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Series GSE253140 Query DataSets for GSE253140
Status Public on Sep 09, 2024
Title Unbiased screening of regulatory insulators
Organism Drosophila melanogaster
Experiment type Expression profiling by high throughput sequencing
Other
Summary Long-range transcriptional activation of gene promoters by abundant enhancers in animal genomes raises the need for mechanisms to limit inappropriate gene regulation. DNA elements known as insulators provide one such mechanism by shielding promoters from an enhancer when placed in between these two elements. Whereas promoters and enhancers have been extensively characterized in animal genomes, insulators have not because of the lack of a high-throughput screening assay. In the absence of unbiased insulator screening in a genome, basic questions remain about the identity, density and diversity of genomic insulators. Here, we establish “insulator-seq” as a plasmid-based massively parallel reporter assay in Drosophila tissue culture cells to perform the first unbiased screen for insulators in any genome. By screening developmental gene loci, we found that not all characterized insulator protein binding sites are able to block enhancer-promoter communication. By dissecting the sequence determinants of functional insulators, we found that an unexpectedly broad sequence context around the central insulator protein binding motif is required for functionality. The ability to screen millions of DNA sequences in parallel without any positional effect has enabled the first functional mapping of insulators and provided further insights into the determinants of functional insulators.
 
Overall design For insulator-seq, test fragments were cloned into a barcoded insulator reporter acceptor plasmid library, then transfected into Drosophila S2R+ tissue culture cells. 72 hours post-transfection, total RNA was isolated and PCR amplicon libraries of two reporter genes (eGFP and mCherry) with a shared unique transcribed barcode were generated. In parallel, DNA-sequencing libraries were generated to link a transcribed barcode in a reporter gene to a cloned test fragment. Three screens were performed to test fragments from the abdominal HOX gene locus (BX-C), an extended region around the even-skipped (eve) gene, or synthetic CTCF-dependent insulators and systematically mutated variants thereof. Also, wildtype or BEAF-320 mutant Drosophila melanogaster pupal heads were subjected to Hi-C.
 
Contributor(s) Semenova A, Cousin P, Jankowski A, Wang B, Dorier J, Barraud J, Zunjarrao S, Gambetta M
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Submission date Jan 12, 2024
Last update date Sep 10, 2024
Contact name Maria Cristina Gambetta
E-mail(s) mariacristina.gambetta@unil.ch
Organization name University of Lausanne
Department Center for Integrative Genomics
Street address Genopode Building
City Lausanne
ZIP/Postal code 1015
Country Switzerland
 
Platforms (2)
GPL25244 Illumina NovaSeq 6000 (Drosophila melanogaster)
GPL34080 DNBSEQ-G50 (Drosophila melanogaster)
Samples (16)
GSM8014841 DNA-seq from BX-C screen, rep1
GSM8014842 DNA-seq from BX-C screen, rep2
GSM8014843 Reporter RNA-seq from BX-C screen, rep1
Relations
BioProject PRJNA1064098

Download family Format
SOFT formatted family file(s) SOFTHelp
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Supplementary file Size Download File type/resource
GSE253140_BEAF-32.mcool 126.3 Mb (ftp)(http) MCOOL
GSE253140_BXC_locus_screen_Rep1_raw_counts.tsv.gz 3.2 Mb (ftp)(http) TSV
GSE253140_BXC_locus_screen_Rep2_raw_counts.tsv.gz 3.8 Mb (ftp)(http) TSV
GSE253140_RAW.tar 350.0 Mb (http)(custom) TAR (of BEDGRAPH, MCOOL)
GSE253140_Synthetic_insulator_screen_Rep1_raw_counts.tsv.gz 921.9 Kb (ftp)(http) TSV
GSE253140_Synthetic_insulator_screen_Rep2_raw_counts.tsv.gz 999.1 Kb (ftp)(http) TSV
GSE253140_WT.mcool 134.4 Mb (ftp)(http) MCOOL
GSE253140_eve_locus_screen_Rep1_raw_counts.tsv.gz 6.1 Mb (ftp)(http) TSV
GSE253140_eve_locus_screen_Rep2_raw_counts.tsv.gz 6.4 Mb (ftp)(http) TSV
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