dCas9-targeted locus-specific protein isolation method identifies histone gene regulators

Abstract

Eukaryotic gene regulation is a complex process, often coordinated by the action of tens to hundreds of proteins. Although previous biochemical studies have identified many components of the basal machinery and various ancillary factors involved in gene regulation, numerous gene-specific regulators remain undiscovered. To comprehensively survey the proteome directing gene expression at a specific genomic locus of interest, we developed an in vitro nuclease-deficient Cas9 (dCas9)-targeted chromatin-based purification strategy, called "CLASP" (Cas9 locus-associated proteome), to identify and functionally test associated gene-regulatory factors. Our CLASP method, coupled to mass spectrometry and functional screens, can be efficiently adapted for isolating associated regulatory factors in an unbiased manner targeting multiple genomic loci across different cell types. Here, we applied our method to isolate the histone cluster in S2 cells to identify several factors including Vig and Vig2, two proteins that bind and regulate core histone and mRNA via interaction with their 3' UTRs.

CRISPR Mutants of Three Y Chromosome Genes Suggest Gradual Evolution of Fertility Functions inDrosophila melanogaster

“…We do not see a fertility defect in FDY mutants, though we have not tested the mutants in 482 cluster (Tsui et al 2018). FDY has a more limited tissue expression pattern than vig2.…”

Adapting dCas9-APEX2 for subnuclear proteomic profiling

“…C-BERST can be successfully applied to identify protein factors associated with repetitive DNA elements using relatively few cells (~40 million) with high specificity compared to similar methods (Déjardin & Kingston, 2009; Garcia-Exposito et al, 2016; Liu et al, 2017; Schmidtmann et al, 2016; Tsui et al, 2018). The rapid kinetics of the APEX2 rapid system allow C-BERST to characterize a broad range of dynamic nuclear processes (e.g., cellular differentiation, extracellular stimulus response, and cell-cycle progression).…”

“…Proteomics of isolated chromatin segments (PICh), for example, isolates cross-linked chromatin using an oligonucleotide probe hybridized to a genomic region of interest (Déjardin & Kingston, 2009). The use of nuclease-dead CRISPR–Cas9 (dCas9) probes in place of modified oligonucleotides provides an additional, streamlined approach to isolating cross-linked chromatin (Fujita & Fujii, 2013; Liu et al, 2017; Tsui et al, 2018). However, a substantial hurdle posed by formaldehyde cross-linking is the nonspecific enrichment of spatially distant proteins.…”

TINC - a method to dissect transcriptional complexes at single locus resolution - reveals novelNanogregulators in mouse embryonic stem cells

“…These include nucleic acid hybridization-based approaches (Ide and Dejardin 2015;Déjardin and Kingston 2009;Antão et al 2012;Kennedy-Darling et al 2014) and approaches that utilize DNA-binding proteins including LexA (Byrum et al 2012;Fujita and Fujii 2011), TetR (Pourfarzad et al 2013), Cas9 (Waldrip et al 2014;Fujita and Fujii 2013;X. Liu et al 2017;Gao et al 2018;Schmidtmann et al 2016;Tsui et al 2018;Qiu et al 2019) or TALE proteins (Fujita et al 2013;Byrum, Taverna, and Tackett 2013;Fang et al 2018). Although nucleic acid hybridization-based approaches pioneered the field, they require intensive probe-specific optimizations (Ide and Dejardin 2015;Déjardin and Kingston 2009;Antão et al 2012), which may account for why this approach has not yet been widely adopted nor translated to single copy elements in mammalian cells.…”