Blocks Of Chromatin Research Articles

Molecular Dynamics Simulations of Nucleosomes Containing Histone Variant H2A.J

Histone proteins form the building blocks of chromatin—nucleosomes. Incorporation of alternative histone variants instead of the major (canonical) histones into nucleosomes is a key mechanism enabling epigenetic regulation of genome functioning. In humans, H2A.J is a constitutively expressed histone variant whose accumulation is associated with cell senescence, inflammatory gene expression, and certain cancers. It is sequence-wise very similar to the canonical H2A histones, and its effects on the nucleosome structure and dynamics remain elusive. This study employed all-atom molecular dynamics simulations to reveal atomistic mechanisms of structural and dynamical effects conferred by the incorporation of H2A.J into nucleosomes. We showed that the H2A.J C-terminal tail and its phosphorylated form have unique dynamics and interaction patterns with the DNA, which should affect DNA unwrapping and the availability of nucleosomes for interactions with other chromatin effectors. The dynamics of the L1-loop and the hydrogen bonding patterns inside the histone octamer were shown to be sensitive to single amino acid substitutions, potentially explaining the higher thermal stability of H2A.J nucleosomes. Taken together, our study demonstrated unique dynamical features of H2A.J-containing nucleosomes, which contribute to further understanding of the molecular mechanisms employed by H2A.J in regulating genome functioning.

On the Hunt for the Histone Code

Our genome is not made of naked DNA, but a fiber (chromatin) composed of DNA and proteins packaged into our chromosomes. The basic building block of chromatin is the nucleosome, which has two copies of each of the proteins called histones (H2A, H2B, H3, and H4) wrapped by 146 base pairs of DNA. Regions of our genetic material are found between the more open (euchromatin) and more compact (heterochromatin) regions of the genome that can be variably accessible to the underlying genes. Furthermore, post-translational modifications (PTMs) on histones, such as on H3, are critical for regulating chromatin accessibility and gene expression. While site specific antibodies were the tool of choice for histone PTM analysis in the early days (pre-2000s), enter Don Hunt changing the histone PTM field forever. Don’s clever thinking brought new innovative mass spectrometry-based approaches to the epigenetics field. His lab’s effort led to the discovery of many new histone modifications and methods to facilitate the detection and quantification of histone PTMs, which are still considered state of the art in the proteomics field today. Due to Don’s pioneering work in this area, many labs have been able to jump into the epigenetics field and “Hunt” down their own histone targets. A walkthrough of those early histone years in the Hunt Lab are described by three of us who were fortunate enough to be at the right place, at the right time.

Molecular dynamics simulations of nucleosomes are coming of age

AbstractUnderstanding the function of eukaryotic genomes, including the human genome, is undoubtedly one of the major scientific challenges of the 21st century. The cornerstone of eukaryotic genome organization is nucleosomes—elementary building blocks of chromatin about 10 nm in size that wrap DNA around an octamer of histone proteins. Nucleosomes are integral players in all genomic processes, including transcription, DNA replication and repair. They mediate genome regulation at the epigenetic level, bridging the discrete nature of the genetic information encoded in DNA with the analog physical nature of the intermolecular interactions required to access that information. Due to their relatively large size and dynamic nature, nucleosomes are difficult objects for experimental characterization. Molecular dynamics (MD) simulations have emerged over the years as a useful tool to complement experimental studies. Particularly in recent years, advances in computing power, refinement of MD force fields and codes have opened up new frontiers in terms of simulation timescales and quality for nucleosomes and related systems. It has become possible to elucidate in atomistic detail their functional dynamics modes such as DNA unwrapping and sliding, to characterize the effects of epigenetic modifications, DNA and protein sequence variation on nucleosome structure and stability, to describe the mechanisms governing nucleosome interactions with chromatin‐associated proteins and the formation of supranucleosome structures. In this review, we systematically analyzed all‐atom MD simulation studies of nucleosomes and related structures published since 2018 and discussed their relevance in the context of older studies, experimental data, and related coarse‐grained and multiscale studies.This article is categorized under: Software > Molecular Modeling Molecular and Statistical Mechanics > Molecular Dynamics and Monte‐Carlo Methods Structure and Mechanism > Computational Biochemistry and Biophysics

Nucleosomal asymmetry: a novel mechanism to regulate nucleosome function.

Nucleosomes constitute the fundamental building blocks of chromatin. They are comprised of DNA wrapped around a histone octamer formed of two copies each of the four core histones H2A, H2B, H3, and H4. Nucleosomal histones undergo a plethora of posttranslational modifications that regulate gene expression and other chromatin-templated processes by altering chromatin structure or by recruiting effector proteins. Given their symmetric arrangement, the sister histones within a nucleosome have commonly been considered to be equivalent and to carry the same modifications. However, it is now clear that nucleosomes can exhibit asymmetry, combining differentially modified sister histones or different variants of the same histone within a single nucleosome. Enabled by the development of novel tools that allow generating asymmetrically modified nucleosomes, recent biochemical and cell-based studies have begun to shed light on the origins and functional consequences of nucleosomal asymmetry. These studies indicate that nucleosomal asymmetry represents a novel regulatory mechanism in the establishment and functional readout of chromatin states. Asymmetry expands the combinatorial space available for setting up complex sets of histone marks at individual nucleosomes, regulating multivalent interactions with histone modifiers and readers. The resulting functional consequences of asymmetry regulate transcription, poising of developmental gene expression by bivalent chromatin, and the mechanisms by which oncohistones deregulate chromatin states in cancer. Here, we review recent progress and current challenges in uncovering the mechanisms and biological functions of nucleosomal asymmetry.

DNA Repair in Nucleosomes: Insights from Histone Modifications and Mutants.

DNA repair pathways play a critical role in genome stability, but in eukaryotic cells, they must operate to repair DNA lesions in the compact and tangled environment of chromatin. Previous studies have shown that the packaging of DNA into nucleosomes, which form the basic building block of chromatin, has a profound impact on DNA repair. In this review, we discuss the principles and mechanisms governing DNA repair in chromatin. We focus on the role of histone post-translational modifications (PTMs) in repair, as well as the molecular mechanisms by which histone mutants affect cellular sensitivity to DNA damage agents and repair activity in chromatin. Importantly, these mechanisms are thought to significantly impact somatic mutation rates in human cancers and potentially contribute to carcinogenesis and other human diseases. For example, a number of the histone mutants studied primarily in yeast have been identified as candidate oncohistone mutations in different cancers. This review highlights these connections and discusses the potential importance of DNA repair in chromatin to human health.

HDAC7: a promising target in cancer.

Histones have a vital function as components of nucleosomes, which serve as the fundamental building blocks of chromatin. Histone deacetylases (HDACs), which target histones, suppress gene transcription by compacting chromatin. This implies that HDACs have a strong connection to the suppression of gene transcription. Histone deacetylase 7 (HDAC7), a member of the histone deacetylase family, may participate in multiple cellular pathophysiological processes and activate relevant signaling pathways to facilitate the progression of different tumors by exerting deacetylation. In recent years, HDAC7 has been increasingly studied in the pathogenesis of tumors. Studies that are pertinent have indicated that it has a significant impact on the growth and metastasis of tumors, the formation of the vascular microenvironment, and the emergence of resistance to drugs. Therefore, HDAC7 could potentially function as a potent predictor for tumor prognosis and a promising target for mitigating drug resistance in tumors. This review primarily concentrates on elucidating the structure and function of HDAC7, its involvement in the development of various tumors, and its interplay with relevant signaling pathways. Meanwhile, we briefly discuss the research direction and prospect of HDAC7.

Nucleosome Dynamics Derived at the Single-Molecule Level Bridges Its Structures and Functions.

Nucleosome, the building block of chromatin, plays pivotal roles in all DNA-related processes. While cryogenic-electron microscopy (cryo-EM) has significantly advanced our understanding of nucleosome structures, the emerging field of single-molecule force spectroscopy is illuminating their dynamic properties. This technique is crucial for revealing how nucleosome behavior is influenced by chaperones, remodelers, histone variants, and post-translational modifications, particularly in their folding and unfolding mechanisms under tension. Such insights are vital for deciphering the complex interplay in nucleosome assembly and structural regulation, highlighting the nucleosome's versatility in response to DNA activities. In this Perspective, we aim to consolidate the latest advancements in nucleosome dynamics, with a special focus on the revelations brought forth by single-molecule manipulation. Our objective is to highlight the insights gained from studying nucleosome dynamics through this innovative approach, emphasizing the transformative impact of single-molecule manipulation techniques in the field of chromatin research.

Multiscale modeling reveals the ion-mediated phase separation of nucleosome core particles

Due to the vast length scale inside the cell nucleus, multiscale models are required to understand chromatin folding, structure, and dynamics and how they regulate genomic activities such as DNA transcription, replication, and repair. We study the interactions and structure of condensed phases formed by the universal building block of chromatin, the nucleosome core particle (NCP), using bottom-up multiscale coarse-grained (CG) simulations with a model extracted from all-atom MD simulations. In the presence of the multivalent cations Mg(H2O)62+ or CoHex3+, we analyze the internal structures of the NCP aggregates and the contributions of histone tails and ions to the aggregation patterns. We then derive a "super" coarse-grained (SCG) NCP model to study the macroscopic scale phase separation of NCPs. The SCG simulations show the formation of NCP aggregates with Mg(H2O)62+ concentration-dependent densities and sizes. Variation of the CoHex3+ concentrations results in highly ordered lamellocolumnar and hexagonal columnar phases in agreement with experimental data. The results give detailed insights into nucleosome interactions and for understanding chromatin folding in the cell nucleus.

Methods to investigate nucleosome structure and dynamics with single-molecule FRET.

The nucleosome is the fundamental building block of chromatin. Changes taking place at the nucleosome level are the molecular basis of chromatin transactions with various enzymes and factors. These changes are directly and indirectly regulated by chromatin modifications such as DNA methylation and histone post-translational modifications including acetylation, methylation, and ubiquitylation. Nucleosomal changes are often stochastic, unsynchronized, and heterogeneous, making it very difficult to monitor with traditional ensemble averaging methods. Diverse single-molecule fluorescence approaches have been employed to investigate the structure and structural changes of the nucleosome in the context of its interactions with various enzymes such as RNA Polymerase II, histone chaperones, transcription factors, and chromatin remodelers. We utilize diverse single-molecule fluorescence methods to study the nucleosomal changes accompanying these processes, elucidate the kinetics of these processes, and eventually learn the implications of various chromatin modifications in directly regulating these processes. The methods include two- and three-color single-molecule fluorescence resonance energy transfer (FRET), single-molecule fluorescence correlation spectroscopy, and fluorescence (co-)localization. Here we report the details of the two- and three-color single-molecule FRET methods we currently use. This report will help researchers design their single-molecule FRET approaches to investigating chromatin regulation at the nucleosome level.

Genome-wide maps of rare and atypical UV photoproducts reveal distinct patterns of damage formation and mutagenesis in yeast chromatin

Ultraviolet (UV) light induces different classes of mutagenic photoproducts in DNA, namely cyclobutane pyrimidine dimers (CPDs), 6-4 photoproducts (6-4PPs), and atypical thymine-adenine photoproducts (TA-PPs). CPD formation is modulated by nucleosomes and transcription factors (TFs), which has important ramifications for Ultraviolet (UV) mutagenesis. How chromatin affects the formation of 6-4PPs and TA-PPs is unclear. Here, we use UV damage endonuclease-sequencing (UVDE-seq) to map these UV photoproducts across the yeast genome. Our results indicate that nucleosomes, the fundamental building block of chromatin, have opposing effects on photoproduct formation. Nucleosomes induce CPDs and 6-4PPs at outward rotational settings in nucleosomal DNA but suppress TA-PPs at these settings. Our data also indicate that DNA binding by different classes of yeast TFs causes lesion-specific hotspots of 6-4PPs or TA-PPs. For example, DNA binding by the TF Rap1 generally suppresses CPD and 6-4PP formation but induces a TA-PP hotspot. Finally, we show that 6-4PP formation is strongly induced at the binding sites of TATA-binding protein (TBP), which is correlated with higher mutation rates in UV-exposed yeast. These results indicate that the formation of 6-4PPs and TA-PPs is modulated by chromatin differently than CPDs and that this may have important implications for UV mutagenesis.

SMC complexes can traverse physical roadblocks bigger than their ring size.

Ring-shaped structural maintenance of chromosomes (SMC) complexes like condensin and cohesin extrude loops of DNA. It remains, however, unclear how they can extrude DNA loops in chromatin that is bound with proteins. Here, we use invitro single-molecule visualization to show that nucleosomes, RNA polymerase, and dCas9 pose virtually no barrier to loop extrusion by yeast condensin. We find that even DNA-bound nanoparticles as large as 200nm, much bigger than the SMC ring size, also translocate into DNA loops during extrusion by condensin and cohesin. This even occurs for a single-chain version of cohesin in which the ring-forming subunits are covalently linked and cannot open to entrap DNA. The data show that SMC-driven loop extrusion has surprisingly little difficulty in accommodating large roadblocks into the loop. The findings also show that the extruded DNA does not pass through the SMC ring (pseudo)topologically, hence pointing to a nontopological mechanism for DNA loop extrusion.

Nucleosome sliding can influence the spreading of histone modifications.

Nucleosomes are the fundamental building blocks of chromatin that not only help in the folding of chromatin, but also in carrying epigenetic information. It is known that nucleosome sliding is responsible for dynamically organizing chromatin structure and the resulting gene regulation. Since sliding can move two neighboring nucleosomes physically close or away, can it play a role in the spreading of histone modifications? We investigate this by simulating a stochastic model that couples nucleosome dynamics with the kinetics of histone modifications. We show that the sliding of nucleosomes can affect the modification pattern as well as the time it takes to modify a given region of chromatin. Exploring different nucleosome densities and modification kinetic parameters, we show that nucleosome sliding can be important for creating histone modification domains. Our model predicts that nucleosome density coupled with sliding dynamics can create an asymmetric histone modification profile around regulatory regions. We also compute the probability distribution of modified nucleosomes and relaxation kinetics of modifications. Our predictions are comparable with known experimental results.

A Bottom-Up Coarse-Grained Model for Nucleosome-Nucleosome Interactions with Explicit Ions.

The nucleosome core particle (NCP) is a large complex of 145–147 base pairs of DNA and eight histone proteins and is the basic building block of chromatin that forms the chromosomes. Here, we develop a coarse-grained (CG) model of the NCP derived through a systematic bottom-up approach based on underlying all-atom MD simulations to compute the necessary CG interactions. The model produces excellent agreement with known structural features of the NCP and gives a realistic description of the nucleosome–nucleosome attraction in the presence of multivalent cations (Mg(H2O)62+ or Co(NH3)63+) for systems comprising 20 NCPs. The results of the simulations reveal structural details of the NCP–NCP interactions unavailable from experimental approaches, and this model opens the prospect for the rigorous modeling of chromatin fibers.

Abstract IA009: Cancer is a disease of epigenetic stochasticity

Abstract My colleagues and I proposed in 2006 (1) that increased epigenetic stochasticity is a driving force of tumor progression from its origin to metastasis and would allow rapid selection for tumor cell survival at the expense of the host. More recently (2), we have been pursuing an idea that natural selection will favor the emergence of genetic loci for epigenetic variance, not just mean, for loci in which the environment changes unpredictably but often enough, and these epigenetically variable loci are critical to normal embryonic development and injury response. The idea is also relevant to cancer, in that increased epigenetic stochasticity would allow rapid selection for tumor cell survival at the expense of the host. This model puts epigenetic instability at the heart of tumor progression and is the primary target of cancer mutations. Several recent observations from the laboratory point to a genome-scale disruption of the epigenome that involves large blocks of variable DNA methylation and chromatin affecting shores and islands and increasing gene expression variability (3,4). We also find large-scale reprogramming of chromatin and DNA modifications during the natural evolution of distant metastasis, with dependence on the oxidative branch of the pentose phosphate pathway (5). We have also developed mathematically rigorous Gibbs-Boltzmann-style epigenetic landscapes incorporating stochasticity, and we have shown its relationship to entropy in information theory (6). Recent data show that this approach identifies epigenetic and genetic drivers of cancer, using acute lymphoblastic leukemia as a model (7).

DNP-enhanced solid-state NMR spectroscopy of chromatin polymers

Chromatin is a DNA-protein polymer that represents the functional form of the genome. The main building block of chromatin is the nucleosome, a structure that contains 147 base pairs of DNA and two copies each of the histone proteins H2A, H2B, H3 and H4. Previous work has shown that magic angle spinning (MAS) NMR spectroscopy can capture the nucleosome at high resolution although studies have been challenging due to low sensitivity, the presence of dynamic and rigid components, and the complex interaction networks of nucleosomes within the chromatin polymer. Here, we use dynamic nuclear polarization (DNP) to enhance the sensitivity of MAS NMR experiments of nucleosome arrays at 100 K and show that well-resolved 13C-13C MAS NMR correlations can be obtained much more efficiently. We evaluate the effect of temperature on the chemical shifts and linewidths in the spectra and demonstrate that changes are relatively minimal and clustered in regions of histone-DNA or histone-histone contacts. We also compare samples prepared with and without DNA and show that the low temperature 13C-13C correlations exhibit sufficient resolution to detect chemical shift changes and line broadening for residues that form the DNA-histone interface. On the other hand, we show that the measurement of DNP-enhanced 15N-13C histone-histone interactions within the nucleosome core is complicated by the natural 13C abundance network in the sample. Nevertheless, the enhanced sensitivity afforded by DNP can be used to detect long-range correlations between histone residues and DNA. Overall, our experiments demonstrate that DNP-enhanced MAS NMR spectroscopy of chromatin samples yields spectra with high resolution and sensitivity and can be used to capture functionally relevant protein-DNA interactions that have implications for gene regulation and genome organization.

Real-time single-molecule sequencing of nucleosomal DNA and combinatory histone modifications detection with zero-mode waveguides

Nucleosome, the building block of chromatin, contains regulatory information apart from the 147bp DNA that wraps around it, such as the modifications on the histone tails which have been proven to play an important role in nucleosome dynamics and gene regulation. The prevalent method for measuring the presence of a certain histone modification across the genome is Chromatin immunoprecipitation sequencing (ChIP-seq), which has limitations in sensitivity, throughput, and signal-to-noise ratio as well as the inability to identify individual nucleosomes with combinatorial modifications such as the bivalent nucleosomes.

Synthesis of Oriented Hexasomes and Asymmetric Nucleosomes Using a Template Editing Process.

Nucleosomes, the structural building blocks of chromatin, possess 2-fold pseudo symmetry which can be broken through differential modification or removal of one copy of a pair of sister histones. The resultant asymmetric nucleosomes and hexasomes have been implicated in gene regulation, yet the use of these noncanonical substrates in chromatin biochemistry is limited, owing to the lack of efficient methods for their preparation. Here, we report a strategy that allows the orientation of these asymmetric species to be tightly controlled relative to the underlying DNA sequence. Our approach is based on the use of truncated DNA templates to assemble oriented hexasomes followed by DNA ligation and, in the case of asymmetric nucleosomes, addition of the missing heterotypic histones. We show that this approach is compatible with multiple nucleosome positioning sequences, allowing the generation of desymmetrized mononucleosomes and oligonucleosomes with varied DNA overhangs and heterotypic histone H2A/H2B dimer compositions. Using this technology, we examine the functional consequences of asymmetry on BRG1/BRM associated factor (BAF) complex-mediated chromatin remodeling. Our results indicate that cancer-associated histone mutations can reprogram the inherent activity of BAF chromatin remodeling to induce aberrant chromatin structure.

Molecular mechanism of UV damage modulation in nucleosomes

Exposure to ultraviolet (UV) light causes the formation of mutagenic cyclobutane pyrimidine dimers (CPDs) in cellular DNA. Previous studies have revealed that CPD formation in nucleosomes, the building blocks of chromatin, shows a striking ∼10 base pair (bp) periodic pattern. CPD formation is suppressed at positions where the DNA minor groove faces toward the histone octamer (minor-in) and elevated CPD formation at positions where the minor groove faces away from the histone octamer (minor-out). However, the molecular mechanism underlying this nucleosome photofootprint is unclear. Here, we analyzed ∼180 high-resolution nucleosome structures to characterize whether differences in DNA mobility or conformation are responsible for the CPD modulation in nucleosomes. Our results indicate that differences in DNA mobility cannot explain CPD modulation in nucleosome. Instead, we find that the sharp DNA bending around the histone octamer results in DNA conformations with structural parameters more susceptible to UV damage formation at minor-out positions and more resistant to CPD formation at minor-in positions. This analysis reveals the molecular mechanism responsible for periodic modulation of CPD formation and UV mutagenesis in nucleosomal DNA.

Abstract 2116: Promoter enrichment of subnucleosomes using breast cancer cell-free DNA

Abstract Breast cancer is the leading cancer diagnosis and the second major cause of cancer-related deaths in women worldwide. The use of non-invasive cell-free DNA (cfDNA) to detect and assess cancer dynamics has recently gained traction with a focus on targeted mutation panels. However, mutation panels have to contend with non-tumor origin of mutations like clonal hematopoiesis and the emergence of novel mutations in the tumor. A potential alternative is to harness the epigenomic, or non-genetic, features of cfDNA. In a healthy individual, blood cell turnover is the major contributor to the cfDNA pool. In cancer, tumor DNA adds to the cfDNA pool, which represents the end product of endogenous nucleases acting on accessible genomic DNA. Since genomic DNA is protected by histone proteins, cfDNA maps the chromatin structure of individual cells that turn over. Subnucleosomes (partially formed structures of nucleosomes that are the building blocks of chromatin) are by-products of transcription and thus reflect small protections enriched in active genes. The enrichment of subnucleosomes at promoters correlate with gene expression profiles of cfDNA tissues-of-origin and based on this knowledge, we hypothesized that we could reconstruct the transcriptional profiles of cancer cells by selectively targeting promoter regions. We first extracted cfDNA from 9 healthy female donors and 8 patient donors with various stages of breast cancer (Stage I, n=1; Stage III, n=1; Stage IV, n=4). We used a single-stranded DNA library protocol that robustly captures highly nicked cfDNA, enabling the genome-wide mapping of both nucleosomes and subnucleosomes. We performed targeted capture of promoter-proximal, nucleosome-protected sequences in cfDNA using non-overlapping tiling probes from transcription start sites to 300 bp downstream. After whole-genome sequencing of pooled libraries, we show that 76.4% (n=13/17) of samples exhibited >10X enrichment compared to unenriched samples, and 64.7% (n=11/17) of samples exhibited >100X enrichment. The enriched samples allowed us to profile >10,000 genes, whereas unenriched samples enabled profiling between 100-10,000 genes at an average sequencing depth of 40 million reads. Overall, we obtain higher depth per sequencing read after enrichment resulting in a high-resolution snapshot of promoter-nucleosome dynamics derived by probing only ~0.18% of the genome. We believe that this significant advancement will allow us not only to identify presence of cancer but also detect distinct tumor states. Citation Format: Alexis Zukowski, Amy Han, Peter Kabos, Srinivas Ramachandran. Promoter enrichment of subnucleosomes using breast cancer cell-free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2116.

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.