H3 N-terminal Tail Research Articles

A circular engineered sortase for interrogating histone H3 in chromatin.

Reversible modification of the histone H3 N-terminal tail is critical in regulating chromatin structure, gene expression, and cell states, while its dysregulation contributes to disease pathogenesis. Understanding the crosstalk between H3 tail modifications in nucleosomes constitutes a central challenge in epigenetics. Here we describe an engineered sortase transpeptidase, cW11, that displays highly favorable properties for introducing scarless H3 tails onto nucleosomes. This approach significantly accelerates the production of both symmetrically and asymmetrically modified nucleosomes. We demonstrate the utility of asymmetrically modified nucleosomes produced in this way in dissecting the impact of multiple modifications on eraser enzyme processing and molecular recognition by a reader protein. Moreover, we show that cW11 sortase is very effective at cutting and tagging histone H3 tails from endogenous histones, facilitating multiplex "cut-and-paste" middle down proteomics with tandem mass tags. This cut-and- paste proteomics approach permits the quantitative analysis of histone H3 modification crosstalk after treatment with different histone deacetylase inhibitors. We propose that these chemoenzymatic tail isolation and modification strategies made possible with cW11 sortase will broadly power epigenetics discovery and therapeutic development.

Histone H3K18 & H3K23 acetylation directs establishment of MLL-mediated H3K4 methylation

In an unmodified state, positively charged histone N-terminal tails engage nucleosomal DNA in a manner which restricts access to not only the underlying DNA, but also key tail residues subject to binding and/or modification. Charge-neutralizing modifications, such as histone acetylation, serve to disrupt this DNA-tail interaction, facilitating access to such residues. We previously showed that a polyacetylation-mediated chromatin “switch” governs the read-write capability of H3K4me3 by the MLL1 methyltransferase complex. Here, we discern the relative contributions of site-specific acetylation states along the H3 tail and extend our interrogation to other chromatin modifiers. We show that the contributions of H3 tail acetylation to H3K4 methylation by MLL1 are highly variable, with H3K18 and H3K23 acetylation exhibiting robust stimulatory effects, and that this extends to the related H3K4 methyltransferase complex, MLL4. We show that H3K4me1 and H3K4me3 are found preferentially co-enriched with H3 N-terminal tail proteoforms bearing dual H3K18 and H3K23 acetylation (H3{K18acK23ac}). We further show that this effect is specific to H3K4 methylation, while methyltransferases targeting other H3 tail residues (H3K9, H3K27, & H3K36), a methyltransferase targeting the nucleosome core (H3K79), and a kinase targeting a residue directly adjacent to H3K4 (H3T3) are insensitive to tail acetylation. Together, these findings indicate a unique and robust stimulation of H3K4 methylation by H3K18 and H3K23 acetylation and provide key insight into why H3K4 methylation is often associated with histone acetylation in the context of active gene expression.

Cancer-associated Histone H3 N-terminal arginine mutations disrupt PRC2 activity and impair differentiation

Dysregulated epigenetic states are a hallmark of cancer and often arise from genetic alterations in epigenetic regulators. This includes missense mutations in histones, which, together with associated DNA, form nucleosome core particles. However, the oncogenic mechanisms of most histone mutations are unknown. Here, we demonstrate that cancer-associated histone mutations at arginines in the histone H3 N-terminal tail disrupt repressive chromatin domains, alter gene regulation, and dysregulate differentiation. We find that histone H3R2C and R26C mutants reduce transcriptionally repressive H3K27me3. While H3K27me3 depletion in cells expressing these mutants is exclusively observed on the minor fraction of histone tails harboring the mutations, the same mutants recurrently disrupt broad H3K27me3 domains in the chromatin context, including near developmentally regulated promoters. H3K27me3 loss leads to de-repression of differentiation pathways, with concordant effects between H3R2 and H3R26 mutants despite different proximity to the PRC2 substrate, H3K27. Functionally, H3R26C-expressing mesenchymal progenitor cells and murine embryonic stem cell-derived teratomas demonstrate impaired differentiation. Collectively, these data show that cancer-associated H3 N-terminal arginine mutations reduce PRC2 activity and disrupt chromatin-dependent developmental functions, a cancer-relevant phenotype.

Bioorthogonal Labeling and Enrichment of Histone Monoaminylation Reveal Its Accumulation and Regulatory Function in Cancer Cell Chromatin.

Histone monoaminylation (i.e., serotonylation and dopaminylation) is an emerging category of epigenetic mark occurring on the fifth glutamine (Q5) residue of H3 N-terminal tail, which plays significant roles in gene transcription. Current analysis of histone monoaminylation is mainly based on site-specific antibodies and mass spectrometry, which either lacks high resolution or is time-consuming. In this study, we report the development of chemical probes for bioorthogonal labeling and enrichment of histone serotonylation and dopaminylation. These probes were successfully applied for the monoaminylation analysis of in vitro biochemical assays, cells, and tissue samples. The enrichment of monoaminylated histones by the probes further confirmed the crosstalk between H3Q5 monoaminylation and H3K4 methylation. Finally, combining the ex vivo and in vitro analyses based on the developed probes, we have shown that both histone serotonylation and dopaminylation are highly enriched in tumor tissues that overexpress transglutaminase 2 (TGM2) and regulate the three-dimensional architecture of cellular chromatin.

MMP-9-dependent proteolysis of the histone H3 N-terminal tail: a critical epigenetic step in driving oncogenic transcription and colon tumorigenesis.

Matrix metalloproteinase 9 (MMP-9) is a member of the MMP family and has been recently identified as a nuclear protease capable of clipping histone H3 N-terminal tails (H3NT). This MMP-9-dependent H3NT proteolysis is critical for establishing an active state of gene transcription during osteoclast differentiation and melanoma development. However, whether H3NT cleavage by MMP-9 plays a similar role in other cellular events has not been explored. Here, we dissect the functional contribution of MMP-9-dependent H3NT clipping to colonic tumorigenesis by using a combination of genome-wide transcriptome data, ChIP/ChIPac-qPCR, CRISPR/dCas9 gene-targeting system, and in vivo xenograft models. We show that MMP-9 is overexpressed in colon cancer cells and catalyzes H3NT proteolysis to drive transcriptional activation of growth stimulatory genes. Our studies using knockdown and inhibition approaches clearly indicate that MMP-9 mediates transcriptional activation and promotes colonic tumorigenesis in a manner dependent on its protease activity toward H3NT. Remarkably, artificial H3NT proteolysis at target gene promoters with dCAS9-MMP-9 is sufficient for establishing their transcriptional competence in colon cancer cells, underscoring the importance of MMP-9-dependent H3NT proteolysis per se in the transactivation process. Our data establish new functions and mechanisms for MMP-9 in driving the oncogenic transcription program in colon cancer through H3NT proteolysis, and demonstrate how this epigenetic pathway can be exploited as a potential therapeutic target for cancer treatment.

Histone H3 N-terminal tail provides tolerance to tartrazine induced stress in Saccharomyces cerevisiae

Histone H3 N-terminal tail provides tolerance to tartrazine induced stress in Saccharomyces cerevisiae

Investigation of the acetic acid stress response in Saccharomyces cerevisiae with mutated H3 residues.

Enhanced levels of acetic acid reduce the activity of yeast strains employed for industrial fermentation-based applications. Therefore, unraveling the genetic factors underlying the regulation of the tolerance and sensitivity of yeast towards acetic acid is imperative for optimising various industrial processes. In this communication, we have attempted to decipher the acetic acid stress response of the previously reported acetic acid-sensitive histone mutants. Revalidation using spot-test assays and growth curves revealed that five of these mutants, viz., H3K18Q, H3S28A, H3K42Q, H3Q68A, and H3F104A, are most sensitive towards the tested acetic acid concentrations. These mutants demonstrated enhanced acetic acid stress response as evidenced by the increased expression levels of AIF1, reactive oxygen species (ROS) generation, chromatin fragmentation, and aggregated actin cytoskeleton. Additionally, the mutants exhibited active cell wall damage response upon acetic acid treatment, as demonstrated by increased Slt2-phosphorylation and expression of cell wall integrity genes. Interestingly, the mutants demonstrated increased sensitivity to cell wall stress-causing agents. Finally, screening of histone H3 N-terminal tail truncation mutants revealed that the tail truncations exhibit general sensitivity to acetic acid stress. Some of these N-terminal tail truncation mutants viz., H3 [del 1-24], H3 [del 1-28], H3 [del 9-24], and H3 [del 25-36] are also sensitive to cell wall stress agents such as Congo red and caffeine suggesting that their enhanced acetic acid sensitivity may be due to cell wall stress induced by acetic acid.

Histone mutations in cancer.

Genes encoding histone proteins are recurrently mutated in tumor samples, and these mutations may impact nucleosome stability, histone post-translational modification, or chromatin dynamics. The prevalence of histone mutations across diverse cancer types suggest that normal chromatin structure is a barrier to tumorigenesis. Oncohistone mutations disrupt chromatin structure and gene regulatory mechanisms, resulting in aberrant gene expression and the development of cancer phenotypes. Examples of oncohistones include the histone H3 K27M mutation found in pediatric brain cancers that blocks post-translational modification of the H3 N-terminal tail and the histone H2B E76K mutation found in some solid tumors that disrupts nucleosome stability. Oncohistones may comprise a limited fraction of the total histone pool yet cause global effects on chromatin structure and drive cancer phenotypes. Here, we survey histone mutations in cancer and review their function and role in tumorigenesis.

Contributions of histone tail clipping and acetylation in nucleosome transcription by RNA polymerase II.

The N-terminal tails of histones protrude from the nucleosome core and are target sites for histone modifications, such as acetylation and methylation. Histone acetylation is considered to enhance transcription in chromatin. However, the contribution of the histone N-terminal tail to the nucleosome transcription by RNA polymerase II (RNAPII) has not been clarified. In the present study, we reconstituted nucleosomes lacking the N-terminal tail of each histone, H2A, H2B, H3or H4, and performed RNAPII transcription assays. We found that the N-terminal tail of H3, but not H2A, H2Band H4, functions in RNAPII pausing at the SHL(-5) position of the nucleosome. Consistently, the RNAPII transcription assay also revealed that the nucleosome containing N-terminally acetylated H3 drastically alleviates RNAPII pausing at the SHL(-5) position. In addition, the H3 acetylated nucleosome produced increased amounts of the run-off transcript. These results provide important evidence that the H3 N-terminal tail plays a role in RNAPII pausing at the SHL(-5) position of the nucleosome, and its acetylation directly alleviates this nucleosome barrier.

Structural insight into H4K20 methylation on H2A.Z-nucleosome by SUV420H1

DNA replication ensures the accurate transmission of genetic information during the cell cycle. Histone variant H2A.Z is crucial for early replication origins licensing and activation in which SUV420H1 preferentially recognizes H2A.Z-nucleosome and deposits H4 lysine 20 dimethylation (H4K20me2) on replication origins. Here, we report the cryo-EM structures of SUV420H1 bound to H2A.Z-nucleosome or H2A-nucleosome and demonstrate that SUV420H1 directly interacts with H4 N-terminal tail, the DNA, and the acidic patch in the nucleosome. The H4 (1-24) forms a lasso-shaped structure that stabilizes the SUV420H1-nucleosome complex and precisely projects the H4K20 residue into the SUV420H1 catalytic center. Invitro and invivo analyses reveal a crucial role of the SUV420H1 KR loop (residues 214-223), which lies close to the H2A.Z-specific residues D97/S98, in H2A.Z-nucleosome preferential recognition. Together, our findings elucidate how SUV420H1 recognizes nucleosomes to ensure site-specific H4K20me2 modification and provide insights into how SUV420H1 preferentially recognizes H2A.Z nucleosome.

Diverse modes of H3K36me3-guided nucleosomal deacetylation by Rpd3S

Context-dependent dynamic histone modifications constitute a key epigenetic mechanism in gene regulation1–4. The Rpd3 small (Rpd3S) complex recognizes histone H3 trimethylation on lysine 36 (H3K36me3) and deacetylates histones H3 and H4 at multiple sites across transcribed regions5–7. Here we solved the cryo-electron microscopy structures of Saccharomyces cerevisiae Rpd3S in its free and H3K36me3 nucleosome-bound states. We demonstrated a unique architecture of Rpd3S, in which two copies of Eaf3–Rco1 heterodimers are asymmetrically assembled with Rpd3 and Sin3 to form a catalytic core complex. Multivalent recognition of two H3K36me3 marks, nucleosomal DNA and linker DNAs by Eaf3, Sin3 and Rco1 positions the catalytic centre of Rpd3 next to the histone H4 N-terminal tail for deacetylation. In an alternative catalytic mode, combinatorial readout of unmethylated histone H3 lysine 4 and H3K36me3 by Rco1 and Eaf3 directs histone H3-specific deacetylation except for the registered histone H3 acetylated lysine 9. Collectively, our work illustrates dynamic and diverse modes of multivalent nucleosomal engagement and methylation-guided deacetylation by Rpd3S, highlighting the exquisite complexity of epigenetic regulation with delicately designed multi-subunit enzymatic machineries in transcription and beyond.

Epigenetic mechanisms to propagate histone acetylation by p300/CBP

Histone acetylation is important for the activation of gene transcription but little is known about its direct read/write mechanisms. Here, we report cryogenic electron microscopy structures in which a p300/CREB-binding protein (CBP) multidomain monomer recognizes histone H4 N-terminal tail (NT) acetylation (ac) in a nucleosome and acetylates non-H4 histone NTs within the same nucleosome. p300/CBP not only recognized H4NTac via the bromodomain pocket responsible for reading, but also interacted with the DNA minor grooves via the outside of that pocket. This directed the catalytic center of p300/CBP to one of the non-H4 histone NTs. The primary target that p300 writes by reading H4NTac was H2BNT, and H2BNTac promoted H2A-H2B dissociation from the nucleosome. We propose a model in which p300/CBP replicates histone N-terminal tail acetylation within the H3-H4 tetramer to inherit epigenetic storage, and transcribes it from the H3-H4 tetramer to the H2B-H2A dimers to activate context-dependent gene transcription through local nucleosome destabilization.

The MMP-2 histone H3 N-terminal tail protease is selectively targeted to the transcription start sites of active genes

BackgroundProteolysis of the histone H3 N-terminal tail (H3NT) is an evolutionarily conserved epigenomic feature of nearly all eukaryotes, generating a cleaved H3 product that is retained in ~ 5–10% of the genome. Although H3NT proteolysis within chromatin was first reported over 60 years ago, the genomic sites targeted for H3NT proteolysis and the impact of this histone modification on chromatin structure and function remain largely unknown. The goal of this study was to identify the specific regions targeted for H3NT proteolysis and investigate the consequence of H3NT “clipping” on local histone post-translational modification (PTM) dynamics.ResultsLeveraging recent findings that matrix metalloproteinase 2 (MMP-2) functions as the principal nuclear H3NT protease in the human U2OS osteosarcoma cell line, a ChIP-Seq approach was used to map MMP-2 localization genome wide. The results indicate that MMP-2 is selectively targeted to the transcription start sites (TSSs) of protein coding genes, primarily at the + 1 nucleosome. MMP-2 localization was exclusive to highly expressed genes, further supporting a functional role for H3NT proteolysis in transcriptional regulation. MMP-2 dependent H3NT proteolysis at the TSSs of these genes resulted in a > twofold reduction of activation-associated histone H3 PTMs, including H3K4me3, H3K9ac and H3K18ac. One of genes requiring MMP-2 mediated H3NT proteolysis for proficient expression was the lysosomal cathepsin B protease (CTSB), which we discovered functions as a secondary nuclear H3NT protease in U2OS cells.ConclusionsThis study revealed that the MMP-2 H3NT protease is selectively targeted to the TSSs of active protein coding genes in U2OS cells. The resulting H3NT proteolysis directly alters local histone H3 PTM patterns at TSSs, which likely functions to regulate transcription. MMP-2 mediated H3NT proteolysis directly activates CTSB, a secondary H3NT protease that generates additional cleaved H3 products within chromatin.

The effects of RNA.DNA-DNA triple helices on nucleosome structures and dynamics

Noncoding RNAs (ncRNAs) are an emerging epigenetic factor and have been recognized as playing a key role in many gene expression pathways. Structurally, binding of ncRNAs to isolated DNA is strongly dependent on sequence complementary and results in the formation of an RNA.DNA-DNA (RDD) triple helix. However, in vivo DNA is not isolated but is rather packed in chromatin fibers, the fundamental unit of which is the nucleosome. Biochemical experiments have shown that ncRNA binding to nucleosomal DNA is elevated at DNA entry and exit sites and is dependent on the presence of the H3 N-terminal tails. However, the structural and dynamical bases for these mechanisms remain unknown. Here, we have examined the mechanisms and effects of RDD formation in the context of the nucleosome using a series of all-atom molecular dynamics simulations. Results highlight the importance of DNA sequence on complex stability, elucidate the effects of the H3 tails on RDD structures, show how RDD formation impacts the structure and dynamics of the H3 tails, and show how RNA alters the local and global DNA double-helical structure. Together, our results suggest ncRNAs can modify nucleosome, and potentially higher-order chromatin, structures and dynamics as a means of exerting epigenetic control.

Histone H3 core domain in chromatin with different DNA linker lengths studied by 1H-Detected solid-state NMR spectroscopy.

Chromatin, a dynamic protein-DNA complex that regulates eukaryotic genome accessibility and essential functions, is composed of nucleosomes connected by linker DNA with each nucleosome consisting of DNA wrapped around an octamer of histones H2A, H2B, H3 and H4. Magic angle spinning solid-state nuclear magnetic resonance (NMR) spectroscopy can yield unique insights into histone structure and dynamics in condensed nucleosomes and nucleosome arrays representative of chromatin at physiological concentrations. Recently we used J-coupling-based solid-state NMR methods to investigate with residue-specific resolution the conformational dynamics of histone H3 N-terminal tails in 16-mer nucleosome arrays containing 15, 30 or 60 bp DNA linkers. Here, we probe the H3 core domain in the 16-mer arrays as a function of DNA linker length via dipolar coupling-based 1H-detected solid-state NMR techniques. Specifically, we established nearly complete assignments of backbone chemical shifts for H3 core residues in arrays with 15-60 bp DNA linkers reconstituted with 2H,13C,15N-labeled H3. Overall, these chemical shifts were similar irrespective of the DNA linker length indicating no major changes in H3 core conformation. Notably, however, multiple residues at the H3-nucleosomal DNA interface in arrays with 15 bp DNA linkers exhibited relatively pronounced differences in chemical shifts and line broadening compared to arrays with 30 and 60 bp linkers. These findings are consistent with increased heterogeneity in nucleosome packing and structural strain within arrays containing short DNA linkers that likely leads to side-chains of these interfacial residues experiencing alternate conformations or shifts in their rotamer populations relative to arrays with the longer DNA linkers.

The MECP2-TRD domain interacts with the DNMT3A-ADD domain at the H3-tail binding site.

The DNMT3A DNA methyltransferase and MECP2 methylation reader are highly expressed in neurons. Both proteins interact via their DNMT3A-ADD and MECP2-TRD domains, and the MECP2 interaction regulates the activity and subnuclear localization of DNMT3A. Here, we mapped the interface of both domains using peptide SPOT array binding, protein pull-down, equilibrium peptide binding assays, and structural analyses. The region D529-D531 on the surface of the ADD domain was identified as interaction point with the TRD domain. This includes important residues of the histone H3 N-terminal tail binding site to the ADD domain, explaining why TRD and H3 binding to the ADD domain is competitive. On the TRD domain, residues 214-228 containing K219 and K223 were found to be essential for the ADD interaction. This part represents a folded patch within the otherwise largely disordered TRD domain. A crystal structure analysis of ADD revealed that the identified H3/TDR lysine binding pocket is occupied by an arginine residue from a crystallographic neighbor in the ADD apoprotein structure. Finally, we show that mutations in the interface of ADD and TRD domains disrupt the cellular interaction of both proteins in NIH3T3 cells. In summary, our data show that the H3 peptide binding cleft of the ADD domain also mediates the interaction with the MECP2-TRD domain suggesting that this binding site may have a broader role also in the interaction of DNMT3A with other proteins leading to complex regulation options by competitive and PTM specific binding.

Structure of IMPORTIN-4 bound to the H3–H4–ASF1 histone–histone chaperone complex

IMPORTIN-4, the primary nuclear import receptor of core histones H3 and H4, binds the H3-H4 dimer and histone chaperone ASF1 prior to nuclear import. However, how H3-H3-ASF1 is recognized for transport cannot be explained by available crystal structures of IMPORTIN-4-histone tail peptide complexes. Our 3.5-Å IMPORTIN-4-H3-H4-ASF1 cryoelectron microscopy structure reveals the full nuclear import complex and shows a binding mode different from suggested by previous structures. The N-terminal half of IMPORTIN-4 clamps the globular H3-H4 domain and H3 αN helix, while its C-terminal half binds the H3 N-terminal tail weakly; tail contribution to binding energy is negligible. ASF1 binds H3-H4 without contacting IMPORTIN-4. Together, ASF1 and IMPORTIN-4 shield nucleosomal H3-H4 surfaces to chaperone and import it into the nucleus where RanGTP binds IMPORTIN-4, causing large conformational changes to release H3-H4-ASF1. This work explains how full-length H3-H4 binds IMPORTIN-4 in the cytoplasm and how it is released in the nucleus.

FACT modulates the conformations of histone H2A and H2B N-terminal tails within nucleosomes

Gene expression is regulated by the modification and accessibility of histone tails within nucleosomes. The histone chaperone FACT (facilitate chromatin transcription), comprising SPT16 and SSRP1, interacts with nucleosomes through partial replacement of DNA with the phosphorylated acidic intrinsically disordered (pAID) segment of SPT16; pAID induces an accessible conformation of the proximal histone H3 N-terminal tail (N-tail) in the unwrapped nucleosome with FACT. Here, we use NMR to probe the histone H2A and H2B tails in the unwrapped nucleosome. Consequently, both the H2A and H2B N-tails on the pAID-proximal side bind to pAID with robust interactions, which are important for nucleosome assembly with FACT. Furthermore, the conformations of these N-tails on the distal DNA-contact site are altered from those in the canonical nucleosome. Our findings highlight that FACT both proximally and distally regulates the conformations of the H2A and H2B N-tails in the asymmetrically unwrapped nucleosome.

A Saccharomyces cerevisiae model and screen to define the functional consequences of oncogenic histone missense mutations.

Somatic missense mutations in histone genes turn these essential proteins into oncohistones, which can drive oncogenesis. Understanding how missense mutations alter histone function is challenging in mammals as mutations occur in a single histone gene. For example, described oncohistone mutations predominantly occur in the histone H3.3 gene, despite the human genome encoding 15 H3 genes. To understand how oncogenic histone missense mutations alter histone function, we leveraged the budding yeast model, which contains only 2 H3 genes, to explore the functional consequences of oncohistones H3K36M, H3G34W, H3G34L, H3G34R, and H3G34V. Analysis of cells that express each of these variants as the sole copy of H3 reveals that H3K36 mutants show different drug sensitivities compared to H3G34 mutants. This finding suggests that changes to proximal amino acids in the H3 N-terminal tail alter distinct biological pathways. We exploited the caffeine-sensitive growth of H3K36-mutant cells to perform a high copy suppressor screen. This screen identified genes linked to histone function and transcriptional regulation, including Esa1, a histone H4/H2A acetyltransferase; Tos4, a forkhead-associated domain-containing gene expression regulator; Pho92, an N6-methyladenosine RNA-binding protein; and Sgv1/Bur1, a cyclin-dependent kinase. We show that the Esa1 lysine acetyltransferase activity is critical for suppression of the caffeine-sensitive growth of H3K36R-mutant cells while the previously characterized binding interactions of Tos4 and Pho92 are not required for suppression. This screen identifies pathways that could be altered by oncohistone mutations and highlights the value of yeast genetics to identify pathways altered by such mutations.

Peptide Dynamics and Metadynamics: Leveraging Enhanced Sampling Molecular Dynamics to Robustly Model Long-Timescale Transitions.

Molecular dynamics simulations can in theory reveal the thermodynamics and kinetics of peptide conformational transitions at atomic-level resolution. However, even with modern computing power, they are limited in the timescales they can sample, which is especially problematic for peptides that are fully or partially disordered. Here, we discuss how the enhanced sampling methods accelerated molecular dynamics (aMD) and metadynamics can be leveraged in a complementary fashion to quickly explore conformational space and then robustly quantify the underlying free energy landscape. We apply these methods to two peptides that have an intrinsically disordered nature, the histone H3 and H4 N-terminal tails, and use metadynamics to compute the free energy landscape along collective variables discerned from aMD simulations. Results show that these peptides are largely disordered, with a slight preference for α-helical structures.