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

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 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-bound complex. BioTAP-XL is a technique that complements native pulldowns. BioTAP-XL uses formaldehyde to inactivate endogenous enzymes and to cross-link labile interactions with the bait, sonication to solubilize the genomic chromatin content, and high affinity tags to capture the bait and its associated factors (K_d = 10⁻⁹ and 10⁻¹⁵ M for the bipartite BioTAP tag compared with 10⁻⁸ M for the FLAG tag). Thus, BioTAP-XL captures interactions that may be inaccessible or disrupted using native pulldowns.BioTAP-XL has successfully identified key interactors of the male-specific lethal (MSL) dosage compensation and heterochromatin protein (HP1a) complexes in Drosophila (5) and revealed novel components of the Polycomb Repressive Complex 2 in human tissue culture cells (6) and fruit flies (7). Despite the aforementioned advantages, BioTAP-XL suffers from several limitations. First, the cell number requirement far exceeds the amounts typically used in standard affinity experiments, namely with 10⁸ cells for native pulldowns (8) compared with 10¹⁰ cells for BioTAP pulldown (9). Second, the high detergent concentrations used during the procedure risks interfering with liquid chromatography mass spectrometry (LC-MS). Third, our analysis of the associated histone PTMs is complicated by both the nearly irreversible binding of the tagged complexes to streptavidin and the inability to obtain histone peptides of comparable ionization efficiencies across modified states using trypsin alone.We sought to address these limitations using Drosophila and human cells expressing BioTAP-tagged factors. To demonstrate scalability, we performed parallel tandem affinity pulldowns of the same bait across various initial chromatin amounts. We recovered the core interactors of MSL3 at even the lowest tested amount of Drosophila cells. To improve detergent removal, we compared a sample processed either with conventional spin columns or hydrophilic interaction chromatography (HILIC). We observed significant improvement in LC-MS quality of the MSL3 elutions prepared using HILIC over spin columns. To facilitate histone modification analysis, we derivatized the associated histones directly on the streptavidin beads. We successfully quantified histone modifications enriched with MSL3 and HP1a from Drosophila cells and with chromobox homolog 4 (CBX4) and bromodomain-containing protein 4 (BRD4) from human cells. In short, this report streamlines BioTAP-XL for biochemically characterizing chromatin complexes and their associated histone modifications.