Histone H3 Tail Research Articles

A Chemical Proteomics Approach to Reveal Direct Protein-Protein Interactions in Living Cells

Protein-protein interactions mediate essential cellular processes, however the detection of native interactions is challenging since they are often low affinity and context dependent. Here, we develop a chemical proteomics approach in vivo CLASPI [iCLASPI] (invivo crosslinking-assisted and stable isotope labeling by amino acids in cell culture [SILAC]-based protein identification) relying uponphoto-crosslinking, amber suppression, and SILAC-based quantitative proteomics to profile context-dependent protein-protein interactions in living cells. First, we use iCLASPI to profile invivo binding partners of the N-terminal tails of soluble histone H3 or H4. We identify known histone chaperones and modifying proteins, thereby validating our approach, and find an interaction between soluble histone H3 and UBR7, an E3 ubiquitin ligase, mediated by UBR7's PHD domain. Furthermore, we apply iCLASPI to profile the context-dependent protein-protein interactions of chromatin-associated histone H3 at different cell-cycle stages, and identify ANP32A as a mitosis-specific interactor. Our results demonstrate that the iCLASPI approach can provide a general strategy for identifying native, context-dependent direct protein-protein interactions using photo-crosslinking and quantitative proteomics.

In silico evidence for sequence-dependent nucleosome sliding

Nucleosomes represent the basic building block of chromatin and provide an important mechanism by which cellular processes are controlled. The locations of nucleosomes across the genome are not random but instead depend on both the underlying DNA sequence and the dynamic action of other proteins within the nucleus. These processes are central to cellular function, and the molecular details of the interplay between DNA sequence and nucleosome dynamics remain poorly understood. In this work, we investigate this interplay in detail by relying on a molecular model, which permits development of a comprehensive picture of the underlying free energy surfaces and the corresponding dynamics of nucleosome repositioning. The mechanism of nucleosome repositioning is shown to be strongly linked to DNA sequence and directly related to the binding energy of a given DNA sequence to the histone core. It is also demonstrated that chromatin remodelers can override DNA-sequence preferences by exerting torque, and the histone H4 tail is then identified as a key component by which DNA-sequence, histone modifications, and chromatin remodelers could in fact be coupled.

Effects of charge-modifying mutations in histone H2A \u03b13-domain on nucleosome stability assessed by single-pair FRET and MD simulations

Nucleosomes are important for chromatin compaction and gene regulation; their integrity depends crucially on the structural properties of the histone tails. Recent all-atom molecular dynamics simulations revealed that removal of the N-terminal tails of histone H3, known to destabilize nucleosomes, causes a rearrangement of two arginines of histone H2A, namely R81 and R88 by altering the electrostatic environment of the H2A α3 domain. Whether this rearrangement is the cause or the effect of decreased stability, is unclear. Here, we emulate the altered electrostatic environment that was found after H3 tail clipping through charge-modifying mutations to decouple its impact on intranucleosomal interactions from that of the histone tails. Förster resonance energy transfer experiments on recombinant nucleosomes and all-atom molecular dynamics simulations reveal a compensatory role of those amino acids in nucleosome stability. The simulations indicate a weakened interface between H2A-H2B dimers and the (H3-H4)2 tetramer, as well as between dimers and DNA. These findings agree with the experimental observations of position and charge dependent decreased nucleosome stability induced by the introduced mutations. This work highlights the importance of the H2A α3 domain and suggests allosteric effects between this domain and the outer DNA gyre as well as the H3 N-terminal tail.

Spatially restricted loading of BRD2 at DNA double-strand breaks protects H4 acetylation domains and promotes DNA repair

The n-terminal tail of histone H4 recruits repair proteins, including 53BP1, to DNA double-strand breaks (DSB) and undergoes dynamic acetylation during DSB repair. However, how H4 acetylation (H4Ac) recruits repair proteins and reorganizes chromatin during DNA repair is unclear. Here, we show that the bromodomain protein BRD2 is recruited to DSBs. This recruitment requires binding of BRD2’s tandem bromodomains to H4Ac, which is generated at DSBs by the Tip60/KAT5 acetyltransferase. Binding of BRD2 to H4Ac protects the underlying acetylated chromatin from attack by histone deacetylases and allows acetylation to spread along the flanking chromatin. However, BRD2 recruitment is spatially restricted to a chromatin domain extending only 2 kb either side of the DSB, and BRD2 does not spread into the chromatin domains flanking the break. Instead, BRD2 facilitates recruitment of a second bromodomain protein, ZMYND8, which spreads along the flanking chromatin, but is excluded from the DSB region. This creates a spatially restricted H4Ac/BRD2 domain which reorganizes chromatin at DSBs, limits binding of the L3MBTL1 repressor and promotes 53BP1 binding, while limiting end-resection of DSBs. BRD2 therefore creates a restricted chromatin environment surrounding DSBs which facilitates DSB repair and which is framed by extensive ZMYND8 domains on the flanking chromatin.

Single-molecule force spectroscopy on histone H4 tail-cross-linked chromatin reveals fiber folding

The eukaryotic genome is highly compacted into a protein-DNA complex called chromatin. The cell controls access of transcriptional regulators to chromosomal DNA via several mechanisms that act on chromatin-associated proteins and provide a rich spectrum of epigenetic regulation. Elucidating the mechanisms that fold chromatin fibers into higher-order structures is therefore key to understanding the epigenetic regulation of DNA accessibility. Here, using histone H4-V21C and histone H2A-E64C mutations, we employed single-molecule force spectroscopy to measure the unfolding of individual chromatin fibers that are reversibly cross-linked through the histone H4 tail. Fibers with covalently linked nucleosomes featured the same folding characteristics as fibers containing wild-type histones but exhibited increased stability against stretching forces. By stabilizing the secondary structure of chromatin, we confirmed a nucleosome repeat length (NRL)-dependent folding. Consistent with previous crystallographic and cryo-EM studies, the obtained force-extension curves on arrays with 167-bp NRLs best supported an underlying structure consisting of zig-zag, two-start fibers. For arrays with 197-bp NRLs, we previously inferred solenoidal folding, which was further corroborated by force-extension curves of the cross-linked fibers. The different unfolding pathways exhibited by these two types of arrays and reported here extend our understanding of chromatin structure and its potential roles in gene regulation. Importantly, these findings imply that chromatin compaction by nucleosome stacking protects nucleosomal DNA from external forces up to 4 piconewtons.

Covalent Modifications of Histone H3K9 Promote Binding of CHD3

Chromatin remodeling is required for genome function and is facilitated by ATP-dependent complexes, such as nucleosome remodeling and deacetylase (NuRD). Among its core components is the chromodomain helicase DNA binding protein 3 (CHD3) whose functional significance is not well established. Here, we show that CHD3 co-localizes with the other NuRD subunits, including HDAC1, near the H3K9ac-enriched promoters of the NuRD target genes. The tandem PHD fingers of CHD3 bind histone H3 tails and posttranslational modifications that increase hydrophobicity of H3K9-methylation or acetylation (H3K9me3 or H3K9ac)-enhance this interaction. Binding of CHD3 PHDs promotes H3K9Cme3-nucleosome unwrapping invitro and perturbs the pericentric heterochromatin structure invivo. Methylation oracetylation of H3K9 uniquely alleviates the intra-nucleosomal interaction of histone H3 tails, increasing H3K9 accessibility. Collectively, our data suggest that the targeting of covalently modified H3K9 by CHD3 might be essential in diverse functions of NuRD.

Nucleosome-Chd1 structure and implications for chromatin remodelling.

Chromatin remodelling factors change nucleosome positioning and facilitate DNA transcription, replication, and repair1. The conserved remodelling factor Chd12 can shift nucleosomes and induce a regular nucleosome spacing3–5. Chd1 is required for RNA polymerase II passage through nucleosomes6 and for cellular pluripotency7. Chd1 contains the DNA-binding domains SANT and SLIDE, a bilobal motor domain that hydrolyses adenosine triphosphate (ATP), and a regulatory double chromodomain. Here we report the cryo-electron microscopy (cryo-EM) structure of Chd1 from the yeast S. cerevisiae bound to a nucleosome at a resolution of 4.8 Å. Chd1 detaches two turns of DNA from the histone octamer and binds between the two DNA gyres in a state poised for catalysis. The SANT and SLIDE domains contact detached DNA around superhelical location (SHL) -7 of the first DNA gyre. The ATPase motor binds the second DNA gyre at SHL +2 and is anchored to the N-terminal tail of histone H4 as in a recent nucleosome-Snf2 ATPase structure8. Comparison with published results9 reveals that the double chromodomain swings towards nucleosomal DNA at SHL +1, resulting in ATPase closure. The ATPase can then promote translocation of DNA towards the nucleosome dyad, thereby loosening the first DNA gyre and remodelling the nucleosome. Translocation may involve ratcheting of the two lobes of the ATPase, which is trapped in a pre- or post-translocated state in the absence8 or presence, respectively, of transition state-mimicking compounds.

RFTS-dependent negative regulation of Dnmt1 by nucleosome structure and histone tails.

DNA methylation in promoter regions represses gene expression and is copied over mitotic divisions by Dnmt1. Dnmt1 activity is regulated by its replication foci targeting sequence (RFTS) domain which masks the catalytic pocket. It has been shown that Dnmt1 activity on unmethylated DNA is inhibited in nucleosome cores. In the present study, we aimed to assess the effect of nuclesome formation on maintenance methylation at single CpG resolution. We show that Dnmt1 fully methylates naked linker DNA in dinucleosomes, whereas maintenance methylation was repressed at all CpG sites in nucleosome core particles. Deletion of RFTS partly released obstruction of Dnmt1 activity in core particles. Histone H3 tail peptides inhibited Dnmt1 in an RFTS-dependent manner and repression was modulated by acetylation or methylation. We propose a novel function of RFTS to regulate Dnmt1 activity in nucleosomes.

A Histone Code Reader and a Transcriptional Activator Interact to Regulate Genes for Salt Tolerance.

Plant homeodomain (PHD) finger proteins are involved in various developmental processes and stress responses. They recognize and bind to epigenetically modified histone H3 tail and function as histone code readers. Here we report that GmPHD6 reads low methylated histone H3K4me0/1/2 but not H3K4me3 with its N-terminal domain instead of the PHD finger. GmPHD6 does not possess transcriptional regulatory ability but has DNA-binding ability. Through the PHD finger, GmPHD6 interacts with its coactivator, LHP1-1/2, to form a transcriptional activation complex. Using a transgenic hairy root system, we demonstrate that overexpression of GmPHD6 improves stress tolerance in soybean (Glycinemax) plants. Knocking down the LHP1 expression disrupts this role of GmPHD6, indicating that GmPHD6 requires LHP1 functions during stress response. GmPHD6 influences expression of dozens of stress-related genes. Among these, we identified three targets of GmPHD6, including ABA-stress-ripening-induced CYP75B1 and CYP82C4 Overexpression of each gene confers stress tolerance in soybean plants. GmPHD6 is recruited to H3K4me0/1/2 marks and recognizes the G-rich elements in target gene promoters, whereas LHP1 activates expression of these targets. Our study reveals a mechanism involving two partners in a complex. Manipulation of the genes in this pathway should improve stress tolerance in soybean or other legumes/crops.

The STAT4/MLL1 Epigenetic Axis Regulates the Antimicrobial Functions of Murine Macrophages

Macrophages are critical immune cells for the clearance of microbial pathogens and cellular debris from peripheral tissues. Macrophage inflammatory responses are governed by gene expression patterns, and these patterns are often subject to epigenetic control. Chromatin modifications, such as histone methylation, regulate gene accessibility in macrophages, and macrophage polarization is governed in part by the expression and function of chromatin-modifying enzymes. The histone methyltransferase mixed-lineage leukemia 1 (MLL1) preferentially modifies lysine residue 4 on the unstructured protein tail of histone H3. MLL1 expression and function have been shown to be governed by signal transduction pathways that are activated by inflammatory stimuli, such as NF-κB. Therefore, we sought to investigate the role of MLL1 in mediating macrophage inflammatory responses. Bone marrow-derived macrophages from mice with a targeted MLL1 gene knockout (Lys2-Cre+/- MLL1fx/fx) exhibited decreased proinflammatory gene expression with concurrent decreases in activating histone methylation. However, MLL1-deficient macrophages also exhibited increased phagocytic and bacterial killing activity in vitro. RNA profiling of MLL1-knockout macrophages identified numerous genes involved with inflammatory responses whose expression was altered in response to TLR ligands or proinflammatory cytokines, including STAT4. STAT4-dependent cytokines, such as type I IFNs were able to drive MLL1 expression in macrophages, and MLL1-knockout macrophages exhibited decreased activating histone methylation in the STAT4 promoter. These results implicate an important role for MLL1-dependent epigenetic regulation of macrophage antimicrobial functions.

The tale of a tail: histone H4 acetylation and the repair of DNA breaks

The ability of cells to detect and repair DNA double-strand breaks (DSBs) within the complex architecture of the genome requires co-ordination between the DNA repair machinery and chromatin remodelling complexes. This co-ordination is essential to process damaged chromatin and create open chromatin structures which are required for repair. Initially, there is a PARP-dependent recruitment of repressors, including HP1 and several H3K9 methyltransferases, and exchange of histone H2A.Z by the NuA4-Tip60 complex. This creates repressive chromatin at the DSB in which the tail of histone H4 is bound to the acidic patch on the nucleosome surface. These repressor complexes are then removed, allowing rapid acetylation of the H4 tail by Tip60. H4 acetylation blocks interaction between the H4 tail and the acidic patch on adjacent nucleosomes, decreasing inter-nucleosomal interactions and creating open chromatin. Further, the H4 tail is now free to recruit proteins such as 53BP1 to DSBs, a process modulated by H4 acetylation, and provides binding sites for bromodomain proteins, including ZMYND8 and BRD4, which are important for DSB repair. Here, we will discuss how the H4 tail functions as a dynamic hub that can be programmed through acetylation to alter chromatin packing and recruit repair proteins to the break site.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'.

Intricate Effects of α-Amino and Lysine Modifications on Arginine Methylation of the N-Terminal Tail of Histone H4.

Chemical modifications of the DNA and nucleosomal histones tightly control the gene transcription program in eukaryotic cells. The "histone code" hypothesis proposes that the frequency, combination, and location of post-translational modifications (PTMs) of the core histones compose a complex network of epigenetic regulation. Currently, there are at least 23 different types and >450 histone PTMs that have been discovered, and the PTMs of lysine and arginine residues account for a crucial part of the histone code. Although significant progress has been achieved in recent years, the molecular basis for the histone code is far from being fully understood. In this study, we investigated how naturally occurring N-terminal acetylation and PTMs of histone H4 lysine-5 (H4K5) affect arginine-3 methylation catalyzed by both type I and type II PRMTs at the biochemical level. Our studies found that acylations of H4K5 resulted in decreased levels of arginine methylation by PRMT1, PRMT3, and PRMT8. In contrast, PRMT5 exhibits an increased rate of arginine methylation upon H4K5 acetylation, propionylation, and crotonylation, but not upon H4K5 methylation, butyrylation, or 2-hydroxyisobutyrylation. Methylation of H4K5 did not affect arginine methylation by PRMT1 or PRMT5. There was a small increase in the rate of arginine methylation by PRMT8. Strikingly, a marked increase in the rate of arginine methylation was observed for PRMT3. Finally, N-terminal acetylation reduced the rate of arginine methylation by PRMT3 but had little influence on PRMT1, -5, and -8 activity. These results together highlight the underlying mechanistic differences in substrate recognition among different PRMTs and pave the way for the elucidation of the complex interplay of histone modifications.

Crosstalk within a functional INO80 complex dimer regulates nucleosome sliding.

Several chromatin remodellers have the ability to space nucleosomes on DNA. For ISWI remodellers, this involves an interplay between H4 histone tails, the AutoN and NegC motifs of the motor domains that together regulate ATPase activity and sense the length of DNA flanking the nucleosome. By contrast, the INO80 complex also spaces nucleosomes but is not regulated by H4 tails and lacks the AutoN and NegC motifs. Instead nucleosome sliding requires cooperativity between two INO80 complexes that monitor DNA length simultaneously on either side of the nucleosome during sliding. The C-terminal domain of the human Ino80 subunit (Ino80CTD) binds cooperatively to DNA and dimerisation of these domains provides crosstalk between complexes. ATPase activity, rather than being regulated, instead gradually becomes uncoupled as nucleosome sliding reaches an end point and this is controlled by the Ino80CTD. A single active ATPase motor within the dimer is sufficient for sliding.

WDR5 positively regulates p53 stability by inhibiting p53 ubiquitination

WD40 repeat protein WDR5 is a core component of the Set/MLL histone methyltransferase complex which catalyzes histone H3 Lys4 trimethylation and activates gene transcription in human cells. WDR5 promotes Set/MLL complex assembly and mediates the complex binding to Lys4-dimethylated histone H3 tail. Most earlier studies report that WDR5 exerts profound effects on various cellular and organismal processes mainly through epigenetic regulation of gene transcription. However, the functions of WDR5 in lung cancer remain largely unknown. Here, we report that WDR5 positively regulates p53 stability by inhibiting p53 ubiquitination in human lung cancer A549 cells. Overexpression of WDR5 dramatically increases p53 protein levels and its half-life in A549 cells, while depletion of WDR5 with WDR5-specific siRNAs significantly decreases p53 protein levels. We also observe that WDR5 is required for p53 induction in response to cisplatin treatment. Mechanistically, WDR5 colocalizes with p53 and inhibits p53 ubiquitination, resulting in p53 stabilization. Consequently, overexpression of WDR5 induces G1 phase arrest in A549 cells, and knocking down WDR5 by siRNAs reduces the population at G1 phase. Furthermore, p53 expression levels is at least in part determined by the p53 positive regulator WDR5 in some cancer cells. Taken together, these data suggest that WDR5 is directly involved in p53 signaling pathway. Our studies provide a new insight into WDR5 functions in A549 cells.

The effect of epigenetic modifications on the secondary structures and possible binding positions of the N-terminal tail of histone H3 in the nucleosome: a computational study

The roles of histone tails as substrates for reversible chemical modifications and dynamic cognate surfaces for the binding of regulatory proteins are well established. Despite these crucial roles, experimentally derived knowledge of the structure and possible binding sites of histone tails in chromatin is limited. In this study, we utilized molecular dynamics of isolated histone H3 N-terminal peptides to investigate its structure as a function of post-translational modifications that are known to be associated with defined chromatin states. We observed a structural preference for α-helices in isoforms associated with an inactive chromatin state, while isoforms associated with active chromatin states lacked α-helical content. The physicochemical effect of the post-translational modifications was highlighted by the interaction of arginine side-chains with the phosphorylated serine residues in the inactive isoform. We also showed that the isoforms exhibit different tail lengths, and, using molecular docking of the first 15 N-terminal residues of an H3 isoform, identified potential binding sites between the superhelical gyres on the octamer surface, close to the site of DNA entry/exit in the nucleosome. We discuss the possible functional role of the binding of the H3 tail within the nucleosome on both nucleosome and chromatin structure and stability.

Xeroderma pigmentosum group C protein interacts with histones: regulation by acetylated states of histone H3.

In the mammalian global genome nucleotide excision repair pathway, two damage recognition factors, XPC and UV-DDB, play pivotal roles in the initiation of the repair reaction. However, the molecular mechanisms underlying regulation of the lesion recognition process in the context of chromatin structures remain to be understood. Here, we show evidence that damage recognition factors tend to associate with chromatin regions devoid of certain types of acetylated histones. Treatment of cells with histone deacetylase inhibitors retarded recruitment of XPC to sites of UV-induced DNA damage and the subsequent repair process. Biochemical studies showed novel multifaceted interactions of XPC with histone H3, which were profoundly impaired by deletion of the N-terminal tail of histone H3. In addition, histone H1 also interacted with XPC. Importantly, acetylation of histone H3 markedly attenuated the interaction with XPC invitro, and local UV irradiation of cells decreased the level of H3K27ac in the damaged areas. Our results suggest that histone deacetylation plays a significant role in the process of DNA damage recognition for nucleotide excision repair and that the localization and functions of XPC can be regulated by acetylated states of histones.

The cysteine-rich domain of TET2 binds preferentially to mono- and dimethylated histone H3K36.

Missense mutations in Ten-eleven translocation 2 (TET2) gene are frequently found in leukaemia patients. Although mutations span the entire coding region, they tend to cluster in the C-terminal enzymatic domain and a cysteine-rich (CR) domain of unknown function. Herein, we found the CR domain binds chromatin preferentially at the histone H3 tail by recognising H3 lysine 36 mono- and dimethylation (H3K36me1/2). Importantly, missense mutations in the CR domain perturbed TET2 recruitment to the target locus and its enzymatic activities. Our findings identify a novel H3K36me recognition domain and uncover a critical link between histone modification and DNA hydroxylation in leukaemogenesis.

ヌクレオソーム中におけるヒストンテールの特性とアセチル化の影響

ヌクレオソーム中におけるヒストンテールの特性とアセチル化の影響

Probing lysine mono-methylation in histone H3 tail peptides with an abiotic receptor coupled to a non-plasmonic resonator.

Binder and effector molecules that allow studying and manipulating epigenetic processes are of biological relevance and pose severe technical challenges. We report the first example of a synthetic receptor able to recognize mono-methylated lysines in a histone H3 tail peptide, which has relevant functions in epigenetic regulation. Recognition is robust and specific regardless of the position and the number of mono-methylated lysines along the polypeptide chain. The peptide is first captured in solution by a tetraphosphonate cavitand (Tiiii) that selectively binds its Lys-NMe+ moieties. Separation from solution and detection of the peptide-Tiiii complexes is then enabled in one single step by an all dielectric SiO2-TiO2 core-shell resonator (T-rex), which captures the complex and operates fully reproducible signal transduction by non-plasmonic surface enhanced Raman scattering (SERS) without degrading the complex. The realized abiotic probe is able to distinguish multiple mono-methylated peptides from the single mono-methylated ones.

Probing Enhanced Double-Strand Break Formation at Abasic Sites within Clustered Lesions in Nucleosome Core Particles.

DNA is rapidly cleaved under mild alkaline conditions at apyrimidinic/apurinic sites, but the half-life is several weeks in phosphate buffer (pH 7.5). However, abasic sites are ∼100-fold more reactive within nucleosome core particles (NCPs). Histone proteins catalyze the strand scission, and at superhelical location 1.5, the histone H4 tail is largely responsible for the accelerated cleavage. The rate constant for strand scission at an abasic site is enhanced further in a nucleosome core particle when it is part of a bistranded lesion containing a proximal strand break. Cleavage of this form results in a highly deleterious double-strand break. This acceleration is dependent upon the position of the abasic lesion in the NCP and its structure. The enhancement in cleavage rate at an apurinic/apyrimidinic site rapidly drops off as the distance between the strand break and abasic site increases and is negligible once the two forms of damage are separated by 7 bp. However, the enhancement of the rate of double-strand break formation increases when the size of the gap is increased from one to two nucleotides. In contrast, the cleavage rate enhancement at 2-deoxyribonolactone within bistranded lesions is more modest, and it is similar in free DNA and nucleosome core particles. We postulate that the enhanced rate of double-strand break formation at bistranded lesions containing apurinic/apyrimidinic sites within nucleosome core particles is a general phenomenon and is due to increased DNA flexibility.