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Status |
Public on Jan 20, 2025 |
Title |
Resolution of a human super-enhancer by targeted genome randomisation [Amplicon-seq] |
Organism |
Homo sapiens |
Experiment type |
Other
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Summary |
Human gene expression is controlled from distance via enhancers, which can form longer ‘super-enhancer’-regions of intense regulatory activity. Whether super-enhancers constitute a separate regulatory paradigm remains unclear, largely due to the difficulty of dissecting the contributions and interactions of individual elements within their natural chromosomal context. To address this challenge, we developed enhancer scrambling, a high-throughput strategy to generate stochastic inversions and deletions of targeted enhancer regions by combining CRISPR prime editing insertion of symmetrical loxP sites with Cre recombinase-induced rearrangements. We applied our approach to dissect a distal super-enhancer of the OTX2 gene, generating up to 134 alternative regulatory configurations in a single experiment, and establishing how they drive gene expression and chromatin accessibility, as well as the individual contributions of its elements to this activity. Surprisingly, the presence of the sequence containing a single DNase I hypersensitive site predominantly controls OTX2 expression. Our findings highlight that enhancer-driven regulation of some highly expressed, cell-type-specific genes can rely on an individual element within a cluster of non-interacting, dispensable components, and suggest a simple functional core to a subset of super-enhancers. The targeted randomisation method to scramble enhancers can scale to resolve many super-enhancers and human gene regulatory landscapes.
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Overall design |
Amplicon-seq profiling of engineered HAP1 clones. Engineered HAP1 clones were generated by inducing deletions at TFBS within the R5 super-enhancer domains of the OTX2 gene.
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Contributor(s) |
Murat P, Koeppel J, Girling G, Madli Peets E, Gouley M, Rebernig V, Maheshwari A, Hepkema J, Weller J, Johnkingsly Jebaraj JH, Crawford R, Liberante FG, Parts L |
Citation missing |
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Submission date |
Jan 17, 2025 |
Last update date |
Jan 21, 2025 |
Contact name |
Pierre Murat |
E-mail(s) |
pm23@sanger.ac.uk
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Organization name |
Wellcome Sanger Institute
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Street address |
Wellcome Trust Genome Campus
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City |
Hinxton |
ZIP/Postal code |
CB10 1RQ |
Country |
United Kingdom |
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Platforms (1) |
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Samples (9)
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GSM8742705 |
TFBS screen, round 1, replicate A |
GSM8742706 |
TFBS screen, round 1, replicate B |
GSM8742707 |
TFBS screen, round 1, replicate C |
GSM8742708 |
TFBS screen, round 2, replicate A |
GSM8742709 |
TFBS screen, round 2, replicate B |
GSM8742710 |
TFBS screen, round 2, replicate C |
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Relations |
BioProject |
PRJNA1211996 |
Supplementary file |
Size |
Download |
File type/resource |
GSE287268_RAW.tar |
40.0 Kb |
(http)(custom) |
TAR (of TXT) |
SRA Run Selector |
Raw data are available in SRA |
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