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| Status |
Public on Mar 14, 2020 |
| Title |
Alteration of genome folding via engineered transposon insertion [H3K27Ac ChIP-seq] |
| Organism |
Homo sapiens |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
Mammalian chromosomes are partitioned into contact domains that can be conserved as functional units in evolution. Disruptions of domains can result from perturbed CTCF, cohesin, or chromosomal rearrangements. However, to what extent domains can be created de novo has not been explored in depth. Here, using a gain-of-function approach leveraging genome editing and Hi-C, we examined whether, and how, a putative boundary element can function to organize de novo domains in the context of multiple ectopic insertion sites. We subsequently dissected the distinct roles of the CTCF binding site and the transcription start site within the insertion element in changing genome folding.
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| Overall design |
Using a transposon-based approach, we inserted a 2 kb DNA fragment, which resides within a tissue-invariant domain boundary, into the genomes of near-haploid HAP1 cells. Following transposition, we established clonal lines and prioritized Clones 21 (C21) and 25 (C25), which contained ten and six insertions, respectively, across a total of 11 chromosomes. We then performed in situ Hi-C on edited clones and unedited parental (WT) cells to characterize how the insertion of a boundary-associated DNA fragment shapes human genome architecture. Furthermore, we carried out a series of CRISPR-based editing experiments to dissect how the TSS and the CTCF binding site within the insertion element contributes to new domain formation. Additionally, we used CRISPR to delete the endogenous 2 kb element and characterized its function at its endogenous boundary. In addition to Hi-C, we performed Capture-C, ChIP-seq (CTCF, Rad21, and H3K27Ac), and RNA-seq to further characterize the structural changes resulting from each round of genome editing experiments.
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| Contributor(s) |
Zhang D, Huang P, Malini S, Keller CA, Giardine B, Zhang H, Gilgenast TG, Phillips-Cremins JE, Hardison RC, Blobel GA |
| Citation(s) |
32868908 |
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| Submission date |
Sep 12, 2019 |
| Last update date |
Nov 24, 2020 |
| Contact name |
Ross Hardison |
| E-mail(s) |
rch8@psu.edu
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| Organization name |
Pennsylvania State University
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| Street address |
303 Wartik Lab
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| City |
University Park |
| State/province |
PA |
| ZIP/Postal code |
16802 |
| Country |
USA |
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| Platforms (1) |
| GPL18573 |
Illumina NextSeq 500 (Homo sapiens) |
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| Samples (9)
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| This SubSeries is part of SuperSeries: |
| GSE137376 |
Alteration of genome folding via engineered transposon insertion |
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| Relations |
| BioProject |
PRJNA565258 |
| SRA |
SRP221513 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE137375_ChIP-insertions_or_edited_endo_bedgraphs.tar.gz |
326.5 Mb |
(ftp)(http) |
TAR |
| GSE137375_RAW.tar |
45.5 Gb |
(http)(custom) |
TAR (of BROADPEAK, BW) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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