A novel proteomics approach for the discovery of chromatin-associated protein networks.
Protein-protein interaction mapping has progressed rapidly in recent years, enabling the completion of several high throughput studies. However, knowledge of physical interactions is limited for numerous classes of proteins, such as chromatin-bound proteins, because of their poor solubility when bound to DNA. To address this problem, we have developed a novel method, termed modified chromatin immunopurification (mChIP), that allows for the efficient purification of protein-DNA macromolecules, enabling subsequent protein identification by mass spectrometry. mChIP consists of a single affinity purification step whereby chromatin-bound protein networks are isolated from mildly sonicated and gently clarified cellular extracts using magnetic beads coated with antibodies. We applied the mChIP method in Saccharomyces cerevisiae cells expressing endogenously tandem affinity purification (TAP)-tagged histone H2A or the histone variant Htz1p and successfully co-purified numerous chromatin-bound protein networks as well as DNA. We further challenged the mChIP procedure by purifying three chromatin-bound bait proteins that have proven difficult to purify by traditional methods: Lge1p, Mcm5p, and Yta7p. The protein interaction networks of these three baits dramatically expanded our knowledge of their chromatin environments and illustrate that the innovative mChIP procedure enables an improved characterization of chromatin-associated proteins.
Lambert
Jean-Philippe
JP
Ottawa Institute of Systems Biology and Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, 451 Smyth Road, Ottawa, Ontario K1H 8M5, Canada.
Mitchell
Leslie
L
Rudner
Adam
A
Baetz
Kristin
K
Figeys
Daniel
D
D002843:Chromatin / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D004271:DNA, Fungal / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D009687:Nuclear Proteins / Q000032:analysis* / Q000302:isolation & purification; D011485:Protein Binding; D040901:Proteomics / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000302:isolation & purification
D002843:Chromatin; D004271:DNA, Fungal; D006657:Histones; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins