|CHROMATIN REMODELING COMPLEXES
||31759698, 32188938, 32001526
||31210637, 29664398, 28767641
||Arp5 (yeast, human)
||30759380, 30981630, 30923167, 30765112
||SAGA deubiquitinase subunit
||E3 ubiquitin ligase subunit
|Sir3 BAH (Sir)
||Chromatin reader domain
||Recruits, activates Ran GTPase
||Centromere binding protein
||Nuclear pore complex
|APC/C, BUBR1, BUB3, CDC20
||Anaphase Promoting Complex and associated proteins
||Karposi’s sarcoma-associated herpesvirus
||Prototype foamy virus (PFV)
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 DNA12,17-19. 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 expression20,21.
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:
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!