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

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• Understanding protein-DNA interactions at specific genomic loci is crucial for deciphering cellular processes like transcription, replication, and repair.
• Current methods face challenges in systematically measuring these interactions at a locus-specific level.
• Existing techniques often rely on mass spectrometry (MS), limiting orthogonal approaches.

Purpose of the Study:

• To develop a novel technology for unbiased, systematic identification and quantification of protein-DNA interactions at individual genomic loci.
• To investigate the dynamic changes in the local chromatin proteome under cellular stress conditions.
• To establish a DNA sequencing-based method independent of MS.

Main Methods:

• Development of Tag-chromatin immunoprecipitation-Barcode-Sequencing (TAG-ChIP-Barcode-Seq), named Epi-Decoder, in budding yeast.
• Application of Epi-Decoder to analyze the proteome of a transcribed locus near an origin of replication.
• Utilizing clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) for barcode library construction.

Main Results:

• Epi-Decoder identified over 400 proteins interacting with a specific transcribed locus.
• Replication stress altered the local chromatin proteome composition before origin firing, impacting both replication and transcription proteins.
• Demonstrated efficient decoding of native genomic loci using CRISPR/Cas9-assisted barcode libraries.

Conclusions:

• Epi-Decoder provides an effective, sequencing-based strategy to unbiasedly identify and quantify the proteome of individual genomic loci.
• The technology offers new insights into the dynamic regulation of protein-DNA interactions during cellular processes and stress.
• Epi-Decoder is orthogonal to MS-based proteomics, expanding the toolkit for studying genome regulation.