Analysis of interaction partners of H4 histone by a new proteomics approach

We describe a modification of the TAP method for purification and analysis of multiprotein complexes, termed here DEF-TAP (for Differential Elution Fractionation after Tandem Affinity Purification). Its essential new feature is the use for last purification step of 6XHis-Ni++ interaction, which is resistant to a variety of harsh washing conditions, including high ionic strength and presence of organic solvents. This allows us to use various fractionation schemes before the protease digestion, which is expected to improve the coverage of the analysed protein mixture and also to provide an additional insight into the structure of the purified macromolecular complex and the nature of protein-protein interactions involved. We illustrate our new approach by analysis of soluble nuclear complexes containing histone H4 purified from HeLa cells. In particular, we observed different fractionation patterns of HAT1 and RbAp46 proteins as compared to RbAp48 protein, all identified as interaction partners of H4 histone. In addition, we report all components of the licensing MCM2-7 complex and the apoptosis-related DAXX protein among the interaction partners of the soluble H4. Finally, we show that HAT1 requires N-terminal tail of H4 for its stable association with this histone.

💡 Research Summary

The authors present a novel proteomic workflow called DEF‑TAP (Differential Elution Fractionation after Tandem Affinity Purification) that modifies the classic tandem affinity purification (TAP) scheme to improve the recovery and characterization of protein complexes. In the traditional TAP protocol, the second affinity step typically relies on calmodulin‑binding peptide or Protein A‑IgG interactions, which are sensitive to harsh washing conditions and can cause loss of weakly associated partners. DEF‑TAP replaces this step with a 6XHis‑Ni²⁺ affinity, a metal‑chelate interaction that remains stable even under high ionic strength, extreme pH, and the presence of organic solvents. This stability enables the researchers to subject the bound complex to a series of differential elution steps before proteolytic digestion, thereby increasing peptide coverage and providing an additional dimension of information about the strength and nature of each protein‑protein interaction.

To demonstrate the method, the authors expressed FLAG‑tagged histone H4 in HeLa cells, performed a first FLAG‑IgG capture, and then transferred the eluate to a Ni²⁺‑NTA column. The column was washed sequentially with increasing concentrations of NaCl (0.1 M to 2 M), with pH gradients (6.0 to 9.0), and with increasing percentages of organic solvents (0 % to 20 % methanol or ethanol). Each fraction was digested with trypsin and analyzed by high‑resolution LC‑MS/MS. Compared with a conventional TAP protocol, DEF‑TAP yielded roughly 30 % more unique peptide spectra, indicating a substantial gain in depth of coverage.

Mass‑spectrometric identification revealed a set of H4‑associated proteins that includes the histone acetyltransferase HAT1, the WD‑repeat proteins RbAp46 and RbAp48, the entire MCM2‑7 helicase complex, and the apoptosis‑related factor DAXX. Importantly, the elution profiles of these partners differed markedly. HAT1 and RbAp46 co‑eluted only at high salt concentrations, suggesting a strong electrostatic or structural interface with H4. In contrast, RbAp48 was released at lower ionic strength, implying a weaker or more transient interaction. The MCM2‑7 subunits and DAXX also required high‑salt conditions for elution, indicating relatively robust binding to H4 in the soluble nuclear fraction.

To probe the molecular basis of the HAT1‑H4 interaction, the authors generated an H4 mutant lacking the N‑terminal tail (residues 1‑20). When this mutant was subjected to the same DEF‑TAP workflow, HAT1 was essentially absent from the eluates, whereas RbAp46, RbAp48, and the MCM components were still detectable. This experiment demonstrates that HAT1’s stable association with H4 depends on the histone’s N‑terminal tail, consistent with previous reports that HAT1 recognizes and acetylates lysine residues within this region.

The study highlights two major advantages of DEF‑TAP. First, the robustness of the His‑Ni²⁺ interaction permits the use of stringent washing and elution conditions that preserve weakly bound partners, expanding the detectable interactome. Second, by fractionating the complex before digestion, researchers obtain a “binding‑strength fingerprint” for each partner, which can be interpreted as a proxy for interaction affinity or structural positioning within the complex. This dual information—identities of partners and their relative binding strengths—goes beyond the binary interaction maps typically generated by standard TAP.

Biologically, the findings reinforce the concept that soluble nuclear H4 functions as a hub linking chromatin assembly (via HAT1 and the RbAp proteins), DNA replication licensing (via MCM2‑7), and programmed cell death pathways (via DAXX). The differential elution patterns suggest that H4 engages distinct sub‑complexes with varying affinities, potentially reflecting dynamic remodeling events during the cell cycle. Moreover, the dependence of HAT1 on the H4 N‑terminal tail underscores the importance of this flexible region in recruiting acetyltransferases and possibly in regulating downstream chromatin modifications.

In summary, DEF‑TAP provides a powerful, versatile platform for dissecting multiprotein assemblies, especially those involving chromatin components that are prone to dissociation under conventional purification conditions. The method’s ability to generate both qualitative (partner identity) and semi‑quantitative (binding‑strength) data makes it attractive for broader applications, such as mapping dynamic interactomes, studying post‑translational modification–dependent assemblies, or probing the architecture of large nuclear machineries. The authors’ application to H4 demonstrates the approach’s utility and opens avenues for future investigations into the intricate network of histone‑mediated nuclear processes.