RNA Therapeutics Institute; Proteomics and Mass Spectrometry Facility; Program in Systems Biology; Department of Biochemistry and Molecular Pharmacology; Department of Molecular, Cell and Cancer Biology
Amino Acids, Peptides, and Proteins | Biochemistry | Genetics and Genomics | Molecular Biology | Structural Biology
Mapping proteomic composition at distinct genomic loci and subnuclear landmarks in living cells has been a long-standing challenge. Here we report that dCas9-APEX2 Biotinylation at genomic Elements by Restricted Spatial Tagging (C-BERST) allows the rapid, unbiased mapping of proteomes near defined genomic loci, as demonstrated for telomeres and centromeres. By combining the spatially restricted enzymatic tagging enabled by APEX2 with programmable DNA targeting by dCas9, C-BERST has successfully identified nearly 50% of known telomere-associated factors and many known centromere-associated factors. We also identified and validated SLX4IP and RPA3 as telomeric factors, confirming C-BERST's utility as a discovery platform. C-BERST enables the rapid, high-throughput identification of proteins associated with specific sequences, facilitating annotation of these factors and their roles in nuclear and chromosome biology.
molecular biology, dCas9-APEX2 Biotinylation at genomic Elements by Restricted Spatial Tagging, C-BERST, proteomes, genomic loci
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The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
DOI of Published Version
bioRxiv 171819; doi: https://doi.org/10.1101/171819. Link to preprint on bioRxiv service.
Now published in Nature Methods doi: 10.1038/s41592-018-0006-2.
Gao, Xin D.; Tu, Li-Chun; Mir, Aamir; Tomas Rodriguez; Ding, Yue-He; Leszyk, John D.; Dekker, Job; Shaffer, Scott A.; Zhu, Lihua Julie; Wolfe, Scot A.; and Sontheimer, Erik J., "C-BERST: Defining subnuclear proteomic landscapes at genomic elements with dCas9-APEX2" (2018). University of Massachusetts Medical School Faculty Publications. 1549.
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