Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Quantification of mouse retinal enhancer activity by massively parallel reporter assay

ABSTRACT: Cone-rod homeobox (CRX) is a paired-like homeodomain transcription factor (TF) and a master regulator of photoreceptor development in vertebrates. The in vitro DNA binding preferences of CRX have been described in detail, but the degree to which in vitro binding affinity is correlated with in vivo enhancer activity is not known. In addition, paired-class homeodomain TFs can bind DNA cooperatively as both homodimers and heterodimers at inverted TAAT half-sites separated by two or three nucleotides. This dimeric configuration is thought to mediate target specificity, but whether monomeric and dimeric sites encode distinct levels of activity is not known. Here, we use a massively parallel reporter assay, CRE-seq, to determine how local sequence context shapes the regulatory activity of CRX binding sites in mouse photoreceptors. We assay inactivating mutations in >1700 TFBSs, and we find that dimeric CRX binding sites act as stronger enhancers than monomeric CRX binding sites. Furthermore, the activity of dimeric half-sites is cooperative, dependent on a strict three-base-pair spacing, and tuned by the identity of the spacer nucleotides. Saturating single-nucleotide mutagenesis of 195 CRX binding sites shows that, on average, changes in TFBS affinity are correlated with changes in regulatory activity, but this relationship is obscured when considering mutations across multiple CREs. Taken together, these results demonstrate that the activity of CRX binding sites is highly dependent on sequence context, providing insight into photoreceptor gene regulation and illustrating functional principles of homeodomain binding sites that may be conserved in other cell types.

ORGANISM(S): Mus musculus

PROVIDER: GSE106243 | GEO | 2018/08/26

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Similar Datasets

General and sequence-specific roles for DNA in glucocorticoid receptor DNA-binding stoichiometry

Project description:The glucocorticoid receptor (GR) binds to DNA in at least three stoichiometric ratios to regulate gene expression. The dimeric binding of GR to a 15 base pair core sequence has been well studied, and is associated with activation of gene expression. Monomeric GR has been associated with repression. GR has also been shown to form tetramers on DNA in live cells. The role of DNA sequence in directing DNA-binding stoichiometry and subsequent gene regulation is not clear. We took an unbiased approach using SELEX-seq to calculate comprehensive monomeric- and dimeric-binding profiles for GR. The monomeric site is composed of a canonical half-site, but is distinguished from the dimer site by a downstream TTTg motif and the absence of a second half-site. To probe the mechanism of how this motif favors monomeric binding, we performed Spec-/Coop-seq. The TTTg increases monomeric affinity while decreasing cooperative dimeric binding. Crystal structures indicated that TTTg results in a narrowed minor groove bringing residues Y455 and K471 of GR into the proximity of DNA, increasing affinity. Interestingly, although monomeric binding is more often associated with repression of gene expression than dimeric binding, the trend is not significant. Consistent with live-cell imaging, a meta-analysis of GR:DNA structures reveals that, regardless of binding sequence, GR binds to DNA as a dimer of dimers using highly similar interfaces. This suggests that the functional stoichiometry of GR in the cell is tetrameric, and that sequence may modulate GR activity.

2018-12-01 | GSE107584 | GEO

Deciphering the cis-regulatory architecture of mammalian photoreceptors

Project description: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. Crx ChIP-Seq analysis of C57BL/6 and Nrl-/- retinas. IgG ChIP-Seq was performed as a control. 1-2 replicates each.

2010-06-24 | E-GEOD-20012 | biostudies-arrayexpress

Deciphering the cis-regulatory architecture of mammalian photoreceptors

2010-06-24 | GSE20012 | GEO

Missense mutations in CRX homeodomain cause dominant retinopathies through two distinct mechanisms [Spec-seq]

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

Missense mutations in CRX homeodomain cause dominant retinopathies through two distinct mechanisms [ChIP-seq]

2023-11-08 | GSE223657 | GEO

Missense mutations in CRX homeodomain cause dominant retinopathies through two distinct mechanisms [RNA-seq]

2023-11-08 | GSE223439 | GEO

Mutations in BCOR, a novel co-repressor of OTX2/CRX, cause pediatric inherited retinal degeneration

Project description:Many transcription factors regulating the production, survival and function of photoreceptor cells in the retina have been identified, but little is known about transcriptional co-regulators in retinal health and disease. Here we show that BCL6 co-repressor (BCOR), a polycomb repressive complex I factor mutated in various cancers, negatively regulates photoreceptor gene expression. Using proteomics and promoter assays, we find that BCOR interacts with the photoreceptor transcription factors OTX2 and CRX, and reduces their ability to activate the promoters of photoreceptor-specific genes, such as Rhodopsin and Nrl. CUT&RUN sequencing further shows that BCOR shares genome-wide binding profiles with CRX/OTX2, consistent with a general co-repression activity. Finally, we identify missense variants in the human BCOR gene in six families with early-onset X-linked inherited retinal degeneration (IRD). Together, this work uncovers BCOR as a co-repressor of OTX2/CRX essential for photoreceptor cell biology and survival.

2022-09-10 | GSE168129 | GEO

Genome-wide Binding Potential and Regulatory Activity of the Glucocorticoid Receptor’s Monomeric and Dimeric Forms

Project description:Genome-wide Binding Potential and Regulatory Activity of the Glucocorticoid Receptor’s Monomeric and Dimeric Forms

| PRJNA482932 | ENA

Genome-wide Binding Potential and Regulatory Activity of the Glucocorticoid Receptor’s Monomeric and Dimeric Forms

Project description:A widely regarded model for glucocorticoid receptor (GR) action postulates that dimeric binding to DNA regulates unfavorable metabolic pathways while monomeric receptor binding promotes repressive gene responses related to its anti-inflammatory effects. This model has been built upon the characterization of the GRdim mutant, reported to be incapable of DNA binding and dimerization. Although quantitative live-cell imaging data shows GRdim as mostly dimeric, genomic studies, on the basis of recovery of enriched half-site response elements, suggest monomeric engagement on DNA. Here, we performed genome-wide studies on GRdim and a constitutively monomeric mutant. Our results show that impairing dimerization affects binding to even open chromatin. Also, GRdim does not exclusively bind half-response elements. Our results do not support a physiological role for monomeric GR and are consistent with a common mode of receptor binding via higher order structures that drives both the activating and repressive actions of glucocorticoids.

2021-01-19 | GSE117661 | GEO

Gene expression analysis of embryonic photoreceptor precursor cells using BAC-Crx-EGFP transgenic mouse.

Project description:We generated a transgenic mouse line which express EGFP in the retina driven by the Crx promoter using BAC transgenesis. We sorted EGFP-positive photoreceptor precursors at E17.5 using FACS, and subsequently performed microarray analysis of the FACS-sorted cells. In order to clarify a molecular role of Crx in developing cone photoreceptor precursors, we investigated the expression profile of the BAC-Crx-EGFP-positive cells compared with that of the BAC-Crx-EGFP-negative cells at E17.5.

2010-12-25 | E-GEOD-25607 | biostudies-arrayexpress

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data