Anastasiia Tonelli, Pascal Cousin, Aleksander Jankowski, Bihan Wang, Julien Dorier et al.
Long-range transcriptional activation of gene promoters by abundant enhancers in animal genomes calls for mechanisms to limit inappropriate regulation. DNA elements called insulators serve this purpose by shielding promoters from an enhancer when interposed. Unlike promoters and enhancers, insulators have not been systematically characterized due to lacking high-throughput screening assays, and questions regarding how insulators are distributed and encoded in the genome remain. Here, we establish "insulator-seq" as a plasmid-based massively parallel reporter assay in Drosophila cultured cells to perform a systematic insulator screen of selected genomic loci. Screening developmental gene loci showed that not all insulator protein binding sites effectively block enhancer-promoter communication. Deep insulator mutagenesis identified sequences flexibly positioned around the CTCF insulator protein binding motif that are critical for functionality. The ability to screen millions of DNA sequences without positional effect has enabled functional mapping of insulators and provided further insights into the determinants of insulators.
Anastasiia Tonelli, Pascal Cousin, Aleksander Jankowski, Bihan Wang, Julien Dorier, Jonas Barraud, Sanyami Zunjarrao, Maria Cristina Gambetta. Systematic screening of enhancer-blocking insulators in Drosophila identifies their DNA sequence determinants. 2025-02-24. https://doi.org/10.1016/j.devcel.2024.10.017
A barcoded dual-fluorescent episomal MPRA screened approximately 339 kb spanning the Drosophila Bithorax complex (BX-C) in S2R+ cells. Reporter RNA mCherry/eGFP ratios from two biological replicates were assigned to mapped genomic fragments and summarized as 50-bp genomic-bin insulation scores.
A barcoded dual-fluorescent episomal MPRA screened an approximately 345-kb region around the Drosophila even-skipped (eve) locus in S2R+ cells. Reporter RNA mCherry/eGFP unique-UMI ratios from two biological replicates were assigned to mapped genomic fragments and summarized as 50-bp genomic-bin insulation scores.
An episomal insulator-seq MPRA tested 32 synthetic 190-bp CTCF-centered wild-type regions and their motif-point-mutation, sequence-shuffle, and context-replacement derivatives in Drosophila S2R+ cells. Two biological replicates and independent barcoded copies were collapsed to construct-level median mCherry/eGFP activity ratios.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 13 files (ZIP)1-s2.0-S1534580724006361-mmc1.pdfdash_database_L_and_R.tsvGSE253140_BXC_locus_screen_Rep1_raw_counts.tsv.gzGSE253140_BXC_locus_screen_Rep2_raw_counts.tsv.gzGSE253140_eve_locus_screen_Rep1_raw_counts.tsv.gzGSE253140_eve_locus_screen_Rep2_raw_counts.tsv.gzGSE253140_family.soft.gzGSE253140_Synthetic_insulator_screen_Rep1_raw_counts.tsv.gzGSE253140_Synthetic_insulator_screen_Rep2_raw_counts.tsv.gzinsulator-seq-2024_github_source.zipsix_bp.tsvsource_notes.txtWT_CTCF_seq_190.txt