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Series GSE106243 Query DataSets for GSE106243
Status Public on Aug 26, 2018
Title Quantification of mouse retinal enhancer activity by massively parallel reporter assay
Organism Mus musculus
Experiment type Other
Summary 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.
Overall design CRE-seq of mouse retinal explants. 100,000 100-bp target seqeunces were cloned upstream of a photoreceptor promoter (either pRho or pCrx) driving a DsRed reporter harboring CRE-specific 13-bp barcodes in the 3' UTR. This library was electroporated into intact retinas from newborn (P0) mice. Retinas were grown in culture for eight days, at which point RNA and DNA were harvested and barcodes were amplifieid and sequenced to quantify enhancer activity. Each biological replicate consisted of five electroporated retinas, and three biological replicates were performed for each condition (assaying constructs on either pRho or pCrx).
Contributor(s) Hughes AE, Myers CA, Corbo JC
Citation(s) 30158147
Submission date Oct 26, 2017
Last update date May 15, 2019
Contact name Joseph Corbo
Organization name Washington University School of Medicine
Street address 660 S. Euclid
City St. Louis
ZIP/Postal code 63110
Country USA
Platforms (1)
GPL21493 Illumina HiSeq 3000 (Mus musculus)
Samples (12)
GSM2832679 CRE-seq pRho DNA replicate 1
GSM2832680 CRE-seq pRho cDNA replicate 1
GSM2832681 CRE-seq pRho DNA replicate 2
BioProject PRJNA415987
SRA SRP121792

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Supplementary file Size Download File type/resource
GSE106243_counts.txt.gz 2.8 Mb (ftp)(http) TXT
GSE106243_differential_expression.txt.gz 1.7 Mb (ftp)(http) TXT
GSE106243_expression.txt.gz 791.1 Kb (ftp)(http) TXT
GSE106243_oligos.txt.gz 1.4 Mb (ftp)(http) TXT
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