Transcriptional activity of an MPRA library containing sequences of genomic origin bound by the transcription factor CRX measured in retinas from mice carrying pathogenic CRX variants
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ABSTRACT:
ORGANISM(S): synthetic construct Mus musculus
PROVIDER: GSE230090 | GEO | 2023/05/27
REPOSITORIES: GEO
Similar Datasets
Project description:Leber congenital amaurosis (LCA) includes congenital or early-onset blinding diseases, characterized by vision loss together with nystagmus and nonrecordable electroretinogram (ERG). At least 19 genes are associated with LCA. While most LCA is recessive, mutations in the homeodomain transcription factor gene CRX lead to autosomal dominant LCA. The mechanism of CRX-LCA is not understood. Here, we report a new spontaneous mouse mutant carrying a frameshift mutation in Crx (CrxRip). We show that, unlike Crx-/- mouse retina, the dominant Crx c.763del1 mutation in CrxRip results in congenital blindness with complete loss of ERG, yet the photoreceptors do not degenerate. Dominant CRX frameshift mutations associated with LCA mimic the CrxRip phenotype that can be rescued by Crx. RNA-Seq profiling reveals progressive and complete loss of rod differentiation factor Nrl in CrxRip, while residual Nrl remains in Crx-/- retina. Moreover, Nrl partially restores the rod phenotype in CrxRip/+ mice. We show that the binding of Otx2 to Nrl promoter is obliterated in CrxRip mutant, and ectopic Otx2 can rescue the rod phenotype. Therefore, Otx2 is required to maintain Nrl expression in developing rods to consolidate rod fate. Our studies provide the mechanism of congenital blindness caused by dominant CRX mutations and should assist in therapeutic design. Retinal samples were harvested from WT, CrxRip/+, CrxRip/Rip, Crx-/- and Nrl-/- retina at postnatal days 2 and 21 for whole transcriptome sequencing (RNAseq). Each sample included 2 independent frozen retina and experiments were performed in duplicates. RNA-seq transcriptome libraries were constructed from 1 ?g of total RNA.
2014-01-13 | E-GEOD-52006 | biostudies-arrayexpress
Project description:Leber congenital amaurosis (LCA) includes congenital or early-onset blinding diseases, characterized by vision loss together with nystagmus and nonrecordable electroretinogram (ERG). At least 19 genes are associated with LCA. While most LCA is recessive, mutations in the homeodomain transcription factor gene CRX lead to autosomal dominant LCA. The mechanism of CRX-LCA is not understood. Here, we report a new spontaneous mouse mutant carrying a frameshift mutation in Crx (CrxRip). We show that, unlike Crx-/- mouse retina, the dominant Crx c.763del1 mutation in CrxRip results in congenital blindness with complete loss of ERG, yet the photoreceptors do not degenerate. Dominant CRX frameshift mutations associated with LCA mimic the CrxRip phenotype that can be rescued by Crx. RNA-Seq profiling reveals progressive and complete loss of rod differentiation factor Nrl in CrxRip, while residual Nrl remains in Crx-/- retina. Moreover, Nrl partially restores the rod phenotype in CrxRip/+ mice. We show that the binding of Otx2 to Nrl promoter is obliterated in CrxRip mutant, and ectopic Otx2 can rescue the rod phenotype. Therefore, Otx2 is required to maintain Nrl expression in developing rods to consolidate rod fate. Our studies provide the mechanism of congenital blindness caused by dominant CRX mutations and should assist in therapeutic design.
2014-01-13 | GSE52006 | GEO
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.
2015-07-27 | E-GEOD-65506 | biostudies-arrayexpress
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.
2015-07-27 | GSE65506 | GEO
Project description:P10 retinal gene expression analysis of the homozygous Crx Knock-OUT or Knock-IN mice carrying the mutation E168d2 or R90W. Results provide important information about the retinal genes affected by mutations in the transcription factor Crx, which have been implicated in human retinopathies. PolyA RNA isolated from 3 Sex-matched (1M,1F) pairs of P10 mouse retinas from each genotype (WT, E168d2neo/d2neo, R90Wneo/Wneo, and Crx -/-)
2013-12-01 | E-GEOD-51184 | biostudies-arrayexpress
Project description:P10 retinal gene expression analysis of the homozygous Crx Knock-OUT or Knock-IN mice carrying the mutation E168d2 or R90W. Results provide important information about the retinal genes affected by mutations in the transcription factor Crx, which have been implicated in human retinopathies.
2013-12-01 | GSE51184 | GEO
Project description:Cone-Rod Homeobox, encoded by CRX, is a transcription factor (TF) essential for the terminal differentiation and maintenance of mammalian photoreceptors. Although a handful of human variants in CRX have been shown to cause several different degenerative retinopathies with varying cone and rod predominance, as with most human disease genes the vast majority of observed CRX genetic variants are uncharacterized variants of uncertain significance (VUS). We performed a deep mutational scan (DMS) of nearly all possible single amino acid substitution variants in CRX, using an engineered cell-based transcriptional reporter assay. We measured the ability of each CRX missense variant to transactivate a synthetic fluorescent reporter construct in a pooled fluorescence-activated cell sorting assay and compared the activation strength of each variant to that of wild-type CRX to compute an activity score, identifying thousands of variants with altered transcriptional activity.
2024-03-21 | GSE262060 | GEO
Project description:Mutations in the cone-rod homeobox (CRX) transcription factor lead to distinct retinopathy phenotypes, including early-onset vision impairment in dominant Leber congenital amaurosis (LCA). Using induced pluripotent stem cells (iPSCs) from a patient with CRX-I138fs mutation, we established an in vitro model of CRX-LCA in retinal organoids that exhibit defective photoreceptor maturation by histology and gene profiling including diminished expression of visual opsins. Gene therapy by delivery of an additional correct CRX allele using an AAV vector partially restored photoreceptor phenotype and expression of phototransduction-related genes as revealed by single cell RNA-sequencing. Retinal organoids derived from iPSCs of a second dominant CRX-LCA patient carrying a K88N mutation revealed loss of opsin expression as a common phenotype, which could also be alleviated by AAV-mediated overexpression of CRX. Our studies provide the proof-of-concept for development of gene therapy for dominant CRX-LCA and other CRX-retinopathies.
2021-02-17 | GSE153099 | GEO
Project description:Mutations in the cone-rod homeobox (CRX) transcription factor lead to distinct retinopathy phenotypes, including early-onset vision impairment in dominant Leber congenital amaurosis (LCA). Using induced pluripotent stem cells (iPSCs) from a patient with CRX-I138fs mutation, we established an in vitro model of CRX-LCA in retinal organoids that exhibit defective photoreceptor maturation by histology and gene profiling including diminished expression of visual opsins. Gene therapy by delivery of an additional correct CRX allele using an AAV vector partially restored photoreceptor phenotype and expression of phototransduction-related genes as revealed by single cell RNA-sequencing. Retinal organoids derived from iPSCs of a second dominant CRX-LCA patient carrying a K88N mutation revealed loss of opsin expression as a common phenotype, which could also be alleviated by AAV-mediated overexpression of CRX. Our studies provide the proof-of-concept for development of gene therapy for dominant CRX-LCA and other CRX-retinopathies.
2021-02-17 | GSE152939 | GEO
Project description:Homeodomain transcription factors (HD TFs) are instrumental to vertebrate development. Mutations in HD TFs have been linked to human diseases, but their pathogenic mechanisms remain elusive. Here we use Cone-Rod Homeobox (CRX) as a model to decipher the disease-causing mechanisms of two HD mutations, p.E80A and p.K88N, that produce severe dominant retinopathies. Through integrated analysis of molecular and functional evidence in vitro and in knock-in mouse models, we uncover two novel gain-of-function mechanisms: p.E80A increases transactivation of canonical CRX target genes in developing photoreceptors; p.K88N alters CRX DNA-binding specificity resulting in binding at ectopic sites and severe perturbation of CRX target gene expression. Both mechanisms produce novel retinal morphological defects and hinders photoreceptor maturation distinct from loss-of-function models. This study reveals the distinct roles of E80 and K88 residues in CRX HD regulatory functions and emphasizes the importance of transcriptional precision in normal development.
2023-11-08 | GSE223658 | GEO