Purification of a specific native genomic locus for proteomic analysis

Stephanie D Byrum,Sean D Taverna,Alan J Tackett

doi:10.1093/nar/gkt822

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.

Highlights

One of the most compositionally diverse structures in a eukaryotic cell is a chromosome
To work toward proteomic studies of native chromatin regions, we recently developed a technique termed Chromatin Affinity Purification with Mass Spectrometry (ChAP-mass spectrometric (MS)) that provides for the enrichment of a native 1-kb section of a chromosome for sitespecific identification of protein interactions and associated histone post-translational modifications (PTMs) [13]
To alleviate genomic engineering for affinity enrichment of chromatin sections, we report the use of modified transcription activator-like (TAL) effector proteins to sitespecifically target a native section of a chromosome for purification and proteomic analysis

Summary

Introduction

One of the most compositionally diverse structures in a eukaryotic cell is a chromosome. To work toward proteomic studies of native chromatin regions (i.e. sections of chromatin that are unaltered genetically and spatially the genome), we recently developed a technique termed Chromatin Affinity Purification with Mass Spectrometry (ChAP-MS) that provides for the enrichment of a native 1-kb section of a chromosome for sitespecific identification of protein interactions and associated histone PTMs [13]. This ChAP-MS approach uses the association of an ectopically expressed affinity-tagged LexA protein with a genomically incorporated LexA DNA binding site for site-specific chromatin enrichment. The ChAP-MS approach provides for the isolation of chromatin from the native site in the chromosome; one must genomically engineer a LexA DNA binding site, which could alter the native state of the chromatin and which requires a biological system readily amendable to genomic engineering

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Conclusion