The Nucleosome Acidic Patch: A Master Landing Dock for Chromatin Regulators

The nucleosome is the repeating unit of chromatin and serves as the physiological substrate for diverse chromatin interacting proteins, including reader proteins, chromatin remodeling complexes, and modifying enzymes. Despite their widespread and often distinct functional outputs, there are several common themes that have emerged from recent biochemical studies. The most striking observation is that a large number of chromatin-associated proteins and/or complexes bind the same region of the histone core: the so-called nucleosome acidic patch^1-3. Here, we break down the fundamentals of the nucleosome acidic patch and dive into a few interesting studies that illustrate key roles for this region in chromatin regulator binding.

To enable chromatin research, EpiCypher has launched a new line of recombinant nucleosomes carrying defined mutations in the acidic patch.

A primer on the nucleosome acidic patch

The acidic patch is a negatively charged region of the nucleosome core, located at the interface of histones H2A and H2B.

For a bit more context, the nucleosome is composed of a histone octamer core – one H3/H4 tetramer and two H2A/H2B dimers – wrapped with DNA⁴. The N-terminal tails of each histone are accessible for post-translational modification (i.e. PTMs), and are historically shown as protruding from the nucleosome globular core⁵. The acidic patch resides on the surface of this globular core and is formed by structurally adjacent, negatively charged amino acids from H2A and H2B. Unlike amino acids on the histone tails, which are decorated with PTMs (e.g. methylation, acylation, etc.), these residues are unmodified.

This small patch of amino acids has emerged as a central nucleosome binding “hub,” due to its role in multivalent chromatin interactions as well as its impact on higher-order chromatin structures³. For instance, the acidic patch has been shown to interact with histone H4 tails on adjacent nucleosomes to support chromatin compaction and gene silencing⁴. This interaction is disrupted when H4 tails are acetylated, providing a mechanistic link between histone acetylation and gene expression⁶.

The term multivalency refers to proteins and/or complexes that make multiple contacts with the nucleosome, including histone PTMs, nucleosomal DNA, and/or the histone core. This is particularly important for chromatin remodeling enzymes and other large complexes, which require several binding interactions to stabilize enzymatic and/or other downstream processes. The concept of multivalency in association with the acidic patch has been demonstrated for many proteins, as outlined in Table 1, and includes subunits from multiple chromatin remodeling complexes (e.g. ISWI, SWI/SNF, INO80)^7-15.

But is the acidic patch really a global feature of chromatin binding? How does it compare to other regions of the nucleosome?

Below we detail a few recent studies that shed light on this subject, and underscore how EpiCypher’s new line of acidic patch mutant nucleosomes can be used for advanced chromatin research.

Table 1: Summary of proteins that engage nucleosome by binding the H2A/H2B acidic patch.

The nucleosome acidic patch is essential for many chromatin binding proteins

The central requirement for the nucleosome acidic patch has been hinted at in many publications, but a direct head-to-head comparison of how different histone regions contribute to chromatin binding has been challenging. To test the role of the acidic patch and other conserved nucleosome surface regions as regulators of chromatin binding, Dr. Robert McGinty’s group developed a comprehensive and unbiased affinity proteomics screening approach.

Briefly, they synthesized a set of recombinant nucleosomes carrying histone mutations targeted to various surface regions of the nucleosome core (nucleosome “disk”). Each mutant was used for affinity pulldowns with mouse embryonic stem cell (ESC) nuclear lysates followed by mass spectrometry identification of bound proteins¹⁶. Wild-type nucleosomes and nucleosomes lacking N-terminal histone tails (i.e. “tailless” nucleosomes) were included as controls.

The results of this screen led to several interesting conclusions:

• The nucleosome acidic patch influences >50% of all chromatin interactions. The acidic patch mutant nucleosome recovered substantially less total protein compared to all other mutant and control nucleosomes, strongly supporting its role as a major landing pad for chromatin regulators.
• Conserved residues in histones H3 and H4 are not required for nucleosome binding. In contrast to the results from acidic patch mutants, mutations in highly conserved regions of H3 and H4 only impacted a small number of chromatin interactions. It isn’t clear why, although it is possible that these sites are playing important, yet nonessential and/or redundant roles. Nevertheless, nucleosomes carrying these H3 / H4 mutations would make excellent controls for acidic patch mutants and enable scientists to uncover potential novel functions for such highly conserved regions.
• Histone tails shield the nucleosome core from nonspecific interactions. Tailless nucleosomes recovered more protein vs. wildtype nucleosomes, demonstrating an increase in nonspecific binding to linker DNA and/or the histone core. This is in agreement with NMR studies, which suggest that the histone tails are collapsed on the globular core via interactions with linker DNA^12,17-19. These interactions can be weakened via modification of histone tails (i.e. acetylation), thereby increasing accessibility.

In addition, this work highlights the importance of working with defined nucleosome substrates vs. modified histone peptides when characterizing chromatin interactions. Histone peptides only represent a single linear epitope, and fail to model the complex structure of chromatin, such as histone tail interactions with linker DNA and/or the histone octamer core.

The nucleosome context is crucial when studying acidic patch driven binding mechanisms, which are often multivalent and greatly influenced by the “histone code.” The histone code is a molecular language formed by distinct combinations of histone PTMs and other modifications (e.g. DNA methylation), which together regulate effector binding and downstream processes, such as gene expression^20,21.

For instance, the Sir3 BAH domain makes 5 distinct contacts with the nucleosome, including the acidic patch and histone H3 and H4 tails²². Based on this crystal structure, methylation of H3K79 or acetylation of H4K16 is predicted to block binding. This early study demonstrated how the acidic patch can stabilize and support multivalent chromatin interactions and provided significant insights towards the function of the histone code.

The nucleosome acidic patch regulates chromatin remodeling complexes and is implicated in disease

The nucleosome acidic patch is associated with the function of chromatin remodeling complexes, including the multi-subunit mammalian SWI/SNF remodeling complex. Mutations in SWI/SNF proteins occur in approximately 20% of all cancers, and are also frequently altered in intellectual disorders, such as Coffin-Siris syndrome (CSS)²³. However, the role of the acidic patch in these disease-driving mechanisms is only beginning to be understood.

A 2019 paper from Dr. Cigall Kadoch’s lab at Dana Farber examined the impact of mutations in the C-terminal domain of SMARCB1, one of the core components of the SWI/SNF remodeling complex¹⁴. Notably, SMARCB1 is not the ATPase remodeling enzyme, but is thought to stabilize and support SWI/SNF complex interactions on chromatin²⁴. Mutations in SMARCB1 correlate with severe forms of CSS and intellectual disability, and are also common in malignant rhabdoid tumors, an aggressive cancer that typically occurs in children²⁵.

As part of this work, researchers used several biochemical and structural methods to demonstrate that wildtype SMARCB1 directly binds the acidic patch. The disease-associated mutations in SMARCB1 abrogated this binding, and also inhibited remodeling activity by the SWI/SNF complex. Of note, nucleosome remodeling activity in this study was quantified by restriction enzyme accessibility (REA) assays using EpiCypher’s EpiDyne® chromatin remodeling substrates. Importantly, mutations in the acidic patch replicated this mechanism, showing the importance of SMARCB1 – acidic patch interactions to SWI/SNF remodeling activity.

Acidic patch mutant Nuc substrates: now available!

To empower additional research on acidic patch binding mechanisms and roles in disease, EpiCypher has launched a new line of recombinant nucleosomes carrying defined mutations in the H2A/H2B acidic patch, as well as important controls indicated by Skrajna et al. (see above)¹⁶. This line of Mutant Nucs includes:

• H2AE61A Acidic Patch Mutant Nucleosomes
• H2AE92K Acidic Patch Mutant Nucleosomes
• H2BE105A, E113A Acidic Patch Mutant Nucleosomes
• Fully tailless nucleosomes
• Nucleosomes with defined truncations in histone H3 and H4 (in development)
• Nucleosomes with mutations in conserved residues of histone H3 and H4 (in development)

Mutant Nuc substrates can be customized for your specific experimental needs upon request, or combined with our existing platforms, including EpiDyne® chromatin remodeling assays and services. Application of Acidic Patch Mutant Nucs to the EpiDyne® platform will provide a powerful system for the study of chromatin remodeling enzymes, as many of these complexes rely on acidic patch interactions (Table 1). To complement these assays, EpiCypher also offers full-length recombinant SMARCA2 and SMARCA4 SWI/SNF remodeling enzymes, enabling quantitative research against these valuable drug targets.

Further, our Acidic Patch Mutant Nucs will be integrated into our dCypher™ nucleosome panels and chromatin interaction discovery services, providing comprehensive analysis of your protein or enzyme of interest against a diverse set of histone PTMs, variants, and mutations, all within a nucleosome context. If you have a specific project in mind, let us know – we are here to help!