Abstract

Mapping proteomic composition at distinct genomic loci 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. C-BERST enables the high-throughput identification of proteins associated with specific sequences, facilitating annotation of these factors and their roles in nuclear biology.

Highlights

  • Telomere-associated proteomes from ALT+ cell lines have been defined previously by TERF1-BirA* BioID13 and proteomics of isolated chromatin segments (PICh)[14]

  • To develop and validate this method, we used telomeres for benchmarking and proof-ofconcept because they are associated with a well-defined suite of proteins and can be targeted by a well-established sgRNA8, 9 in human U2OS cells

  • Using intensity-based absolute quantification [a label-free quantification (LFQ) proteomic approach] values to measure enrichment in the sgTelo sample relative to sgNS [Benjamini-Hochberg (BH)-adjusted p < 0.05 and log[2] fold change (FC) ≥ 2.0], we found 30 out of 143 proteins that have been reported to be telomereassociated or otherwise linked to telomere function (Supplementary Fig. 4d, Supplementary Tables 1 and 2, and Supplementary Note 2)

Read more

Summary

Introduction

Telomere-associated proteomes from ALT+ cell lines have been defined previously by TERF1-BirA* BioID13 and proteomics of isolated chromatin segments (PICh)[14]. We use dSpyCas9-APEX2 fusions in the development of C-BERST (Fig. 1a) for genomic element-specific profiling of subnuclear proteomes in live cells.

Results
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call