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Deciphering the cis-regulatory architecture of mammalian photoreceptors

ABSTRACT: In order to define the genomic targets of Crx, we carried out Crx chromatin-immunoprecipitation followed by massively parallel sequencing (ChIP-seq) of eight-week-old mouse retinas using the Solexa platform. Sequence reads were mapped to the genome and 'peaks' were identified. These data were subjected to extensive bioinformatic analysis. In addition, selected peaks were experimentally tested for cis-regulatory activity by electroporation as promoter-reporter fusions into living mouse retinas. Over 5,000 Crx-bound regions (CBRs) were identified throughout the mouse genome. Many of these clusters of CBRs occur specifically around photoreceptor genes. In fact, Crx directly regulates the majority of known photoreceptor transcription factors as well as most known photoreceptor disease genes. Cis-regulatory analysis revealed that individual CBRs contribute in a combinatorial fashion to the overall activity of the gene they control. In addition, multiple Crx-binding sites within CBRs cooperatively interact via precise spacing rules to generate optimal levels of transcription.

ORGANISM(S): Mus musculus

PROVIDER: GSE20012 | GEO | 2010/06/24

SECONDARY ACCESSION(S): PRJNA124083

REPOSITORIES: GEO

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Project description:Background: Mutations in the cone-rod-homeobox protein CRX are typically associated with dominant blinding retinopathies with variable age of onset and severity. Five well-characterized mouse models carrying different Crx mutations show a wide range of disease phenotypes. To determine if the phenotype variability correlates with distinct changes in CRX target gene expression, we perform RNA-seq analyses on three of these models and compare the results with published data. Results: Despite dramatic phenotypic differences between the three models tested, graded expression changes in shared sets of genes are detected. Phenotype severity correlates with the down-regulation of genes encoding key rod and cone phototransduction proteins. Interestingly, in increasingly severe mouse models, the transcription of many rod-enriched genes decreases decrementally, whereas that of cone-enriched genes increases incrementally. Unlike down-regulated genes, which show a high degree of CRX binding and dynamic epigenetic profiles in normal retinas, the up-regulated cone-enriched genes do not correlate with direct activity of CRX, but instead likely reflect a change in rod cell-fate integrity. Furthermore, these analyses describe the impact of minor gene expression changes on the phenotype, as two mutants showed marginally distinguishable expression patterns but huge phenotypic differences, including distinct mechanisms of retinal degeneration. Conclusions: Our results implicate a threshold effect of gene expression level on photoreceptor function and survival, highlight the importance of CRX in photoreceptor subtype development and maintenance, and provide a molecular basis for phenotype variability in CRX-associated retinopathies. All genotypes were analyzed in triplicate. Heterozygous and homozygous mutants were all sequenced at P10, the control for which is the P10 C57BL6/J data. Heterozygous mutants were also analyzed at P21, the control for which is the P21 C57BL6/J data.

Project description:NeuroD1 encodes a basic helix-loop-helix transcription factor involved in the development of neural and endocrine structures. NeuroD1 mRNA is highly abundant in the adult mammalian pineal gland and exhibits a developmental expression pattern similar to the retina. This is consistent with the common evolutionary origin of pinealocytes and retinal photoreceptors. Pinealocytes and retinal photoreceptors express a shared set of phototransduction genes and submammalian pinealocytes are photosensitive. In contrast to the retina, the pineal gland is a relatively homogeneous structure, composed 95% of pinealocytes. This makes the pineal gland a particularly useful model for understanding photoreceptor cell biology. The loss of NeuroD1 in the retina results in progressive photoreceptor degeneration and the molecular mechanisms underlying this retinal degeneration phenotype remain unknown. Similarly, the role that NeuroD1 plays in the pineal gland is unknown. To determine the function of NeuroD1 in the pineal gland and retina, a Cre/loxP recombination strategy was used to selectively target a NeuroD1 floxed allele and generate NeuroD1 conditional knockout (cKO) mice. Tissue specificity was conferred using Cre recombinase expressed under the control of the promoter of Crx, a transcription factor selectively expressed in the pineal gland and retina. Pineal and retinal tissues from two-month-old NeuroD1 cKO and control animals were used in microarray studies to identify candidate genes responsible for the photoreceptor degeneration phenotype. The Cre/loxP recombination strategy was used to target a NeuroD1 floxed allele and generate NeuroD1 conditional knockout mice (NeuroD1floxed::Crx-Cre+/-). NeuroD1 floxed mice (NeuroD1floxed::Crx-Cre-/-) served as the controls. Pineal glands and retinas from two-month-old control and conditional knockout mice were collected at ZT6 and ZT20. 3 pools of 6 pineal glands per genotype and respective time of day were collected for each sample. Similarly, 3 pools of 6 retinas each were also collected. RNA from each pool was extracted and hybridized on the Affymetrix Mouse 430 2.0 array.

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Deciphering the cis-regulatory architecture of mammalian photoreceptors

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