Determination of local chromatin composition by CasID

Schmidtmann, Elisabeth; Anton, Tobias; Rombaut, Pascaline; Herzog, Franz und Leonhardt, Heinrich (2016): Determination of local chromatin composition by CasID. In: Nucleus, Bd. 7, Nr. 5: S. 476-484

Abstract

Chromatin structure and function are determined by a plethora of proteins whose genome-wide distribution is typically assessed by immunoprecipitation (ChIP). Here, we developed a novel tool to investigate the local chromatin environment at specific DNA sequences. We combined the programmable DNA binding of dCas9 with the promiscuous biotin ligase BirA* (CasID) to biotinylate proteins in the direct vicinity of specific loci. Subsequent streptavidin-mediated precipitation and mass spectrometry identified both known and previously unknown chromatin factors associated with repetitive telomeric, major satellite and minor satellite DNA. With super-resolution microscopy, we confirmed the localization of the putative transcription factor ZNF512 at chromocenters. The versatility of CasID facilitates the systematic elucidation of functional protein complexes and locus-specific chromatin composition.

• Dokumententyp: Zeitschriftenartikel
• EU Funded Grant Agreement Number: 638218
• EU-Projekte: Horizon 2020 > ERC Grants > ERC Starting Grant > ERC Grant 638218: MolStruKT - Molecular structure and cell cycle regulated assembly of the kinetochore
• Fakultät: Chemie und Pharmazie > Department Biochemie ; Chemie und Pharmazie > GenZentrum
• Themengebiete: 500 Naturwissenschaften und Mathematik > 540 Chemie
• Sprache: Englisch