Streamlined discovery of cross-linked chromatin complexes and associated histone modifications by mass spectrometry

Abstract

Posttranslational modifications (PTMs) are key contributors to chromatin function. The ability to comprehensively link specific histone PTMs with specific chromatin factors would be an important advance in understanding the functions and genomic targeting mechanisms of those factors. We recently introduced a cross-linked affinity technique, BioTAP-XL, to identify chromatin-bound protein interactions that can be difficult to capture with native affinity techniques. However, BioTAP-XL was not strictly compatible with similarly comprehensive analyses of associated histone PTMs. Here we advance BioTAP-XL by demonstrating the ability to quantify histone PTMs linked to specific chromatin factors in parallel with the ability to identify nonhistone binding partners. Furthermore we demonstrate that the initially published quantity of starting material can be scaled down orders of magnitude without loss in proteomic sensitivity. We also integrate hydrophilic interaction chromatography to mitigate detergent carryover and improve liquid chromatography-mass spectrometric performance. In summary, we greatly extend the practicality of BioTAP-XL to enable comprehensive identification of protein complexes and their local chromatin environment.chromatin complexes | affinity pulldown | liquid chromatography-mass spectrometry | BioTAP-XL | histone modifications C hromatin encompasses the subset of proteins and RNAs in complex with DNA to form the chromosomes within eukaryotic nuclei. As the primary protein constituents of chromatin, histones organize genomic DNA into nucleosomes and display an extensive array of posttranslational modifications (PTMs) (1). It is increasingly evident that specific histone PTMs are selectively recognized by specific nonhistone proteins that are themselves regulators of many nuclear events such as epigenetic silencing (2, 3). Consequently, defects in these chromatin components often manifest in a number of human diseases (4).As specific interactions between modified histones and nonhistone proteins help distinguish complexes participating in distinct processes, the identification of protein and modification-dependent interactions provides key insights into chromatin biology. Various chromatography and pulldown methods have been developed to identify binding partners. In a typical native pulldown experiment, nuclei from hypotonically lysed cells are digested with micrococcal nuclease (MNase). Extracted chromatin is subjected to immunoprecipitation to isolate the desired complexes. If using a tag such as FLAG, complexes are then competitively eluted and identified. Several drawbacks to native pulldowns as described here are that MNase leaves behind a substantial insoluble nuclear pellet and degrades associating RNAs, that salt extraction of the digested nucleosomes risks dissociation of the tagged bait from its interacting partners, and that most tags do not have sufficient affinity for their respective antibody to permit highly stringent washes. The result is often an incomplete picture of the chromatin...

Expanding the Circuitry of Pluripotency by Selective Isolation of Chromatin-Associated Proteins

“…BioTAP-XL (Zee et al., 2016) and a method coined as “chromatin-interaction protein MS,” confusingly also abbreviated as ChIP-MS (Wang et al., 2013), tag a given protein with protein A or a His tag along with a biotin-acceptor sequence. Although this allows for stringent washing after capture on streptavidin beads, introduction of the tag may alter the functionality or expression level of the protein, while requiring a cloning step that may not be suitable or desirable for all cell types.…”

Bivalent complexes of PRC1 with orthologs of BRD4 and MOZ/MORF target developmental genes in Drosophila

“…For protein identification, streptavidin-agarose complexes were digested with trypsin (Promega) while rotating overnight at 37°C, and peptides were desalted with in-house-constructed C 18 (3M) tips as described previously (Zee et al 2016). Peptides were lyophilized with a vacuum centrifuge and stored in −80°C until liquid chromatography-mass spectrometry (LC-MS) analysis as described previously .…”

“…For histone PTM analysis, streptavidin-bound complexes were derivatized with propionic anhydride (Sigma-Aldrich) prior to trypsin digestion as described previously (Zee et al 2016). Another round of derivatization was performed following trypsin digestion before desalting and LC-MS analysis.…”

Unravelling the biology of chromatin in health and cancer using proteomic approaches

“…Crosslinking allows for the preservation of weak and transient interactions during harsh steps, such as sonication and stringent washing and has been optimized for different applications [16, 17, 18, 19]. From this, numerous methods have been developed that allow for proteomic analysis at specific genomic regions.…”