Histone H3 Tail Research Articles

Structural insights into the recognition of histone H3Q5 serotonylation by WDR5.

Serotonylation of histone H3Q5 (H3Q5ser) is a recently identified posttranslational modification of histones that acts as a permissive marker for gene activation in synergy with H3K4me3 during neuronal cell differentiation. However, any proteins that specifically recognize H3Q5ser remain unknown. Here, we found that WDR5 interacts with the N-terminal tail of histone H3 and functions as a "reader" for H3Q5ser. Crystal structures of WDR5 in complex with H3Q5ser and H3K4me3Q5ser peptides revealed that the serotonyl group is accommodated in a shallow surface pocket of WDR5. Experiments in neuroblastoma cells demonstrate that H3K4me3 modification is hampered upon disruption of WDR5-H3Q5ser interaction. WDR5 colocalizes with H3Q5ser in the promoter regions of cancer-promoting genes in neuroblastoma cells, where it promotes gene transcription to induce cell proliferation. Thus, beyond revealing a previously unknown mechanism through which WDR5 reads H3Q5ser to activate transcription, our study suggests that this WDR5-H3Q5ser-mediated epigenetic regulation apparently promotes tumorigenesis.

Progressive Phosphorylation Modulates the Self-Association of a Variably Modified Histone H3 Peptide.

Protein phosphorylation is a key regulatory mechanism in eukaryotic cells. In the intrinsically disordered histone tails, phosphorylation is often a part of combinatorial post-translational modifications and an integral part of the “histone code” that regulates gene expression. Here, we study the association between two histone H3 tail peptides modified to different degrees, using fully atomistic molecular dynamics simulations. Assuming that the initial conformations are either α-helical or fully extended, we compare the propensity of the two peptides to associate with one another when both are unmodified, one modified and the other unmodified, or both modified. The simulations lead to the identification of distinct inter- and intramolecular interactions in the peptide dimer, highlighting a prominent role of a fine-tuned phosphorylation rheostat in peptide association. Progressive phosphorylation appears to modulate peptide charge, inducing strong and specific intermolecular interactions between the monomers, which do not result in the formation of amorphous or ordered aggregates, as documented by experimental evidence derived from Circular Dichroism and NMR spectroscopy. However, upon complete saturation of positive charges by phosphate groups, this effect is reversed: intramolecular interactions prevail and dimerization of zero-charge peptides is markedly reduced. These findings underscore the role of phosphorylation thresholds in the dynamics of intrinsically disordered proteins. Phosphorylation rheostats might account for the divergent effects of histone modifications on the modulation of chromatin structure.

Cystatin B-deficiency triggers ectopic histone H3 tail cleavage during neurogenesis

Cystatin B (CSTB) acts as an inhibitor of cysteine proteases of the cathepsin family and loss-of-function mutations result in human brain diseases with a genotype-phenotype correlation. In the most severe case, CSTB-deficiency disrupts brain development, and yet the molecular basis of this mechanism is missing. Here, we establish CSTB as a regulator of chromatin structure during neural stem cell renewal and differentiation. Murine neural precursor cells (NPCs) undergo transient proteolytic cleavage of the N-terminal histone H3 tail by cathepsins B and L upon induction of differentiation into neurons and glia. In contrast, CSTB-deficiency triggers premature H3 tail cleavage in undifferentiated self-renewing NPCs and sustained H3 tail proteolysis in differentiating neural cells. This leads to significant transcriptional changes in NPCs, particularly of nuclear-encoded mitochondrial genes. In turn, these transcriptional alterations impair the enhanced mitochondrial respiration that is induced upon neural stem cell differentiation. Collectively, our findings reveal the basis of epigenetic regulation in the molecular pathogenesis of CSTB deficiency.

Histone tails cooperate to control the breathing of genomic nucleosomes.

Genomic DNA is packaged in chromatin, a dynamic fiber variable in size and compaction. In chromatin, repeating nucleosome units wrap 145-147 DNA basepairs around histone proteins. Genetic and epigenetic regulation of genes relies on structural transitions in chromatin which are driven by intra- and inter-nucleosome dynamics and modulated by chemical modifications of the unstructured terminal tails of histones. Here we demonstrate how the interplay between histone H3 and H2A tails control ample nucleosome breathing motions. We monitored large openings of two genomic nucleosomes, and only moderate breathing of an engineered nucleosome in atomistic molecular simulations amounting to 24 μs. Transitions between open and closed nucleosome conformations were mediated by the displacement and changes in compaction of the two histone tails. These motions involved changes in the DNA interaction profiles of clusters of epigenetic regulatory aminoacids in the tails. Removing the histone tails resulted in a large increase of the amplitude of nucleosome breathing but did not change the sequence dependent pattern of the motions. Histone tail modulated nucleosome breathing is a key mechanism of chromatin dynamics with important implications for epigenetic regulation.

Role of Brahma-related gene 1 (Brg1) in heart disease

Role of Brahma-related gene 1 (Brg1) in heart disease

DNMT1 reads heterochromatic H4K20me3 to reinforce LINE-1 DNA methylation

DNA methylation and trimethylated histone H4 Lysine 20 (H4K20me3) constitute two important heterochromatin-enriched marks that frequently cooperate in silencing repetitive elements of the mammalian genome. However, it remains elusive how these two chromatin modifications crosstalk. Here, we report that DNA methyltransferase 1 (DNMT1) specifically ‘recognizes’ H4K20me3 via its first bromo-adjacent-homology domain (DNMT1BAH1). Engagement of DNMT1BAH1-H4K20me3 ensures heterochromatin targeting of DNMT1 and DNA methylation at LINE-1 retrotransposons, and cooperates with the previously reported readout of histone H3 tail modifications (i.e., H3K9me3 and H3 ubiquitylation) by the RFTS domain to allosterically regulate DNMT1’s activity. Interplay between RFTS and BAH1 domains of DNMT1 profoundly impacts DNA methylation at both global and focal levels and genomic resistance to radiation-induced damage. Together, our study establishes a direct link between H4K20me3 and DNA methylation, providing a mechanism in which multivalent recognition of repressive histone modifications by DNMT1 ensures appropriate DNA methylation patterning and genomic stability.

Mechanism of non‐coding RNA regulation of DNMT3A and its relation to histones, regulatory proteins, and clinically relevant mutations

De novo DNA methylation by DNMT3A is a fundamental epigenetic modification that contributes to transcriptional regulation during cellular development and differentiation. Recent findings show that Fos Proto-Oncogene (Fos) extra-coding RNA (ecRNA) inhibits DNMT3A activity, Fos ecRNA knockdown results in neuronal hypomethylation and that Fos ecRNA expression is essential for fear memory formation in rats. Thus, further highlighting the role of RNA in the modulation of genomic DNA methylation. We show Fos ecRNA strongly correlates with Fos mRNA synthesis in primary cortical neurons on a single cell level and provide evidence that Fos ecRNA modulation of DNMT3A at these actively transcribed sites occurs in a sequence-independent manner. DNMT3A is also regulated by interactions with histone H3 tails and regulatory proteins, and the crosstalk between these epigenetic systems ensures appropriate epigenetic regulation. We provide insights into this crosstalk by assessing the relationship of regulatory RNAs, histone H3 tails and regulatory proteins in the simultaneous regulation of DNMT3A activity. Using DNMT3L, Fos ecRNA or CHD RNA in the presence of synthetic histone H3 tails or reconstituted polynucleosomes, we show regulatory RNAs play dominant roles in the modulation of DNMT3A activity in DNMT3A-H3 tails-regulatory protein-RNA complexes. Through mutational mapping and computational modeling we show that the interactions between DNMT3A and Fos-1 ecRNA occur at the tetramer interface of DNMT3A. Furthermore, while substitutions to clinically relevant residues on the DNMT3A tetramer interface, such as R771A, disrupt this ecRNA-mediated regulation, formation of DNMT3A_CDR771A-DNMT3L heterotetramersrestores the inhibition of enzymatic activity by Fos-1 ecRNA. The regulation of epigeneticmodifiers by non-coding RNAs is linked to diverse cancers including AML and UCEC. Our results further highlight the significance of RNA molecules in directing DNMT3A function and additionally provide insights into the crosstalk and mechanism for the modulation of DNMT3A by H3 tails, regulatory proteins and RNAs.

The C‐terminus of VRK1 Governs Interactions with Nucleosomes to Influence Histone H3 Threonine 3 (H3T3) Phosphorylation

Vaccinia-related kinase 1 (VRK1) is a highly abundant nuclear kinase that has numerous functions during mitosis, including the phosphorylation of the histone H3 tail at threonine 3 (H3T3ph). This post-translational modification, attributed to both VRK1 and the haspin kinase, is crucial for ensuring appropriate chromosome segregation. Despite the importance of VRK1 to cell cycle regulation, little is known about the biochemical basis of its activity on nucleosomes. Because placement of histone phosphorylation during mitosis is tightly spatiotemporally regulated, we sought to determine the chromatin template itself serves as a regulatory mechanism for VRK1 activity. In this work, we characterize the interaction between VRK1 and nucleosomes and determine how nucleosome binding affects nucleosome-directed kinase activity at H3T3. We find that VRK1 directly and stably interacts with nucleosomes with an apparent Kd of ~200 nM. VRK1 binds nucleosomes via a C-terminal alternating arginine motif, mutations or truncations of which are found in rare cases of genetic spinal muscular atrophy. Mutation or truncation of this motif leads to a 4-50 fold reduction in nucleosome affinity that is correlated with mutation position and severity. C-terminal arginine mutants of VRK1 show reduced capacity for histone H3T3 phosphorylation, suggesting that stable interaction with nucleosomes is required for nucleosome-directed kinase activity at H3T3. The nucleosome acidic patch, a negatively charged region at the dimer interface of H2A/H2B, is a hot spot for arginine-mediated nucleosome interactions. Neutralization of the acidic patch impairs VRK1 binding but surprisingly increases the activity of VRK1 at H3T3. In contrast, the presence of extended lengths of linker DNA promotes both nucleosome binding and phosphorylation of H3T3. Cryo-EM single particle analysis of VRK1-bound nucleosome particles demonstrates both acidic patch binding and heterogenous DNA binding that concentrates around the nucleosome linker DNA. This evidence suggests that VRK1 adopts at least two alternative binding modes on the nucleosome (acidic patch-governed vs. linker DNA-governed) that either promote or inhibit nucleosome-directed kinase activity based on the position and distance of VRK1 from the H3 tail. We suggest a model wherein dynamic access to the nucleosome acidic patch regulates the activity of VRK1 towards H3T3 during mitosis.

Nucleosome composition regulates the histone H3 tail conformational ensemble and accessibility.

Hexasomes and tetrasomes are intermediates in nucleosome assembly and disassembly. Their formation is promoted by histone chaperones, ATP-dependent remodelers, and RNA polymerase II. In addition, hexasomes are maintained in transcribed genes and could be an important regulatory factor. While nucleosome composition has been shown to affect the structure and accessibility of DNA, its influence on histone tails is largely unknown. Here, we investigate the conformational dynamics of the H3 tail in the hexasome and tetrasome. Using a combination of NMR spectroscopy, MD simulations, and trypsin proteolysis, we find that the conformational ensemble of the H3 tail is regulated by nucleosome composition. As has been found for the nucleosome, the H3 tails bind robustly to DNA within the hexasome and tetrasome, but upon loss of the H2A/H2B dimer, we determined that the adjacent H3 tail has an altered conformational ensemble, increase in dynamics, and increase in accessibility. Similar to observations of DNA dynamics, this is seen to be asymmetric in the hexasome. Our results indicate that nucleosome composition has the potential to regulate chromatin signaling and ultimately help shape the chromatin landscape.

Stability and folding pathways of tetra-nucleosome from six-dimensional free energy surface

The three-dimensional organization of chromatin is expected to play critical roles in regulating genome functions. High-resolution characterization of its structure and dynamics could improve our understanding of gene regulation mechanisms but has remained challenging. Using a near-atomistic model that preserves the chemical specificity of protein-DNA interactions at residue and base-pair resolution, we studied the stability and folding pathways of a tetra-nucleosome. Dynamical simulations performed with an advanced sampling technique uncovered multiple pathways that connect open chromatin configurations with the zigzag crystal structure. Intermediate states along the simulated folding pathways resemble chromatin configurations reported from in situ experiments. We further determined a six-dimensional free energy surface as a function of the inter-nucleosome distances via a deep learning approach. The zigzag structure can indeed be seen as the global minimum of the surface. However, it is not favored by a significant amount relative to the partially unfolded, in situ configurations. Chemical perturbations such as histone H4 tail acetylation and thermal fluctuations can further tilt the energetic balance to stabilize intermediate states. Our study provides insight into the connection between various reported chromatin configurations and has implications on the in situ relevance of the 30 nm fiber.

Histone H4 Tails in Nucleosomes: a Fuzzy Interaction with DNA

The interaction of positively charged N-terminal histone tails with nucleosomal DNA plays an important role in chromatin assembly and regulation, modulating their susceptibility to post-translational modifications and recognition by chromatin-binding proteins. Here, we report residue-specific 15 N NMR relaxation rates for histone H4 tails in reconstituted nucleosomes. These data indicate that H4 tails are strongly dynamically disordered, albeit with reduced conformational flexibility compared to a free peptide with the same sequence. Remarkably, the NMR observables were successfully reproduced in a 2-μs MD trajectory of the nucleosome. This is an important step toward resolving an apparent inconsistency where prior simulations were generally at odds with experimental evidence on conformational dynamics of histone tails. Our findings indicate that histone H4 tails engage in a fuzzy interaction with nucleosomal DNA, underpinned by a variable pattern of short-lived salt bridges and hydrogen bonds, which persists at low ionic strength (0-100 mM NaCl).

Histone H4 Tails in Nucleosomes: a Fuzzy Interaction with DNA

AbstractThe interaction of positively charged N‐terminal histone tails with nucleosomal DNA plays an important role in chromatin assembly and regulation, modulating their susceptibility to post‐translational modifications and recognition by chromatin‐binding proteins. Here, we report residue‐specific 15N NMR relaxation rates for histone H4 tails in reconstituted nucleosomes. These data indicate that H4 tails are strongly dynamically disordered, albeit with reduced conformational flexibility compared to a free peptide with the same sequence. Remarkably, the NMR observables were successfully reproduced in a 2‐μs MD trajectory of the nucleosome. This is an important step toward resolving an apparent inconsistency where prior simulations were generally at odds with experimental evidence on conformational dynamics of histone tails. Our findings indicate that histone H4 tails engage in a fuzzy interaction with nucleosomal DNA, underpinned by a variable pattern of short‐lived salt bridges and hydrogen bonds, which persists at low ionic strength (0–100 mM NaCl).

Structural mechanism of bivalent histone H3K4me3K9me3 recognition by the Spindlin1/C11orf84 complex in rRNA transcription activation

Spindlin1 is a unique multivalent epigenetic reader that facilitates ribosomal RNA transcription. In this study, we provide molecular and structural basis by which Spindlin1 acts in complex with C11orf84 to preferentially recognize non-canonical bivalent mark of trimethylated lysine 4 and lysine 9 present on the same histone H3 tail (H3K4me3K9me3). We demonstrate that C11orf84 binding stabilizes Spindlin1 and enhances its association with bivalent H3K4me3K9me3 mark. The functional analysis suggests that Spindlin1/C11orf84 complex can displace HP1 proteins from H3K4me3K9me3-enriched rDNA loci, thereby facilitating the conversion of these poised rDNA repeats from the repressed state to the active conformation, and the consequent recruitment of RNA Polymerase I for rRNA transcription. Our study uncovers a previously unappreciated mechanism of bivalent H3K4me3K9me3 recognition by Spindlin1/C11orf84 complex required for activation of rRNA transcription.

Surprising phenotypic diversity of cancer-associated mutations of Gly 34 in the histone H3 tail.

Sequencing of cancer genomes has identified recurrent somatic mutations in histones, termed oncohistones, which are frequently poorly understood. Previously we showed that fission yeast expressing only the H3.3G34R mutant identified in aggressive pediatric glioma had reduced H3K36 trimethylation and acetylation, increased genomic instability and replicative stress, and defective homology-dependent DNA damage repair. Here we show that surprisingly distinct phenotypes result from G34V (also in glioma) and G34W (giant cell tumors of bone) mutations, differentially affecting H3K36 modifications, subtelomeric silencing, genomic stability; sensitivity to irradiation, alkylating agents, and hydroxyurea; and influencing DNA repair. In cancer, only 1 of 30 alleles encoding H3 is mutated. Whilst co-expression of wild-type H3 rescues most G34 mutant phenotypes, G34R causes dominant hydroxyurea sensitivity, homologous recombination defects, and dominant subtelomeric silencing. Together, these studies demonstrate the complexity associated with different substitutions at even a single residue in H3 and highlight the utility of genetically tractable systems for their analysis.

The N-Terminal Tail of Histone H3 Regulates Copper Homeostasis in Saccharomyces cerevisiae.

Copper homeostasis is crucial for various cellular processes. The balance between nutritional and toxic copper levels is maintained through the regulation of its uptake, distribution, and detoxification via antagonistic actions of two transcription factors, Ace1 and Mac1. Ace1 responds to toxic copper levels by transcriptionally regulating detoxification genes CUP1 and CRS5 Cup1 metallothionein confers protection against toxic copper levels. CUP1 gene regulation is a multifactorial event requiring Ace1, TATA-binding protein (TBP), chromatin remodeler, acetyltransferase (Spt10), and histones. However, the role of histone H3 residues has not been fully elucidated. To investigate the role of the H3 tail in CUP1 transcriptional regulation, we screened the library of histone mutants in copper stress. We identified mutations in H3 (K23Q, K27R, K36Q, Δ5-16, Δ13-16, Δ13-28, Δ25-28, Δ28-31, and Δ29-32) that reduce CUP1 expression. We detected reduced Ace1 occupancy across the CUP1 promoter in K23Q, K36Q, Δ5-16, Δ13-28, Δ25-28, and Δ28-31 mutations correlating with the reduced CUP1 transcription. The majority of these mutations affect TBP occupancy at the CUP1 promoter, augmenting the CUP1 transcription defect. Additionally, some mutants displayed cytosolic protein aggregation upon copper stress. Altogether, our data establish previously unidentified residues of the H3 N-terminal tail and their modifications in CUP1 regulation.

Expeditious Extraction of Histones from Limited Cells or Tissue Samples and Quantitative Top-Down Proteomic Analysis.

Histones are the primary protein component of chromatin and are involved in virtually all DNA-templated processes. Histones are abundantly post-translationally modified by a variety of chromatin-modifying machinery. These post-translational modifications (PTMs) are recognized by a range of "reader" proteins, which recruit additional proteins to specific locations on chromatin and impart precise and powerful effects on gene regulation. Each PTM typically exerts a positive or negative effect on transcription, and recent studies have shown that histone PTMs function in a combinatorial histone code: that is, histone PTMs function in combination to exert precise DNA-templated regulation. Thus, there is a need to identify and understand proteoforms, or unambiguously defined single protein molecules with all combinations of modifications. Top-down proteomics is currently the only viable approach for identifying and quantitating histone proteoforms, and mass spectrometry instruments have become sufficiently powerful to perform these quantitative analyses in a robust and high-throughput fashion. These recent innovations have enabled new experimental directions in chromatin research but have also introduced temporal and other constraints. This has led us to develop the protocols described here, which increase throughput, reduce sample requirements, and maintain robust quantitation. Although originally designed for high-throughput quantitative top-down proteomics, the protocols described here are useful for a wide range of chromatin biology applications. Starting with small amounts of cells or tissue, we describe two basic protocols for exceptionally rapid and efficient nuclei isolation, acid extraction of histones, and high-performance liquid chromatography fractionation of histones into histone families. We additionally describe the quantitative top-down proteomic analysis of histone H4 proteoforms. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Nuclei isolation and acid extraction of histones from mammalian cells in culture/tissues Basic Protocol 2: HPLC fractionation of histones and histone H4 HPLC-MS/MS Support Protocol: Preparation of intact H3 histone tails by Glu-C digestion.

Nucleosome Core Particles Lacking H2B or H3 Tails Are Altered Structurally and Have Differential Base Excision Repair Fingerprints.

A recently discovered post-translational modification of histone proteins is the irreversible proteolytic clipping of the histone N-terminal tail domains. This modification is involved in the regulation of various biological processes, including the DNA damage response. In this work, we used chemical footprinting to characterize the structural alterations to nucleosome core particles (NCPs) that result from a lack of a histone H2B or H3 tail. We also examine the influence of these histone tails on excision of the mutagenic lesion 1,N6-ethenoadenine (εA) by the repair enzyme alkyladenine DNA glycosylase. We found that the absence of the H2B or H3 tail results in altered DNA periodicity relative to that of native NCPs. We correlated these structural alterations to εA excision by utilizing a global analysis of 21 εA sites in NCPs and unincorporated duplex DNA. In comparison to native NCPs, there is enhanced excision of εA in tailless H2B NCPs in regions that undergo DNA unwrapping. This enhanced excision is not observed for tailless H3 NCPs; rather, excision is inhibited in more static areas of the NCP not prone to unwrapping. Our results support in vivo observations of alkylation damage profiles and the potential role of tail clipping as a mechanism for overcoming physical obstructions caused by packaging in NCPs but also reveal the potential inhibition of repair by tail clipping in some locations. Taken together, these results further our understanding of how base excision repair can be facilitated or diminished by histone tail removal and contribute to our understanding of the underlying mechanism that leads to mutational hot spots.

Intestinal differentiation involves cleavage of histone H3 N-terminal tails by multiple proteases.

The proteolytic cleavage of histone tails, also termed histone clipping, has been described as a mechanism for permanent removal of post-translational modifications (PTMs) from histone proteins. Such activity has been ascribed to ensure regulatory function in key cellular processes such as differentiation, senescence and transcriptional control, for which different histone-specific proteases have been described. However, all these studies were exclusively performed using cell lines cultured in vitro and no clear evidence that histone clipping is regulated in vivo has been reported. Here we show that histone H3 N-terminal tails undergo extensive cleavage in the differentiated cells of the villi in mouse intestinal epithelium. Combining biochemical methods, 3D organoid cultures and in vivo approaches, we demonstrate that intestinal H3 clipping is the result of multiple proteolytic activities. We identified Trypsins and Cathepsin L as specific H3 tail proteases active in small intestinal differentiated cells and showed that their proteolytic activity is differentially affected by the PTM pattern of histone H3 tails. Together, our findings provide in vivo evidence of H3 tail proteolysis in mammalian tissues, directly linking H3 clipping to cell differentiation.

Is the H4 histone tail intrinsically disordered or intrinsically multifunctional?

The structural versatility of histone tails is one of the key elements in the organisation of chromatin, which allows for the compact storage of genomic information. However, this structural diversity also complicates experimental and computational studies. Here, the potential and free energy landscape for the isolated and bound H4 histone tail are explored. The landscapes exhibit a set of distinct structural ensembles separated by high energy barriers, with little difference between isolated and bound tails. This consistency is a desirable feature that facilitates the formation of transient interactions, which are required for the liquid-like chromatin organisation. The existence of multiple, distinct structures on a multifunnel energy landscape is likely to be associated with multifunctionality, i.e. a set of evolved, distinct functions. Contrasting it with previously reported results for other disordered peptides, this type of landscape may be associated with a conformational selection based binding mechanism. Given the similarity to other systems exhibiting similar multifunnel energy landscapes, the disorder in histone tails might be better described in context of multifunctionality.

Nuclear Condensates of p300 Formed Though the Structured Catalytic Core Can Act as a Storage Pool of p300 with Reduced HAT Activity

The transcriptional co-activator and acetyltransferase p300 is required for fundamental cellular processes, including differentiation and growth. Here, we report that p300 forms phase separated condensates in the cell nucleus. The phase separation ability of p300 is regulated by autoacetylation and relies on its catalytic core, representing a rare example of biomolecular phase separation occurred through structured regions - the catalytic HAT domain, the autoinhibition loop, and bromodomain. p300 condensates sequester chromatin components, such as histone H3 tail and DNA, and are amplified through binding of p300 to the nucleosome. The catalytic HAT activity of p300 is decreased in the phase separated droplets, a large portion of which co-localizes with chromatin regions enriched in H3K27me3. Our findings suggest a model in which p300 condensates can act as a storage pool of the protein with reduced HAT activity, allowing p300 to be compartmentalized and concentrated at poised or repressed chromatin regions.