Determination of local chromatin composition by CasID.
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.
a Department of Biology II and Center for Integrated Protein Science Munich (CIPSM) , LMU Munich , Martinsried , Germany.
Elisabeth
E
Schmidtmann
a Department of Biology II and Center for Integrated Protein Science Munich (CIPSM) , LMU Munich , Martinsried , Germany.
Tobias
T
Anton
b Gene Center and Department of Biochemistry , LMU Munich , Munich , Germany.
Pascaline
P
Rombaut
b Gene Center and Department of Biochemistry , LMU Munich , Munich , Germany.
Franz
F
Herzog
a Department of Biology II and Center for Integrated Protein Science Munich (CIPSM) , LMU Munich , Martinsried , Germany.
Heinrich
H
Leonhardt
D000818:Animals; D001483:Base Sequence; D019731:Carbon-Nitrogen Ligases / Q000378:metabolism*; D002460:Cell Line; D002843:Chromatin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D023281:Genomics; D013058:Mass Spectrometry; D051379:Mice; D008856:Microscopy, Fluorescence
D002843:Chromatin; D004247:DNA; D019731:Carbon-Nitrogen Ligases
CRISPR/Cas; CasID; biotinylation; chromatin composition; repetitive elements
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