Proteomics to study DNA-bound and chromatin-associated gene regulatory complexes

Area of Science:

• Proteomics
• Epigenetics
• Molecular Biology

Background:

• Studying chromatin-associated protein complexes is challenging due to low abundance and purification difficulties.
• High-resolution mass spectrometry (MS) has revolutionized soluble protein complex identification.
• Advances in proteomics are overcoming previous limitations in chromatin protein analysis.

Purpose of the Study:

• To highlight recent methodological and technological advances in MS-based proteomics for analyzing chromatin-associated proteins.
• To discuss novel techniques for identifying proteins interacting with chromatin marks and DNA alterations.
• To explore the potential of locus-specific chromatin immunoprecipitation coupled with mass spectrometry (ChIP-MS) in epigenetics.

Main Methods:

• Pulldowns using synthesized histone tails with posttranslational modifications (PTMs).
• Pulldowns with DNA baits containing single nucleotide polymorphisms or DNA modifications.
• Chromatin immunoprecipitation coupled with mass spectrometry (ChIP-MS) using various baits (proteins, histone PTMs, lncRNAs).
• Locus-specific ChIP-MS targeting individual genomic loci via engineered DNA-binding proteins or sequence-specific factors (TAL, Cas9).

Main Results:

• Accurate quantitation (isotope-based or label-free) identifies significantly enriched proteins.
• ChIP-MS enables the study of gene regulatory complexes in their native in vivo context.
• Locus-specific ChIP-MS allows for the purification and analysis of proteins associated with a single genomic locus.

Conclusions:

• Recent proteomics advancements facilitate the study of challenging chromatin-associated protein complexes.
• ChIP-MS and locus-specific ChIP-MS are powerful tools for dissecting epigenetic regulation.
• Future MS technology developments will likely make these approaches standard in epigenetics research.