Interactions of Nucleosomes with Acidic Patch-Binding Peptides: A Combined Structural Bioinformatics, Molecular Modeling, Fluorescence Polarization, and Single-Molecule FRET Study.

In eukaryotic organisms, genomic DNA associates with histone proteins to form nucleosomes. Nucleosomes provide a basis for genome compaction, epigenetic markup, and mediate interactions of nuclear proteins with their target DNA loci. A negatively charged (acidic) patch located on the H2A-H2B histone dimer is a characteristic feature of the nucleosomal surface. The acidic patch is a common site in the attachment of various chromatin proteins, including viral ones. Acidic patch-binding peptides present perspective compounds that can be used to modulate chromatin functioning by disrupting interactions of nucleosomes with natural proteins or alternatively targeting artificial moieties to the nucleosomes, which may be beneficial for the development of new therapeutics. In this work, we used several computational and experimental techniques to improve our understanding of how peptides may bind to the acidic patch and what are the consequences of their binding. Through extensive analysis of the PDB database, histone sequence analysis, and molecular dynamic simulations, we elucidated common binding patterns and key interactions that stabilize peptide-nucleosome complexes. Through MD simulations and FRET measurements, we characterized changes in nucleosome dynamics conferred by peptide binding. Using fluorescence polarization and gel electrophoresis, we evaluated the affinity and specificity of the LANA1-22 peptide to DNA and nucleosomes. Taken together, our study provides new insights into the different patterns of intermolecular interactions that can be employed by natural and designed peptides to bind to nucleosomes, and the effects of peptide binding on nucleosome dynamics and stability.

Structure and Dynamics of Compact Dinucleosomes: Analysis by Electron Microscopy and spFRET.

Formation of compact dinucleosomes (CODIs) occurs after collision between adjacent nucleosomes at active regulatory DNA regions. Although CODIs are likely dynamic structures, their structural heterogeneity and dynamics were not systematically addressed. Here, single-particle Förster resonance energy transfer (spFRET) and electron microscopy were employed to study the structure and dynamics of CODIs. spFRET microscopy in solution and in gel revealed considerable uncoiling of nucleosomal DNA from the histone octamer in a fraction of CODIs, suggesting that at least one of the nucleosomes is destabilized in the presence of the adjacent closely positioned nucleosome. Accordingly, electron microscopy analysis suggests that up to 30 bp of nucleosomal DNA are involved in transient uncoiling/recoiling on the octamer. The more open and dynamic nucleosome structure in CODIs cannot be stabilized by histone chaperone Spt6. The data suggest that proper internucleosomal spacing is an important determinant of chromatin stability and support the possibility that CODIs could be intermediates of chromatin disruption.

Nucleosomes and their complexes in the cryoEM era: Trends and limitations.

Twenty-five years have passed since the appearance of the first atomistic model of the nucleosome structure, and since then the number of new structures has gradually increased. With the advent of cryo-microscopy, the rate of accumulation of models has increased significantly. New structures are emerging with different histone variants and a variety of proteins that bind to nucleosomes. At the moment, there are more than four hundred structures containing nucleosomes in the Protein Data Bank. Many of these structures represent similar complexes, others differ in composition, conformation and quality. In this perspective, we investigate the diversity of known nucleosome structures, analyze data and model quality, variations in histone/DNA content of nucleosomes and spectrum of their interactors. We outline those parts of the nucleosome "structurome" that are already explored and those awaiting further exploration.

A standardized nomenclature for mammalian histone genes.

Histones have a long history of research in a wide range of species, leaving a legacy of complex nomenclature in the literature. Community-led discussions at the EMBO Workshop on Histone Variants in 2011 resulted in agreement amongst experts on a revised systematic protein nomenclature for histones, which is based on a combination of phylogenetic classification and historical symbol usage. Human and mouse histone gene symbols previously followed a genome-centric system that was not applicable across all vertebrate species and did not reflect the systematic histone protein nomenclature. This prompted a collaboration between histone experts, the Human Genome Organization (HUGO) Gene Nomenclature Committee (HGNC) and Mouse Genomic Nomenclature Committee (MGNC) to revise human and mouse histone gene nomenclature aiming, where possible, to follow the new protein nomenclature whilst conforming to the guidelines for vertebrate gene naming. The updated nomenclature has also been applied to orthologous histone genes in chimpanzee, rhesus macaque, dog, cat, pig, horse and cattle, and can serve as a framework for naming other vertebrate histone genes in the future.

Structure of an Intranucleosomal DNA Loop That Senses DNA Damage during Transcription.

Transcription through chromatin by RNA polymerase II (Pol II) is accompanied by the formation of small intranucleosomal DNA loops containing the enzyme (i-loops) that are involved in survival of core histones on the DNA and arrest of Pol II during the transcription of damaged DNA. However, the structures of i-loops have not been determined. Here, the structures of the intermediates formed during transcription through a nucleosome containing intact or damaged DNA were studied using biochemical approaches and electron microscopy. After RNA polymerase reaches position +24 from the nucleosomal boundary, the enzyme can backtrack to position +20, where DNA behind the enzyme recoils on the surface of the histone octamer, forming an i-loop that locks Pol II in the arrested state. Since the i-loop is formed more efficiently in the presence of SSBs positioned behind the transcribing enzyme, the loop could play a role in the transcription-coupled repair of DNA damage hidden in the chromatin structure.

H2A-H2B Histone Dimer Plasticity and Its Functional Implications.

The protein core of the nucleosome is composed of an H3-H4 histone tetramer and two H2A-H2B histone dimers. The tetramer organizes the central 60 DNA bp, while H2A-H2B dimers lock the flanking DNA segments. Being positioned at the sides of the nucleosome, H2A-H2B dimers stabilize the overall structure of the nucleosome and modulate its dynamics, such as DNA unwrapping, sliding, etc. Such modulation at the epigenetic level is achieved through post-translational modifications and the incorporation of histone variants. However, the detailed connection between the sequence of H2A-H2B histones and their structure, dynamics and implications for nucleosome functioning remains elusive. In this work, we present a detailed study of H2A-H2B dimer dynamics in the free form and in the context of nucleosomes via atomistic molecular dynamics simulations (based on X. laevis histones). We supplement simulation results by comparative analysis of information in the structural databases. Particularly, we describe a major dynamical mode corresponding to the bending movement of the longest H2A and H2B α-helices. This overall bending dynamics of the H2A-H2B dimer were found to be modulated by its interactions with DNA, H3-H4 tetramer, the presence of DNA twist-defects with nucleosomal DNA and the amino acid sequence of histones. Taken together, our results shed new light on the dynamical mechanisms of nucleosome functioning, such as nucleosome sliding, DNA-unwrapping and their epigenetic modulation.

N-Terminal Tails of Histones H2A and H2B Differentially Affect Transcription by RNA Polymerase II In Vitro.

Histone N-terminal tails and their post-translational modifications affect various biological processes, often in a context-specific manner; the underlying mechanisms are poorly studied. Here, the role of individual N-terminal tails of histones H2A/H2B during transcription through chromatin was analyzed in vitro. spFRET data suggest that the tail of histone H2B (but not of histone H2A) affects nucleosome stability. Accordingly, deletion of the H2B tail (amino acids 1-31, but not 1-26) causes a partial relief of the nucleosomal barrier to transcribing RNA polymerase II (Pol II), likely facilitating uncoiling of DNA from the histone octamer during transcription. Taken together, the data suggest that residues 27-31 of histone H2B stabilize DNA-histone interactions at the DNA region localized ~25 bp in the nucleosome and thus interfere with Pol II progression through the region localized 11-15 bp in the nucleosome. This function of histone H2B requires the presence of the histone H2A N-tail that mediates formation of nucleosome-nucleosome dimers; however, nucleosome dimerization per se plays only a minimal role during transcription. Histone chaperone FACT facilitates transcription through all analyzed nucleosome variants, suggesting that H2A/H2B tails minimally interact with FACT during transcription; therefore, an alternative FACT-interacting domain(s) is likely involved in this process.

Electron microscopy analysis of ATP-independent nucleosome unfolding by FACT.

FACT is a histone chaperone that participates in nucleosome removal and reassembly during transcription and replication. We used electron microscopy to study FACT, FACT:Nhp6 and FACT:Nhp6:nucleosome complexes, and found that all complexes adopt broad ranges of configurations, indicating high flexibility. We found unexpectedly that the DNA binding protein Nhp6 also binds to the C-terminal tails of FACT subunits, inducing more open geometries of FACT even in the absence of nucleosomes. Nhp6 therefore supports nucleosome unfolding by altering both the structure of FACT and the properties of nucleosomes. Complexes formed with FACT, Nhp6, and nucleosomes also produced a broad range of structures, revealing a large number of potential intermediates along a proposed unfolding pathway. The data suggest that Nhp6 has multiple roles before and during nucleosome unfolding by FACT, and that the process proceeds through a series of energetically similar intermediate structures, ultimately leading to an extensively unfolded form.

Histone dynamics mediate DNA unwrapping and sliding in nucleosomes.

Nucleosomes are elementary building blocks of chromatin in eukaryotes. They tightly wrap ∼147 DNA base pairs around an octamer of histone proteins. How nucleosome structural dynamics affect genome functioning is not completely clear. Here we report all-atom molecular dynamics simulations of nucleosome core particles at a timescale of 15 microseconds. At this timescale, functional modes of nucleosome dynamics such as spontaneous nucleosomal DNA breathing, unwrapping, twisting, and sliding were observed. We identified atomistic mechanisms of these processes by analyzing the accompanying structural rearrangements of the histone octamer and histone-DNA contacts. Octamer dynamics and plasticity were found to enable DNA unwrapping and sliding. Through multi-scale modeling, we showed that nucleosomal DNA dynamics contribute to significant conformational variability of the chromatin fiber at the supranucleosomal level. Our study further supports mechanistic coupling between fine details of histone dynamics and chromatin functioning, provides a framework for understanding the effects of various chromatin modifications.

Molecular Mechanisms of Oncogenesis through the Lens of Nucleosomes and Histones.

At the cellular level, cancer is the disease of both the genome and the epigenome, and the interplay between genetic mutations and epigenetic states may occur at the level of elementary chromatin units, the nucleosomes. They are formed by a segment of DNA wrapped around an octamer of histone proteins. In this review, we survey various mechanisms of cancer etiology and progression mediated by histones and nucleosomes. In particular, we discuss the effects of mutations in histones, changes in their expression and slicing on epigenetic dysregulation and carcinogenesis. The links between cancer phenotypes and differential expression of histone variants and isoforms are summarized. Finally, we discourse the geometric and steric effects of DNA compaction in nucleosomes on DNA mutation rate, interactions with transcription factors, including pioneer transcription factors, and prospects of cancer cells' genome and epigenome editing.

Histone Octamer Structure Is Altered Early in ISW2 ATP-Dependent Nucleosome Remodeling.

Nucleosomes are the fundamental building blocks of chromatin that regulate DNA access and are composed of histone octamers. ATP-dependent chromatin remodelers like ISW2 regulate chromatin access by translationally moving nucleosomes to different DNA regions. We find that histone octamers are more pliable than previously assumed and distorted by ISW2 early in remodeling before DNA enters nucleosomes and the ATPase motor moves processively on nucleosomal DNA. Uncoupling the ATPase activity of ISW2 from nucleosome movement with deletion of the SANT domain from the C terminus of the Isw2 catalytic subunit traps remodeling intermediates in which the histone octamer structure is changed. We find restricting histone movement by chemical crosslinking also traps remodeling intermediates resembling those seen early in ISW2 remodeling with loss of the SANT domain. Other evidence shows histone octamers are intrinsically prone to changing their conformation and can be distorted merely by H3-H4 tetramer disulfide crosslinking.

Linking chromatin composition and structural dynamics at the nucleosome level.

Nucleosomes are fundamental units of chromatin compaction, which organize ∼200 DNA base pairs using an octamer of histone proteins. Their ubiquitous presence in the cell nucleus since the first eukaryotes compelled the chromatin machinery to coevolve and learn how to exploit various modes of nucleosome dynamics and sense differences in nucleosome composition. Alterations to histone or DNA sequences, post-translational modifications (PTM) of histones, recruitment of chromatin proteins modulate nucleosome dyn amics and provide epigenetic regulation to the DNA processing pathways (transcription, replication, repair, etc.). Our understanding of this complex interplay between nucleosome composition, dynamics and functioning is constantly evolving through new insights and discoveries. In this review, we highlight recent contributions to the field while attempting to organize them in a unified framework.

Structural interpretation of DNA-protein hydroxyl-radical footprinting experiments with high resolution using HYDROID.

Hydroxyl-radical footprinting (HRF) is a powerful method for probing structures of nucleic acid-protein complexes with single-nucleotide resolution in solution. To tap the full quantitative potential of HRF, we describe a protocol, hydroxyl-radical footprinting interpretation for DNA (HYDROID), to quantify HRF data and integrate them with atomistic structural models. The stages of the HYDROID protocol are extraction of the lane profiles from gel images, quantification of the DNA cleavage frequency at each nucleotide and theoretical estimation of the DNA cleavage frequency from atomistic structural models, followed by comparison of experimental and theoretical results. Example scripts for each step of HRF data analysis and interpretation are provided for several nucleosome systems; they can be easily adapted to analyze user data. As input, HYDROID requires polyacrylamide gel electrophoresis (PAGE) images of HRF products and optionally can use a molecular model of the DNA-protein complex. The HYDROID protocol can be used to quantify HRF over DNA regions of up to 100 nucleotides per gel image. In addition, it can be applied to the analysis of RNA-protein complexes and free RNA or DNA molecules in solution. Compared with other methods reported to date, HYDROID is unique in its ability to simultaneously integrate HRF data with the analysis of atomistic structural models. HYDROID is freely available. The complete protocol takes ~3 h. Users should be familiar with the command-line interface, the Python scripting language and Protein Data Bank (PDB) file formats. A graphical user interface (GUI) with basic functionality (HYDROID_GUI) is also available.

Molecular basis of CENP-C association with the CENP-A nucleosome at yeast centromeres.

Histone CENP-A-containing nucleosomes play an important role in nucleating kinetochores at centromeres for chromosome segregation. However, the molecular mechanisms by which CENP-A nucleosomes engage with kinetochore proteins are not well understood. Here, we report the finding of a new function for the budding yeast Cse4/CENP-A histone-fold domain interacting with inner kinetochore protein Mif2/CENP-C. Strikingly, we also discovered that AT-rich centromere DNA has an important role for Mif2 recruitment. Mif2 contacts one side of the nucleosome dyad, engaging with both Cse4 residues and AT-rich nucleosomal DNA. Both interactions are directed by a contiguous DNA- and histone-binding domain (DHBD) harboring the conserved CENP-C motif, an AT hook, and RK clusters (clusters enriched for arginine-lysine residues). Human CENP-C has two related DHBDs that bind preferentially to DNA sequences of higher AT content. Our findings suggest that a DNA composition-based mechanism together with residues characteristic for the CENP-A histone variant contribute to the specification of centromere identity.

Hydroxyl-radical footprinting combined with molecular modeling identifies unique features of DNA conformation and nucleosome positioning.

Nucleosomes are the most abundant protein-DNA complexes in eukaryotes that provide compaction of genomic DNA and are implicated in regulation of transcription, DNA replication and repair. The details of DNA positioning on the nucleosome and the DNA conformation can provide key regulatory signals. Hydroxyl-radical footprinting (HRF) of protein-DNA complexes is a chemical technique that probes nucleosome organization in solution with a high precision unattainable by other methods. In this work we propose an integrative modeling method for constructing high-resolution atomistic models of nucleosomes based on HRF experiments. Our method precisely identifies DNA positioning on nucleosome by combining HRF data for both DNA strands with the pseudo-symmetry constraints. We performed high-resolution HRF for Saccharomyces cerevisiae centromeric nucleosome of unknown structure and characterized it using our integrative modeling approach. Our model provides the basis for further understanding the cooperative engagement and interplay between Cse4p protein and the A-tracts important for centromere function.

MS_HistoneDB, a manually curated resource for proteomic analysis of human and mouse histones.

BACKGROUND: Histones and histone variants are essential components of the nuclear chromatin. While mass spectrometry has opened a large window to their characterization and functional studies, their identification from proteomic data remains challenging. Indeed, the current interpretation of mass spectrometry data relies on public databases which are either not exhaustive (Swiss-Prot) or contain many redundant entries (UniProtKB or NCBI). Currently, no protein database is ideally suited for the analysis of histones and the complex array of mammalian histone variants. RESULTS: We propose two proteomics-oriented manually curated databases for mouse and human histone variants. We manually curated >1700 gene, transcript and protein entries to produce a non-redundant list of 83 mouse and 85 human histones. These entries were annotated in accordance with the current nomenclature and unified with the "HistoneDB2.0 with Variants" database. This resource is provided in a format that can be directly read by programs used for mass spectrometry data interpretation. In addition, it was used to interpret mass spectrometry data acquired on histones extracted from mouse testis. Several histone variants, which had so far only been inferred by homology or detected at the RNA level, were detected by mass spectrometry, confirming the existence of their protein form. CONCLUSIONS: Mouse and human histone entries were collected from different databases and subsequently curated to produce a non-redundant protein-centric resource, MS_HistoneDB. It is dedicated to the proteomic study of histones in mouse and human and will hopefully facilitate the identification and functional study of histone variants.

Genomic profiling of multiple sequentially acquired tumor metastatic sites from an "exceptional responder" lung adenocarcinoma patient reveals extensive genomic heterogeneity and novel somatic variants driving treatment response.

We used next-generation sequencing to identify somatic alterations in multiple metastatic sites from an "exceptional responder" lung adenocarcinoma patient during his 7-yr course of ERBB2-directed therapies. The degree of heterogeneity was unprecedented, with ∼1% similarity between somatic alterations of the lung and lymph nodes. One novel translocation, PLAG1-ACTA2, present in both sites, up-regulated ACTA2 expression. ERBB2, the predominant driver oncogene, was amplified in both sites, more pronounced in the lung, and harbored an L869R mutation in the lymph node. Functional studies showed increased proliferation, migration, metastasis, and resistance to ERBB2-directed therapy because of L869R mutation and increased migration because of ACTA2 overexpression. Within the lung, a nonfunctional CDK12, due to a novel G879V mutation, correlated with down-regulation of DNA damage response genes, causing genomic instability, and sensitivity to chemotherapy. We propose a model whereby a subclone metastasized early from the primary site and evolved independently in lymph nodes.

Large-scale ATP-independent nucleosome unfolding by a histone chaperone.

DNA accessibility to regulatory proteins is substantially influenced by nucleosome structure and dynamics. The facilitates chromatin transcription (FACT) complex increases the accessibility of nucleosomal DNA, but the mechanism and extent of its nucleosome reorganization activity are unknown. Here we determined the effects of FACT from the yeast Saccharomyces cerevisiae on single nucleosomes by using single-particle Förster resonance energy transfer (spFRET) microscopy. FACT binding results in dramatic ATP-independent, symmetrical and reversible DNA uncoiling that affects at least 70% of the DNA within a nucleosome, occurs without apparent loss of histones and proceeds via an 'all-or-none' mechanism. A mutated version of FACT is defective in uncoiling, and a histone mutation that suppresses phenotypes caused by this FACT mutation in vivo restores the uncoiling activity in vitro. Thus, FACT-dependent nucleosome unfolding modulates the accessibility of nucleosomal DNA, and this activity is an important function of FACT in vivo.

Nucleosomal Barrier to Transcription: Structural Determinants and Changes in Chromatin Structure.

Packaging of DNA into chromatin affects all processes on DNA. Nucleosomes present a strong barrier to transcription, raising important questions about the nature and the mechanisms of overcoming the barrier. Recently it was shown that DNA sequence, DNA-histone interactions and backtracking by RNA polymerase II (Pol II) all contribute to formation of the barrier. After partial uncoiling of nucleosomal DNA from histone octamer by Pol II and backtracking of the enzyme, nucleosomal DNA recoils on the octamer, locking Pol II in the arrested state. Histone chaperones and transcription factors TFIIS, TFIIF and FACT facilitate transcription through chromatin using different molecular mechanisms.