Nucleosome Acidic Patch Research Articles

Phosphorylation of the HMGN1 Nucleosome Binding Domain Decreases Helicity and Interactions with the Acidic Patch.

Intrinsically disordered proteins are abundant in the nucleus and are prime sites for posttranslational modifications that modulate transcriptional regulation. Lacking a defined three-dimensional structure, intrinsically disordered proteins populate an ensemble of several conformational states, which are dynamic and often altered by posttranslational modifications, or by binding to interaction partners. Although there is growing appreciation for the role that intrinsically disordered regions have in regulating protein-protein interactions, we still have a poor understanding of how to determine conformational population shifts, their causes under various conditions, and how to represent and model conformational ensembles. Here, we study the effects of serine phosphorylation in the nucleosome-binding domain of an intrinsically disordered protein - HMGN1 - using NMR spectroscopy, circular dichroism and modelling of protein complexes. We show that phosphorylation induces local conformational changes in the peptide backbone and decreases the helical propensity of the nucleosome binding domain. Modelling studies using AlphaFold3 suggest that phosphorylation disrupts the interface between HMGN1 and the nucleosome acidic patch, but that the models over-predict helicity in comparison to experimental data. These studies help us to build a picture of how posttranslational modifications might shift the conformational populations of disordered regions, alter access to histones, and regulate chromatin compaction.

Cancer-associated DNA hypermethylation of Polycomb targets requires DNMT3A dual recognition of histone H2AK119 ubiquitination and the nucleosome acidic patch.

During tumor development, promoter CpG islands that are normally silenced by Polycomb repressive complexes (PRCs) become DNA-hypermethylated. The molecular mechanism by which de novo DNA methyltransferase(s) [DNMT(s)] catalyze CpG methylation at PRC-regulated regions remains unclear. Here, we report a cryo-electron microscopy structure of the DNMT3A long isoform (DNMT3A1) amino-terminal region in complex with a nucleosome carrying PRC1-mediated histone H2A lysine-119 monoubiquitination (H2AK119Ub). We identify regions within the DNMT3A1 amino terminus that bind H2AK119Ub and the nucleosome acidic patch. This bidentate interaction is required for effective DNMT3A1 engagement with H2AK119Ub-modified chromatin in cells. Further, aberrant redistribution of DNMT3A1 to Polycomb target genes recapitulates the cancer-associated DNA hypermethylation signature and inhibits their transcriptional activation during cell differentiation. This effect is rescued by disruption of the DNMT3A1-acidic patch interaction. Together, our analyses reveal a binding interface critical for mediating promoter CpG island DNA hypermethylation, a major molecular hallmark of cancer.

Ubiquitinated histone H2B as gatekeeper of the nucleosome acidic patch.

Monoubiquitination of histones H2B-K120 (H2BK120ub) and H2A-K119 (H2AK119ub) play opposing roles in regulating transcription and chromatin compaction. H2BK120ub is a hallmark of actively transcribed euchromatin, while H2AK119ub is highly enriched in transcriptionally repressed heterochromatin. Whereas H2BK120ub is known to stimulate the binding or activity of various chromatin-modifying enzymes, this post-translational modification (PTM) also interferes with the binding of several proteins to the nucleosome H2A/H2B acidic patch via an unknown mechanism. Here, we report cryoEM structures of an H2BK120ub nucleosome showing that ubiquitin adopts discrete positions that occlude the acidic patch. Molecular dynamics simulations show that ubiquitin remains stably positioned over this nucleosome region. By contrast, our cryoEM structures of H2AK119ub nucleosomes show ubiquitin adopting discrete positions that minimally occlude the acidic patch. Consistent with these observations, H2BK120ub, but not H2AK119ub, abrogates nucleosome interactions with acidic patch-binding proteins RCC1 and LANA, and single-domain antibodies specific to this region. Our results suggest a mechanism by which H2BK120ub serves as a gatekeeper to the acidic patch and point to distinct roles for histone H2AK119 and H2BK120 ubiquitination in regulating protein binding to nucleosomes.

In silico screening identifies SHPRH as a novel nucleosome acidic patch interactor.

https://predictomes.org/view/acidicpatch.

Dual engagement of the nucleosomal acidic patches is essential for deposition of histone H2A.Z by SWR1C

The yeast SWR1C chromatin remodeling enzyme catalyzes the ATP-dependent exchange of nucleosomal histone H2A for the histone variant H2A.Z, a key variant involved in a multitude of nuclear functions. How the 14-subunit SWR1C engages the nucleosomal substrate remains largely unknown. Studies on the ISWI, CHD1, and SWI/SNF families of chromatin remodeling enzymes have demonstrated key roles for the nucleosomal acidic patch for remodeling activity, however a role for this nucleosomal epitope in nucleosome editing by SWR1C has not been tested. Here, we employ a variety of biochemical assays to demonstrate an essential role for the acidic patch in the H2A.Z exchange reaction. Utilizing asymmetrically assembled nucleosomes, we demonstrate that the acidic patches on each face of the nucleosome are required for SWR1C-mediated dimer exchange, suggesting SWR1C engages the nucleosome in a ‘pincer-like’ conformation, engaging both patches simultaneously. Loss of a single acidic patch results in loss of high affinity nucleosome binding and nucleosomal stimulation of ATPase activity. We identify a conserved arginine-rich motif within the Swc5 subunit that binds the acidic patch and is key for dimer exchange activity. In addition, our cryoEM structure of a Swc5–nucleosome complex suggests that promoter proximal, histone H2B ubiquitylation may regulate H2A.Z deposition. Together these findings provide new insights into how SWR1C engages its nucleosomal substrate to promote efficient H2A.Z deposition.

Dual engagement of the nucleosomal acidic patches is essential for deposition of histone H2A.Z by SWR1C.

The yeast SWR1C chromatin remodeling enzyme catalyzes the ATP-dependent exchange of nucleosomal histone H2A for the histone variant H2A.Z, a key variant involved in a multitude of nuclear functions. How the 14-subunit SWR1C engages the nucleosomal substrate remains largely unknown. Studies on the ISWI, CHD1, and SWI/SNF families of chromatin remodeling enzymes have demonstrated key roles for the nucleosomal acidic patch for remodeling activity, however a role for this nucleosomal epitope in nucleosome editing by SWR1C has not been tested. Here, we employ a variety of biochemical assays to demonstrate an essential role for the acidic patch in the H2A.Z exchange reaction. Utilizing asymmetrically assembled nucleosomes, we demonstrate that the acidic patches on each face of the nucleosome are required for SWR1C-mediated dimer exchange, suggesting SWR1C engages the nucleosome in a 'pincer-like' conformation, engaging both patches simultaneously. Loss of a single acidic patch results in loss of high affinity nucleosome binding and nucleosomal stimulation of ATPase activity. We identify a conserved arginine-rich motif within the Swc5 subunit that binds the acidic patch and is key for dimer exchange activity. In addition, our cryoEM structure of a Swc5-nucleosome complex suggests that promoter proximal, histone H2B ubiquitylation may regulate H2A.Z deposition. Together these findings provide new insights into how SWR1C engages its nucleosomal substrate to promote efficient H2A.Z deposition.

Structural basis of the histone ubiquitination read–write mechanism of RYBP–PRC1

Histone H2A monoubiquitination (H2Aub1) by the PRC1 subunit RING1B entails a positive feedback loop, mediated by the RING1B-interacting protein RYBP. We uncover that human RYBP–PRC1 binds unmodified nucleosomes via RING1B but H2Aub1-modified nucleosomes via RYBP. RYBP interactions with both ubiquitin and the nucleosome acidic patch create the high binding affinity that favors RYBP- over RING1B-directed PRC1 binding to H2Aub1-modified nucleosomes; this enables RING1B to monoubiquitinate H2A in neighboring unmodified nucleosomes.

Mechanism of histone H2B monoubiquitination by Bre1.

Monoubiquitination of histone H2B-K120/123 plays several roles in regulating transcription, DNA replication and the DNA damage response. The structure of a nucleosome in complex with the dimeric RING E3 ligase Bre1 reveals that one RING domain binds to the nucleosome acidic patch, where it can position the E2 ubiquitin conjugating enzyme Rad6, while the other RING domain contacts the DNA. Comparisons with H2A-specific E3 ligases suggest a general mechanism of tuning histone specificity via the non-E2-binding RING domain.

UBA80 and UBA52 fine-tune RNF168-dependent histone ubiquitination and DNA repair

The ubiquitin signaling pathway is crucial for the DNA damage response pathway. More specifically, RNF168 is integral in regulating DNA repair proteins at damaged chromatin. However, the detailed mechanism by which RNF168 is regulated in cells is not fully understood. Here, we identify the ubiquitin-ribosomal fusion proteins UBA80 (also known as RPS27A) and UBA52 (also known as RPL40) as interacting proteins for H2A/H2AX histones and RNF168. Both UBA80 and UBA52 are recruited to laser-induced micro-irradiation DNA damage sites and are required for DNA repair. Ectopic expression of UBA80 and UBA52 inhibits RNF168-mediated H2A/H2AX ubiquitination at K13/15 and impairs 53BP1 recruitment to DNA lesions. Mechanistically, the C-terminal ribosomal fragments of UBA80 and UBA52, S27A and L40, respectively, limit RNF168-nucleosome engagement by masking the regulatory acidic residues at E143/E144 and the nucleosome acidic patch. Together, our results reveal that UBA80 and UBA52 antagonize the ubiquitination signaling pathway and fine-tune the spatiotemporal regulation of DNA repair proteins at DNA damage sites.

Development of multifunctional synthetic nucleosomes to interrogate chromatin-mediated protein interactions

Various proteins bind to chromatin to regulate DNA and its associated processes such as replication, transcription, and damage repair. The identification and characterization of these chromatin-associating proteins remain a challenge, as their interactions with chromatin often occur within the context of the local nucleosome or chromatin structure, which makes conventional peptide-based strategies unsuitable. Here, we developed a simple and robust protein labeling chemistry to prepare synthetic multifunctional nucleosomes that carry a photoreactive group, a biorthogonal handle, and a disulfide moiety to examine chromatin-protein interactions in a nucleosomal context. Using the prepared protein- and nucleosome-based photoaffinity probes, we examined a number of protein-protein and protein-nucleosome interactions. In particular, we (i) mapped the binding sites for the HMGN2-nucleosome interaction, (ii) provided the evidence for transition between the active and poised states of DOT1L in recognizing H3K79 within the nucleosome, and (iii) identified OARD1 and LAP2α as nucleosome acidic patch-associating proteins. This study provides powerful and versatile chemical tools for interrogating chromatin-associating proteins.

Structural basis of paralog-specific KDM2A/B nucleosome recognition.

The nucleosome acidic patch is a major interaction hub for chromatin, providing a platform for enzymes to dock and orient for nucleosome-targeted activities. To define the molecular basis of acidic patch recognition proteome wide, we performed an amino acid resolution acidic patch interactome screen. We discovered that the histone H3 lysine 36 (H3K36) demethylase KDM2A, but not its closely related paralog, KDM2B, requires the acidic patch for nucleosome binding. Despite fundamental roles in transcriptional repression in health and disease, the molecular mechanisms governing nucleosome substrate specificity of KDM2A/B, or any related JumonjiC (JmjC) domain lysine demethylase, remain unclear. We used a covalent conjugate between H3K36 and a demethylase inhibitor to solve cryogenic electron microscopy structures of KDM2A and KDM2B trapped in action on a nucleosome substrate. Our structures show that KDM2-nucleosome binding is paralog specific and facilitated by dynamic nucleosomal DNA unwrapping and histone charge shielding that mobilize the H3K36 sequence for demethylation.

Antecedent chromatin organization determines cGAS recruitment to ruptured micronuclei

Micronuclei (MN) are cytosolic bodies that sequester acentric fragments or mis-segregated chromosomes from the primary nucleus. Spontaneous rupture of the MN envelope allows recognition by the viral receptor cyclic GMP-AMP synthase (cGAS), initiating interferon signaling downstream of DNA damage. Here, we demonstrate that MN rupture is permissive but not sufficient for cGAS localization. Chromatin characteristics such as histone 3, lysine 79 dimethylation (H3K79me2) are present in the nucleus before DNA damage, retained in ruptured MN, and regulate cGAS recruitment. cGAS is further responsive to dynamic intra-MN processes occurring prior to rupture, including transcription. MN chromatin tethering via the nucleosome acidic patch is necessary for cGAS-dependent interferon signaling. Our data suggest that both damage-antecedent nuclear chromatin status and MN-contained chromatin organizational changes dictate cGAS recruitment and the magnitude of the cGAS-driven interferon cascade. Our work defines MN as integrative signaling hubs for the cellular response to genotoxic stress.

Time Resolved-Fluorescence Resonance Energy Transfer platform for quantitative nucleosome binding and footprinting.

Quantitative analysis of chromatin protein-nucleosome interactions is essential to understand regulation of genome-templated processes. However, current methods to measure nucleosome interactions are limited by low throughput, low signal-to-noise, and/or the requirement for specialized instrumentation. Here, we report a Lanthanide Chelate Excite Time-Resolved Fluorescence Resonance Energy Transfer (LANCE TR-FRET) assay to efficiently quantify chromatin protein-nucleosome interactions. The system makes use of commercially available reagents, offers robust signal-to-noise with minimal sample requirements, uses a conventional fluorescence microplate reader, and can be adapted for high-throughput workflows. We determined the nucleosome-binding affinities of several chromatin proteins and complexes, which are consistent with measurements obtained through orthogonal biophysical methods. We also developed a TR-FRET competition assay for high-resolution footprinting of chromatin protein-nucleosome interactions. Finally, we set up a TR-FRET competition assay using the LANA peptide to quantitate nucleosome acidic patch binding. We applied this assay to establish a proof-of-principle for regulation of nucleosome acidic patch binding by methylation of chromatin protein arginine anchors. Overall, our TR-FRET assays allow facile, high-throughput quantification of chromatin interactions and are poised to complement mechanistic chromatin biochemistry, structural biology, and drug discovery programs.

Multivalent DNA and nucleosome acidic patch interactions specify VRK1 mitotic localization and activity.

A key role of chromatin kinases is to phosphorylate histone tails during mitosis to spatiotemporally regulate cell division. Vaccinia-related kinase 1 (VRK1) is a serine–threonine kinase that phosphorylates histone H3 threonine 3 (H3T3) along with other chromatin-based targets. While structural studies have defined how several classes of histone-modifying enzymes bind to and function on nucleosomes, the mechanism of chromatin engagement by kinases is largely unclear. Here, we paired cryo-electron microscopy with biochemical and cellular assays to demonstrate that VRK1 interacts with both linker DNA and the nucleosome acidic patch to phosphorylate H3T3. Acidic patch binding by VRK1 is mediated by an arginine-rich flexible C-terminal tail. Homozygous missense and nonsense mutations of this acidic patch recognition motif in VRK1 are causative in rare adult-onset distal spinal muscular atrophy. We show that these VRK1 mutations interfere with nucleosome acidic patch binding, leading to mislocalization of VRK1 during mitosis, thus providing a potential new molecular mechanism for pathogenesis.

Acidic patch histone mutations and their effects on nucleosome remodeling.

Structural and biochemical studies have identified a histone surface on each side of the nucleosome disk termed 'the nucleosome acidic patch' that acts as a regulatory hub for the function of numerous nuclear proteins, including ATP-dependent chromatin complexes (remodelers). Four major remodeler subfamilies, SWI/SNF, ISWI, CHD, and INO80, have distinct modes of interaction with one or both nucleosome acidic patches, contributing to their specific remodeling outcomes. Genome-wide sequencing analyses of various human cancers have uncovered high-frequency mutations in histone coding genes, including some that map to the acidic patch. How cancer-related acidic patch histone mutations affect nucleosome remodeling is mainly unknown. Recent advances in in vitro chromatin reconstitution have enabled access to physiologically relevant nucleosomes, including asymmetric nucleosomes that possess both wild-type and acidic patch mutant histone copies. Biochemical investigation of these substrates revealed unexpected remodeling outcomes with far-reaching implications for alteration of chromatin structure. This review summarizes recent findings of how different remodeler families interpret wild-type and mutant acidic patches for their remodeling functions and discusses models for remodeler-mediated changes in chromatin landscapes as a consequence of acidic patch mutations.

Targeting the Nucleosome Acidic Patch by Viral Proteins: Two Birds with One Stone?

ABSTRACTThe past decade illuminated the H2A-H2B acidic patch as a cornerstone for both nucleosome recognition and chromatin structure regulation. Higher-order folding of chromatin arrays is mediated by interactions of histone H4 tail with an adjacent nucleosome acidic patch. Dynamic chromatin folding ensures a proper regulation of nuclear functions fundamental to cellular homeostasis. Many cellular factors have been shown to act on chromatin by tethering nucleosomes via an arginine anchor binding to the acidic patch. This tethering mechanism has also been described for several viral proteins. In this minireview, we will discuss the structural basis for acidic patch engagement by viral proteins and the implications during respective viral infections. We will also discuss a model in which acidic patch occupancy by these non-self viral proteins alters the local chromatin state by preventing H4 tail-mediated higher-order chromatin folding.

DOT1L activity in leukemia cells requires interaction with ubiquitylated H2B that promotes productive nucleosome binding.

SUMMARYDOT1L methylates histone H3 lysine 79 during transcriptional elongation and is stimulated by ubiquitylation of histone H2B lysine 120 (H2BK120ub) in a classical trans-histone crosstalk pathway. Aberrant genomic localization of DOT1L is implicated in mixed lineage leukemia (MLL)-rearranged leukemias, an aggressive subset of leukemias that lacks effective targeted treatments. Despite recent atomic structures of DOT1L in complex with H2BK120ub nucleosomes, fundamental questions remain as to how DOT1L-ubiquitin and DOT1L-nucleosome acidic patch interactions observed in these structures contribute to nucleosome binding and methylation by DOT1L. Here, we combine bulk and single-molecule biophysical measurements with cancer cell biology to show that ubiquitin and cofactor binding drive conformational changes to stimulate DOT1L activity. Using structure-guided mutations, we demonstrate that ubiquitin and nucleosome acidic patch binding by DOT1L are required for cell proliferation in the MV4; 11 leukemia model, providing proof of principle for MLL targeted therapeutic strategies.

Mapping the electrostatic potential of the nucleosome acidic patch

The nucleosome surface contains an area with negative electrostatic potential known as the acidic patch, which functions as a binding platform for various proteins to regulate chromatin biology. The dense clustering of acidic residues may impact their effective pKa and thus the electronegativity of the acidic patch, which in turn could influence nucleosome-protein interactions. We here set out to determine the pKa values of residues in and around the acidic patch in the free H2A-H2B dimer using NMR spectroscopy. We present a refined solution structure of the H2A-H2B dimer based on intermolecular distance restraints, displaying a well-defined histone-fold core. We show that the conserved histidines H2B H46 and H106 that line the acidic patch have pKa of 5.9 and 6.5, respectively, and that most acidic patch carboxyl groups have pKa values well below 5.0. For H2A D89 we find strong evidence for an elevated pKa of 5.3. Our data establish that the acidic patch is highly negatively charged at physiological pH, while protonation of H2B H106 and H2B H46 at slightly acidic pH will reduce electronegativity. These results will be valuable to understand the impact of pH changes on nucleosome-protein interactions in vitro, in silico or in vivo.

Abstract 2082: Deciphering the nucleosome acidic patch binding at amino acid resolution

Abstract In all eukaryotic cells, nucleosomes provide basic organization of genomic DNA and serve as signaling hubs for gene expression, DNA replication and DNA damage repair. These genome-templated processes are tuned by chromatin enzymes that introduce chemical modifications to nucleosomes and control their positioning and occupancy. Mutations in genes encoding chromatin enzymes are highly correlated with numerous cancer types. A thorough exploration of how these chromatin enzymes act on their physiologic target, the nucleosome, is critical to understanding abnormal DNA methylation, histone posttranslational modifications, and chromatin composition, leading to cancer. In our recent nucleosome interactome proteomic screen, we demonstrated that the vast majority of nuclear proteins require a group of acidic residues on the nucleosome disk surface to interact with nucleosomes. This acidic patch and an adjacent region were recognized by over 50% and 18% of nuclear proteins, respectively, while almost half of the nucleosome disk surface contributed only minimally to nucleosome recognition. Overall, we paired about 300 proteins representing diverse nuclear functions including DNA methylation (DNMT1 and 3, Tet1), histone modification (HAT complexes, kinases), nucleosome remodeling (SWI/SNF), DNA repair (MRN complex), DNA replication (ORC complex), RNA processing, and cell cycle progression (APC/C) with specific nucleosome surface requirements. We have now repeated our screen at amino acid resolution using a library of fifteen nucleosomes containing single point mutations in and around the acid patch, including potential histone oncomutations identified to be overrepresented in cancers. Using our nucleosome library, we preformed pulldowns from human embryonic kidney 293 cell nuclear lysates to comprehensively evaluate the role of single histone residues in nucleosome recognition. Overall, this study dissects the primary nucleosome binding hot-spot at high resolution and provide a robust resource for further exploration of the nucleosome interactome in normal and cancer cell biology. Citation Format: Aleksandra Skrajna, Holly Simmons, Charlotte Smith, Jeanne-Marie E. Mcpherson, Dennis Goldfarb, Robert K. McGinty. Deciphering the nucleosome acidic patch binding at amino acid resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2082.

Interphase epichromatin: last refuge for the 30-nm chromatin fiber?

"Interphase epichromatin" describes the surface of chromatin located adjacent to the interphase nuclear envelope. It was discovered in 2011 using a bivalent anti-nucleosome antibody (mAb PL2-6), now known to be directed against the nucleosome acidic patch. The molecular structure of interphase epichromatin is unknown, but is thought to be heterochromatic with a high density of "exposed" acidic patches. In the 1960s, transmission electron microscopy of fixed, dehydrated, sectioned, and stained inactive chromatin revealed "unit threads," frequently organized into parallel arrays at the nuclear envelope, which were interpreted as regular helices with ~ 30-nm center-to-center distance. Also observed in certain cell types, the nuclear envelope forms a "sandwich" around a layer of closely packed unit threads (ELCS, envelope-limited chromatin sheets). Discovery of the nucleosome in 1974 led to revised helical models of chromatin. But these models became very controversial and the existence of in situ 30-nm chromatin fibers has been challenged. Development of cryo-electron microscopy (Cryo-EM) gave hope that in situ chromatin fibers, devoid of artifacts, could be structurally defined. Combining a contrast-enhancing phase plate and cryo-electron tomography (Cryo-ET), it is now possible to visualize chromatin in a "close-to-native" situation. ELCS are particularly interesting to study by Cryo-ET. The chromatin sheet appears to have two layers of ~ 30-nm chromatin fibers arranged in a criss-crossed pattern. The chromatin in ELCS is continuous with adjacent interphase epichromatin. It appears that hydrated ~ 30-nm chromatin fibers are quite rare in most cells, possibly confined to interphase epichromatin at the nuclear envelope.