Purification of a specific native genomic locus for proteomic analysis

Nucleic Acids Res. 2013 Nov;41(20):e195. doi: 10.1093/nar/gkt822. Epub 2013 Sep 11.

Abstract

Here, we describe an approach to isolate native chromatin sections without genomic engineering for label-free proteomic identification of associated proteins and histone post-translational modifications. A transcription activator-like (TAL) protein A fusion protein was designed to recognize a unique site in the yeast GAL1 promoter. The TAL-PrA fusion enabled chromatin affinity purification (ChAP) of a small section of native chromatin upstream from the GAL1 locus, permitting mass spectrometric (MS) identification of proteins and histone post-translational modifications regulating galactose-induced transcription. This TAL-ChAP-MS approach allows the biochemical isolation of a specific native genomic locus for proteomic studies and will provide for unprecedented objective insight into protein and epigenetic mechanisms regulating site-specific chromosome metabolism.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Chromatin / isolation & purification*
  • Chromosomal Proteins, Non-Histone / analysis*
  • DNA-Binding Proteins / metabolism
  • Galactokinase / genetics
  • Genetic Loci
  • Genomics
  • Histones / metabolism*
  • Mass Spectrometry
  • Protein Processing, Post-Translational
  • Proteomics / methods*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae Proteins / genetics

Substances

  • Chromatin
  • Chromosomal Proteins, Non-Histone
  • DNA-Binding Proteins
  • Histones
  • Saccharomyces cerevisiae Proteins
  • GAL1 protein, S cerevisiae
  • Galactokinase