho-CRE fused to DsRed co-electroporated with CAG-eGFP. All constructs were electroporated at P0, cultured as explants, and harvested at P8. The column labeled ‘CRE architecture’ shows the distribution of Crx (top half) and Nrl (bottom half) sites with the indicated ‘affinity’ as described in the legend at the top of the figure. The orientation of a given site is indicated by the direction of the triangle representing that site. The score on the y-axis is the log odds score for that site which reflects its closeness to consensus. Size bar = 500 µm for flatmount images and 100 µm for cross-sections B-D, Images of retinas electroporated with the indicated synthetic CREs. For example, ‘Syn1-G70’ indicates a sequence corresponding to the genome of the ‘fittest’ organism of generation 70 in evolutionary run 1. The CREs in B-D derive from three separate evolutionary runs. E, Graph of first 700 generations of evolutionary run 3. The four ‘organisms’ from this run whose genomes were tested for CRE activity are circled and images of their expression patterns are given in Fig. S4. F, Graph of expression strength of four synthetic CREs from evolutionary run 3.
Mentions: Comparative analysis of the CREs identified in this study demonstrated a correlation between a closely linked pair of Crx and Nrl sites and strong expression in photoreceptors. In order to test the generality of this association, we created a genetic algorithm to evolve photoreceptor CREs from random DNA sequences in silico using a selection filter based on clustering and affinity of Crx and Nrl binding sites (see MATERIALS AND METHODS for details). The purpose of this experiment was to produce sequences that retain the essential features of photoreceptor-specific CREs (i.e., closely clustered Crx and Nrl binding sites), while randomizing all intervening sequences. We found that in less than 100 generations it was possible to evolve CREs with features highly reminiscent of naturally occurring photoreceptor-specific CREs, including some with a strong resemblance to the well characterized Rho-CRE (compare Fig. 5A with B-D; Table S10). In order to test whether these in silico evolved CREs could drive photoreceptor-specific transcription in vivo, we selected three ‘organisms’ from three independent evolutionary runs which had a distribution of Crx and Nrl binding sites resembling that of Rho-CRE and which contained the cis-regulatory motif described above. None of these evolved sequences had significant linear sequence homology with each other or with Rho-CRE. We then synthesized these three 400 bp sequences and cloned them upstream of a minimal basal promoter driving DsRed. This basal promoter alone does not drive any expression in photoreceptors (Fig. S2). When electroporated into explanted P0 retinas, these synthetic CREs drove photoreceptor-specific expression after several days in culture. All three synthetic CREs were scored as ‘strong’ with syn1-G70 and syn2-G65 being somewhat stronger than syn3-G55. These results strongly suggest that closely clustered Crx and Nrl sites are a critical determinant of strong photoreceptor-specific expression and that there is significant flexibility in the architecture of photoreceptor-specific CREs.

Bottom Line: Examination of these CREs permitted the definition of a simple cis-regulatory grammar rule associated with high-level expression.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, United States of America.

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.

Show MeSH
Related in: MedlinePlus