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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Sep 09, 2024 |
| Title |
Unbiased screening of regulatory insulators |
| Organism |
Drosophila melanogaster |
| Experiment type |
Expression profiling by high throughput sequencing
Other
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| 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.
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| 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.
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| Contributor(s) |
Semenova A, Cousin P, Jankowski A, Wang B, Dorier J, Barraud J, Zunjarrao S, Gambetta M |
| Citation(s) |
39532105 |
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| Submission date |
Jan 12, 2024 |
| Last update date |
Nov 22, 2024 |
| Contact name |
Maria Cristina Gambetta |
| E-mail(s) |
mariacristina.gambetta@unil.ch
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| Organization name |
University of Lausanne
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| Department |
Center for Integrative Genomics
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| Street address |
Genopode Building
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| City |
Lausanne |
| ZIP/Postal code |
1015 |
| Country |
Switzerland |
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| Platforms (2) |
| GPL25244 |
Illumina NovaSeq 6000 (Drosophila melanogaster) |
| GPL34080 |
DNBSEQ-G50 (Drosophila melanogaster) |
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| Samples (16)
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| GSM8014844 |
Reporter RNA-seq from BX-C screen, rep2 |
| GSM8014845 |
DNA-seq from eve screen |
| GSM8014846 |
Reporter RNA-seq from eve screen, rep1 |
| GSM8014847 |
Reporter RNA-seq from eve screen, rep2 |
| GSM8014848 |
DNA-seq from synthetic insulator screen |
| GSM8014849 |
Reporter RNA-seq from synthetic insulator screen, rep1 |
| GSM8014850 |
Reporter RNA-seq from synthetic insulator screen, rep2 |
| GSM8339496 |
Wildtype D. mel. pupal heads, Hi-C rep1 |
| GSM8339497 |
Wildtype D. mel. pupal heads, Hi-C rep2 |
| GSM8339498 |
Wildtype D. mel. pupal heads, Hi-C rep3 |
| GSM8339499 |
BEAF-320 D. mel. pupal heads, Hi-C rep1 |
| GSM8339500 |
BEAF-320 D. mel. pupal heads, Hi-C rep2 |
| GSM8339501 |
BEAF-320 D. mel. pupal heads, Hi-C rep3 |
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| Relations |
| BioProject |
PRJNA1064098 |
| 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 |
SRA Run Selector |
| Raw data are available in SRA |
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