C-Berst: Defining Subnuclear Proteomic Landscapes at Genomic Elements with dCas9-APEX2

Spatial and temporal genome organization is essential to gene regulation and to all aspects of chromosome dynamics¹. Thanks in large part to chromosome conformation capture (3C)², Hi-C, and related techniques, we are able to identify many physical interactions of entire genomes in four dimensions (4D). To further understand the specifications and interactions of the 4D nucleome, it is important to develop minimally biased, high-throughput methods for identifying the proteomic landscapes that shape and reflect subnuclear structure. Recently, nuclease-dead Cas9 (dCas9)-based techniques have enabled locus-specific genome labeling and gene regulation³. Separately, spatially restricted enzymatic tagging (SRET) approaches [such as those involving localized ascorbate peroxidase (APEX)-catalyzed biotinylation] have been applied successfully to map subcellular proteomes⁴. Here we report the development of dCas9-APEX2 Biotinylation at genomic Elements by Restricted Spatial Tagging (C-BERST) for the unbiased mapping of subnuclear proteomes in proximity to defined genomic loci in living U2OS cells⁵. In the presence of a single-guide RNA complementary to telomeric repeats (sgTelo), dCas9-APEX2 enables the specific biotinylation, enrichment and proteomic identification of 192 proteins via mass spectrometry. The striking success of our approach is exemplified by the six subunits of the telomeric shelterin complex, which were among the seven most highly enriched proteins that we identified. Another highly enriched protein was Apollo, a 5’ to 3’ exonuclease that interacts with the shelterin component TRF2 and functions in the alternative lengthening of telomeres (ALT) pathway that maintains telomere length in this cell type⁶. Overall, among the 192 most significantly sgTelo-enriched proteins, 32 have been reported previously to be associated with telomeres or linked to telomere function. These include proteins from complexes known to contribute to ALT-associated pathways or processes. Gene ontology analysis of the 192 C-BERST proteomic hits reveals strong functional associations with terms such as telomere maintenance, DNA replication, DNA repair, and homologous recombination, all of which are important for ALT pathways⁷. By combining the flexibility of RNA-guided dSpyCas9 genome binding with the efficiency and rapid kinetics of APEX2-catalyzed biotinylation, C-BERST extends the unbiased definition of subnuclear proteomes to many genomic elements, and to a range of dynamic processes that occur too rapidly to analyze via the 18-24h labeling procedures inherent to proximity-dependent biotin identification (BioID). Importantly, C-BERST promises to augment and extend Hi-C and related methods by linking conformationally important cis-elements with the factors that associate with them.

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