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| Status |
Public on Apr 25, 2018 |
| Title |
The dTAG system for immediate and target-specific protein degradation |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
The dissection of complex biological systems requires target-specific control of protein function or abundance. Genetic perturbations have markedly advanced science but are variably limited by off-target effects, multi-component complexity and irreversibility. Most limiting to the study of fast biology is the requisite delay from modulation to experimental measurement. To enable the immediate and selective control of single protein abundance, we created a chemical biology system that leverages the potency of cell-permeable heterobifunctional degraders. The dTAG system pairs a novel allele-specific degrader of FKBP12F36V with expression of FKBP12F36V in-frame with a target protein of interest. By transgene expression or CRISPR-mediated locus-specific knock-in, we exemplify a generalizable strategy to study the immediate consequent biology of protein loss. Using dTAG, we observe an unexpected superior anti-proliferative effect of BET bromodomain inhibition and degradation over selective BRD4 degradation, characterize immediate effects of KRASG12V loss on proteomic signaling, and demonstrate rapid degradation in vivo. This technology platform is expected to confer high kinetic resolution to biological investigation and provide early target validation in the context of drug discovery.
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| Overall design |
We performed two ERCC spike-in normalized RNA-sequencing experiments to quantify transcriptional changes upon dTAG-13 treatment. In the first experiment (see datasets identified as associated with 'Control-related experiment'), we profiled control NIH/3T3 cells treated with DMSO, dTAG-13, ortho-AP1867, AP1867, Thalidomide, dBET6, and dTAG-47 for four hours. In the second experiment (see datasets identified as associated with 'KRAS-related experiment'), we profiled NIH/3T3 cells expressing FKBP12F36V-KRASG12V treated with DMSO, dTAG-13 for four and eight hours to induce KRASG12V degradation, or Trametinib (GSK112021) for four and eight hours to inhibit MEK as well as control NIH/3T3 cells with treated with DMSO. All samples were prepared in biological triplicate.
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| Contributor(s) |
Nabet B, Erb MA, Lawlor MA, Bradner JE |
| Citation(s) |
29581585 |
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| Submission date |
Dec 22, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
James Bradner |
| E-mail(s) |
bradner_computation@dfci.harvard.edu
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| Organization name |
Dana-Farber Cancer Institute
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| Department |
Medical Oncology
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| Lab |
Bradner Lab
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| Street address |
450 Brookline
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
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| Platforms (1) |
| GPL19057 |
Illumina NextSeq 500 (Mus musculus) |
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| Samples (39)
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| Relations |
| BioProject |
PRJNA358588 |
| SRA |
SRP095542 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE92775_dTAG_all_fpkm_exprs_norm.txt.gz |
2.6 Mb |
(ftp)(http) |
TXT |
| GSE92775_dTAG_parental_control_all_fpkm_exprs_norm.txt.gz |
3.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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