Decoding the chromatin proteome of a single genomic locus by DNA sequencing

PLoS Biology
Tessy KorthoutFred van Leeuwen

Abstract

Transcription, replication, and repair involve interactions of specific genomic loci with many different proteins. How these interactions are orchestrated at any given location and under changing cellular conditions is largely unknown because systematically measuring protein-DNA interactions at a specific locus in the genome is challenging. To address this problem, we developed Epi-Decoder, a Tag-chromatin immunoprecipitation-Barcode-Sequencing (TAG-ChIP-Barcode-Seq) technology in budding yeast. Epi-Decoder is orthogonal to proteomics approaches because it does not rely on mass spectrometry (MS) but instead takes advantage of DNA sequencing. Analysis of the proteome of a transcribed locus proximal to an origin of replication revealed more than 400 interacting proteins. Moreover, replication stress induced changes in local chromatin proteome composition prior to local origin firing, affecting replication proteins as well as transcription proteins. Finally, we show that native genomic loci can be decoded by efficient construction of barcode libraries assisted by clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9). Thus, Epi-Decoder is an effective strategy to identify and quanti...Continue Reading

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Citations

Jul 20, 2019·The FEBS Journal·Birgit Singer-KrügerRalf-Peter Jansen
Mar 20, 2020·Genome Research·Deepani W Poramba-LiyanageFred van Leeuwen
Mar 11, 2020·Nature Methods·Mathilde GauchierJérôme Déjardin
Sep 12, 2018·Nucleic Acids Research·Tibor van WelsemFred van Leeuwen
Apr 16, 2020·BMC Molecular and Cell Biology·Nastassja L KrielRobin M Warren
Feb 10, 2021·Medicinal Research Reviews·Xianlong ZhangTony D James

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Methods Mentioned

BETA
immunoprecipitation
genetic array
Tandem Affinity Purification
ChIP
SGA
PCR
reverse transcription PCR
affinity capture
flow cytometry
gene knockout

Software Mentioned

custom
Decoder
R scripts
Bioconductor
FlowJo
CellQuest
Limma R /
XCALIBR
R
Perl script eXtracting Counting and LInking to Barcode Referen...

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