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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 115; no. 12; pp. E2734 - E2741
• Main Authors: Tsui, Chiahao, Inouye, Carla, Levy, Michaella, Lu, Andrew, Florens, Laurence, Washburn, Michael P., Tjian, Robert
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 20.03.2018
• Series: PNAS Plus
• Subjects: 3' Untranslated Regions; Animals; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biological Sciences; Chromatin; Chromatin - genetics; Chromatin - isolation & purification; Chromatin Immunoprecipitation; Chromosomal Proteins, Non-Histone - genetics; Chromosomal Proteins, Non-Histone - metabolism; CRISPR; CRISPR-Associated Protein 9; Drosophila melanogaster - genetics; Drosophila Proteins - genetics; Drosophila Proteins - metabolism; Endonucleases - genetics; Endonucleases - metabolism; Fruit flies; Gene Expression; Gene loci; Gene regulation; Genes, Regulator - genetics; Histone H2A; Histones - genetics; Histones - metabolism; Humans; Mass spectrometry; Mass spectroscopy; Nuclease; PNAS Plus; Promoter Regions, Genetic; Proteins; Proteomes; Proteomics; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; RNA, Guide, CRISPR-Cas Systems - genetics; RNA, Guide, CRISPR-Cas Systems - metabolism; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; Screens; histone regulation; CRISPR/Cas9; reverse ChIP; gene expression
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1718844115

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 Drosophila melanogaster histone cluster in S2 cells to identify several factors including Vig and Vig2, two proteins that bind and regulate core histone H2A and H3 mRNA via interaction with their 3′ UTRs.