A novel proteomics approach for the discovery of chromatin-associated protein networks

Abstract

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.

Fig. 1.

Experimental protocol for immunopurification of chromatin-bound protein complexes using the mChIP approach as compared with conventional IP methods.

Fig. 2.

Purification of chromatin-bound protein associated with Hta2p and Htz1p by mChIP. A, silver-stained SDS-PAGE gel of protein network associated with H2A-TAP (YSC1178-7499158), Htz1-TAP (YSC1178-7502629), and Htz1-TAP swr1Δ (YKB963) purified by mChIP compared with an untagged control (YPH499) and with a traditional Htz1-TAP (YSC1178-7502629) purification (IP). An aliquot of the protein samples purified was also transferred on nitrocellulose membrane for Western blot (WB) analysis against TAP-tagged (B) or histone H3 proteins (C). D, PCR analysis for GAL1 locus (+1039 to +1331) following mChIP showing the presence of chromatin co-purifying with the histones H2A and Htz1p. E, DNA isolated before and after DNA shearing by sonication and micrococcal nuclease S7 (MNase) treatment resolved on a 1.5% agarose gel stained with ethidium bromide. The sample prepared for mChIP usually contains chromatin fragments of 500–1500 base pairs. Results shown are representative of three experiments. F, comparison of histone H2A-associated proteins observed by mChIP and those previously reported in IP-MS experiments with H2A as a bait (literature data from BioGRID) and of their nuclear localization (69).

Fig. 3.

The size of chromatin fragments in whole cell extract determines the magnitude of the protein networks co-purifying with Htz1-TAP by mChIP. The mChIP procedure was performed from the Htz1-TAP strain (YSC1178-7502629) or untagged control strain (YPH499) previously treated with sonication, micrococcal nuclease S7 (MNase) or DNase I (see “Materials and Methods” for details). A, 90% of the protein eluted for each sample was resolved on a NuPAGE 4–12% SDS-PAGE gel and silver-stained. B, phenol/chloroform extractions from the whole cell extracts used in A were resolved on a 1% agarose gel, and the chromatin fragments were visualized by ethidium bromide to define their size. 2.5% of each input and mChIP was analyzed by Western blot (WB) using antibodies against histone H3 (C) or the TAP tag (D).

Fig. 4.

mChIP efficiently purifies chromatin-associated protein networks when non-histone protein are used as baits. A, silver-stained 4–12% NuPAGE gels of the protein networks associated with Lge1-TAP (YSC1178-7503047), Mcm5-TAP (YSC1178-7501832), and Yta7-TAP (YSC1178-7500768). mChIP purifications performed according to the “Materials and Methods” are shown as compared with an untagged control (YPH499). The red arrows indicate the TAP-tagged bait protein. Results shown are representative of three experiments. B, summary of the overlap between the associated proteins observed in the high confidence data set (supplemental Table 2) and each mChIP bait (bottom left triangle) and with the chromatin background or Hta2-TAP high confidence data set removed (top right triangle).

Fig. 5.

mChIP improve the depth and nuclear coverage of protein networks associated with chromatin-bound proteins. Shown are Venn diagrams of unique and shared associated proteins found in two of three mChIP purifications and traditional TAPs as curated in the BioGRID (supplemental Table 2). The localization of each associated proteins was assessed by using the yeast green fluorescent protein fusion localization database (69).

Fig. 6.

Distinct protein networks co-purify by mChIP with histone Htz1-TAP, Lge1-TAP, Mcm5-TAP, and Yta7-TAP (baits are shown in bold text). Associated preys displayed are those reproducibly identified (minimum of two of three mChIPs) that were not identified in Hta2p-TAP mChIP (supplemental Table 2). Osprey was used to display proteins identified in one or more mChIP purifications for each bait protein. Gray edges represent physical interaction observed by mChIP, and node color corresponds to gene ontology annotations as listed in the Saccharomyces Genome Database. Some known protein complexes are denoted by a black circle.