Genomics

Dataset Information

43

The dTAG system for immediate and target-specific protein degradation


ABSTRACT: 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. 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.

INSTRUMENT(S): Illumina NextSeq 500 (Mus musculus)

SUBMITTER: James Bradner  

PROVIDER: GSE92775 | GEO | 2018-04-25

REPOSITORIES: GEO

Dataset's files

Source:
Action DRS
GSE92775_dTAG_all_fpkm_exprs_norm.txt.gz Txt
GSE92775_dTAG_parental_control_all_fpkm_exprs_norm.txt.gz Txt
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Publications


Dissection of complex biological systems requires target-specific control of the function or abundance of proteins. Genetic perturbations are limited by off-target effects, multicomponent complexity, and irreversibility. Most limiting is the requisite delay between 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 dTA  ...[more]

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