Nucleosomal Arrays Research Articles

Chromatin Organization and Phase Separation of Histones

A concept that formation of heterochromatin is driven by liquid-liquid phase separation (LLPS) has emerged in recent years. Using live cell fluorescence imaging, we show that the linker histone H1 mediates LLPS of heterochromatin in the human cell nucleus. We also find that the core histone H2A readily undergoes LLPS with DNA and nucleosome arrays of varying length in vitro and in doing so does not disrupt the nucleosome core, as observed by FRET measurements. LLPS with histones is enhanced by the presence of free nucleotides like ATP. Consistently, in cells under ATP depleted conditions, the number of H1 rich liquid-like domains in their nuclei are significantly diminished. These findings add yet another dimension to the role of histones in organizing and maintaining the genome inside the nucleus.

On the role of transcription in positioning nucleosomes.

Nucleosome positioning is crucial for the genome’s function. Though the role of DNA sequence in positioning nucleosomes is well understood, a detailed mechanistic understanding on the impact of transcription remains lacking. Using numerical simulations, we investigated the dependence of nucleosome density profiles on transcription level across multiple species. We found that the low nucleosome affinity of yeast, but not mouse, promoters contributes to the formation of phased nucleosomes arrays for inactive genes. For the active genes, a heterogeneous distribution of +1 nucleosomes, caused by a tug-of-war between two types of remodeling enzymes, is essential for reproducing their density profiles. In particular, while positioning enzymes are known to remodel the +1 nucleosome and align it toward the transcription start site (TSS), spacer enzymes that use a pair of nucleosomes as their substrate can shift the nucleosome array away from the TSS. Competition between these enzymes results in two types of nucleosome density profiles with well- and ill-positioned +1 nucleosome. Finally, we showed that Pol II assisted histone exchange, if occurring at a fast speed, can abolish the impact of remodeling enzymes. By elucidating the role of individual factors, our study reconciles the seemingly conflicting results on the overall impact of transcription in positioning nucleosomes across species.

Chromatin hierarchical branching visualized at the nanoscale by electron microscopy.

Chromatin is spatially organized in a hierarchical manner by virtue of single nucleosomes condensing into higher order chromatin structures, conferring various mechanical properties and biochemical signals. These higher order chromatin structures regulate genomic function by organization of the heterochromatin and euchromatin landscape. Less is known about its transition state from higher order heterochromatin to the lower order nucleosome form, and there is no information on its physical properties. We have developed a facile method of electron microscopy visualization to reveal the interphase chromatin in eukaryotic cells and its organization into hierarchical branching structures. We note that chromatin hierarchical branching can be distinguished at four levels, clearly indicating the stepwise transition from heterochromatin to euchromatin. The protein–DNA density across the chromatin fibers decreases during the transition from compacted heterochromatin to dispersed euchromatin. Moreover, the thickness of the chromatin ranges between 10 to 270 nm, and the controversial 30 nm chromatin fiber exists as a prominent intermediate structure. This study provides important insights into higher order chromatin organization which plays a key role in diseases such as cancer.

Linker DNA Length is a Key to Tri-nucleosome Folding

The folding of a nucleosome array has long been one of the fundamental and unsolved problems in chromatin biology. In this study, we address how nucleosome array folding depends on the length of linker DNA. We performed molecular dynamics simulations of a tri-nucleosome, a minimal model of chromatin folding, with various linker lengths (LLs) ranging from 20 to 40 base pairs (bps). We found that the tri-nucleosome folding strongly depends on LLs, and classified the structure ensemble into five classes, named from trinuc-1 to trinuc-5. As a function of LL, the different classes appear, on average, every 2 bps with a period of 10 bps, and are characterized by distinct inter-nucleosome interactions. The trinuc-1 conformation corresponds to LL ~ 10n, where n is an integer, and is stabilized by the tight packing between the first and the third nucleosomes, consistent with a zigzag fiber form. Structures of the other four classes are more diverse and distributed continuously in the space of possible configurations. Histone-DNA electrostatic interactions in the tri-nucleosome are further analyzed.

H1 histones control the epigenetic landscape by local chromatin compaction.

H1 linker histones are the most abundant chromatin-binding proteins1. In vitro studies indicate that their association with chromatin determines nucleosome spacing and enables arrays of nucleosomes to fold into more compact chromatin structures. However, the in vivo roles of H1 are poorly understood2. Here we show that the local density of H1 controls the balance of repressive and active chromatin domains by promoting genomic compaction. We generated a conditional triple-H1-knockout mouse strain and depleted H1 in haematopoietic cells. H1 depletion in T cells leads to de-repression of T cell activation genes, a process that mimics normal T cell activation. Comparison of chromatin structure in normal and H1-depleted CD8+ T cells reveals that H1-mediated chromatin compaction occurs primarily in regions of the genome containing higher than average levels of H1: the chromosome conformation capture (Hi-C) B compartment and regions of the Hi-C A compartment marked by PRC2. Reduction of H1 stoichiometry leads to decreased H3K27 methylation, increased H3K36 methylation, B-to-A-compartment shifting and an increase in interaction frequency between compartments. In vitro, H1 promotes PRC2-mediated H3K27 methylation and inhibits NSD2-mediated H3K36 methylation. Mechanistically, H1 mediates these opposite effects by promoting physical compaction of the chromatin substrate. Our results establish H1 as a critical regulator of gene silencing through localized control of chromatin compaction, 3D genome organization and the epigenetic landscape.

Condensed Chromatin Behaves like a Solid on the Mesoscale In Vitro and in Living Cells

The association of nuclear DNA with histones to form chromatin is essential for temporal and spatial control of eukaryotic genomes. In this study, we examined the physical state of condensed chromatin invitro and invivo. Our invitro studies demonstrate that self-association of nucleosomal arrays under a wide range of solution conditions produces supramolecular condensates in which the chromatin is physically constrained and solid-like. By measuring DNA mobility in living cells, we show that condensed chromatin also exhibits solid-like behavior invivo. Representative heterochromatin proteins, however, display liquid-like behavior and coalesce around the solid chromatin scaffold. Importantly, euchromatin and heterochromatin show solid-like behavior even under conditions that produce limited interactions between chromatin fibers. Our results reveal that condensed chromatin exists in a solid-like state whose properties resist external forces and create an elastic gel and provides a scaffold that supports liquid-liquid phase separation of chromatin binding proteins.

Abstract PO-10: Histone 1 deficiency drives lymphoma through disruption of 3D chromatin architecture

Abstract Linker histone H1 binds to nucleosomes and causes chromatin compaction, yet little is known about their biologic function. Somatic missense mutations in histone 1 genes (HIST1H1B-E) occur in ~30% of follicular lymphomas and DLBCL, with significant mutual co-occurrence among these alleles, most frequently involving H1C and H1E. Examining whole exomes in 547 DLBCL patients, we found significant enrichment for H1 gene SNVs and copy number loss in the MCD class of ABC-DLBCLs as compared to other subtypes. Next we performed a genetic driver analysis in 101 DLBCLs with matched germline control to identify mutations significantly enriched over background somatic variation. We find that lymphoma-associated H1 alleles H1C and H1E are true genetic driver mutations in lymphomas. Lymphoma H1 mutations affect the H1 globular domain or C-terminus. We found that the globular domain mutants fail to bind to chromatin whereas C-ter mutants fail to compact chromatin as shown by atomic force microscopy, in vitro assembled nucleosome arrays, and FRAP assays in live cells. Hence both types of mutation confer loss of function. Constitutive H1C/E knockout mice are healthy and have no overt phenotype. However, immunization with T cell-dependent antigen caused significant GC hyperplasia (p=0.013) and disruption of polarity due to expansion in the number of centrocytes. Notably, H1C/EDKO GC B-cells readily outcompeted WT GC B-cells in mixed chimera experiments, indicating that they have superior fitness (p=0.0086). We performed RNA-seq in H1C/EDKO GC B-cells, which revealed an aberrant gene derepression signature. Strikingly, these same genes are upregulated during induced pluripotency (iPS cell) reprogramming and are normally silenced during early development by the PRC2 complex (p <0.05 to 1e-05). Indeed, histone mass spectrometry showed reduced H3K27me3 (p=0.0003) and increased H3K36me2 (p =0.001) in H1C/EDKO GC B cells. This prompted us to characterize the epigenome of purified H1C/EDKO vs WT GC B cells using Hi-C, ATAC-seq, and ChIP-seq for multiple histone marks. H1 deficiency causes profound architectural remodeling of the genome characterized by large-scale yet focal compartment shifts from compartment B to compartment A. The degree of decompaction results in distinct epigenetic states, primarily due to gain of H3K36me2 and/or loss of repressive H3K27me3. Strikingly, the H1C/EDKO primitive stem cell gene expression signature was highly significantly enriched (NES 1.24, FDR<0.001) in the RNA-seq profiles of independent cohorts of DLBCL patients with H1C and H1E mutations. We crossed H1C+/-H1E+/- mice with VavP-Bcl2 transgenic mice and observed significant acceleration of lymphomagenesis (p=0.0001). Consistent with acquisition of stem cell characteristics, H1C+/-H1E+/-;VavP-Bcl2 but not VavP-Bcl2 primary lymphoma cells manifested lymphoma-initiating functionality after secondary transplantation into recipient animals. Citation Format: Nevin Z. Yusufova, Matt Teater, Andreas Kloetgen, Alexey Soshnev, Adewola Osunade, Christopher Chin, Ashley Doane, Louis Staudt, David Scott, Neil Kelleher, Aristotelis Tsirigos, Marcin Imielinski, Yael David, David Allis, Ethel Cesarman, Ari Melnick. Histone 1 deficiency drives lymphoma through disruption of 3D chromatin architecture [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-10.

Nucleosome Positioning Regulates the Establishment, Stability, and Inheritance of Heterochromatin in Saccharomyces cerevisiae

Heterochromatic domains are complex structures composed of nucleosome arrays that are bound by silencing factors. This composition raises the possibility that certain configurations of nucleosome arrays facilitate heterochromatic silencing. We tested this possibility in Saccharomyces cerevisiae by systematically altering the distance between heterochromatic nucleosome-depleted regions (NDRs), which is predicted to affect local nucleosome positioning by limiting how nucleosomes can be packed between NDRs. Consistent with this prediction, serial deletions that altered the distance between heterochromatic NDRs revealed a striking oscillatory relationship between inter-NDR distance and defects in nucleosome positioning. Furthermore, conditions that caused poor nucleosome positioning also led to defects in both heterochromatin stability and the ability of cells to generate and inherit epigenetic transcriptional states. These findings strongly suggest that nucleosome positioning can contribute to formation and maintenance of functional heterochromatin and point to previously unappreciated roles of NDR positioning within heterochromatic domains.

Interplay among ATP-Dependent Chromatin Remodelers Determines Chromatin Organisation in Yeast.

Cellular DNA is packaged into chromatin, which is composed of regularly-spaced nucleosomes with occasional gaps corresponding to active regulatory elements, such as promoters and enhancers, called nucleosome-depleted regions (NDRs). This chromatin organisation is primarily determined by the activities of a set of ATP-dependent remodeling enzymes that are capable of moving nucleosomes along DNA, or of evicting nucleosomes altogether. In yeast, the nucleosome-spacing enzymes are ISW1 (Imitation SWitch protein 1), Chromodomain-Helicase-DNA-binding (CHD)1, ISW2 (Imitation SWitch protein 2) and INOsitol-requiring 80 (INO80); the nucleosome eviction enzymes are the SWItching/Sucrose Non-Fermenting (SWI/SNF) family, the Remodeling the Structure of Chromatin (RSC) complexes and INO80. We discuss the contributions of each set of enzymes to chromatin organisation. ISW1 and CHD1 are the major spacing enzymes; loss of both enzymes results in major chromatin disruption, partly due to the appearance of close-packed di-nucleosomes. ISW1 and CHD1 compete to set nucleosome spacing on most genes. ISW1 is dominant, setting wild type spacing, whereas CHD1 sets short spacing and may dominate on highly-transcribed genes. We propose that the competing remodelers regulate spacing, which in turn controls the binding of linker histone (H1) and therefore the degree of chromatin folding. Thus, genes with long spacing bind more H1, resulting in increased chromatin compaction. RSC, SWI/SNF and INO80 are involved in NDR formation, either directly by nucleosome eviction or repositioning, or indirectly by affecting the size of the complex that resides in the NDR. The nature of this complex is controversial: some suggest that it is a RSC-bound “fragile nucleosome”, whereas we propose that it is a non-histone transcription complex. In either case, this complex appears to serve as a barrier to nucleosome formation, resulting in the formation of phased nucleosomal arrays on both sides.

Constructing arrays of nucleosome positioning sequences using Gibson Assembly for single-molecule studies

As the basic building blocks of chromatin, nucleosomes play a key role in dictating the accessibility of the eukaryotic genome. Consequently, nucleosomes are involved in essential genomic transactions such as DNA transcription, replication and repair. In order to unravel the mechanisms by which nucleosomes can influence, or be altered by, DNA-binding proteins, single-molecule techniques are increasingly employed. To this end, DNA molecules containing a defined series of nucleosome positioning sequences are often used to reconstitute arrays of nucleosomes in vitro. Here, we describe a novel method to prepare DNA molecules containing defined arrays of the ‘601’ nucleosome positioning sequence by exploiting Gibson Assembly cloning. The approaches presented here provide a more accessible and efficient means to generate arrays of nucleosome positioning motifs, and facilitate a high degree of control over the linker sequences between these motifs. Nucleosomes reconstituted on such arrays are ideal for interrogation with single-molecule techniques. To demonstrate this, we use dual-trap optical tweezers, in combination with fluorescence microscopy, to monitor nucleosome unwrapping and histone localisation as a function of tension. We reveal that, although nucleosomes unwrap at ~20 pN, histones (at least histone H3) remain bound to the DNA, even at tensions beyond 60 pN.

Global Examination of the Effects of Methamphetamine on Histone Post‐Translational Modifications in the Rat Striatum

Methamphetamine (METH) is a highly addictive psychostimulant that has been associated with deleterious physical and social consequences. A variety of factors ranging from life experiences to genetics are believed to play a role in the addiction process. Post‐translational modifications (PTMs) on histone proteins can lead to alterations in the organization of nucleosomal arrays and the accessibility of transcriptional factors to DNA as a form of epigenetic regulation. This study investigated the effects of chronic METH self‐administration in rats on PTMs of histones H3 and H4 in the striatum using liquid chromatography‐mass spectrometry. Chronic METH use induced group downregulation of the following PTMs on histone H3: K9 dimethylation with unmodified S10 and T11 and K14 acetylation; K18 methylation with K23 acetylation; and K79 dimethylation with T80 phosphorylation. Individual economic demand for METH was positively correlated with H3K79 methylation, H3K14 acetylation, H4K5 acetylation, H4K8 acetylation, H4K12 acetylation, and H4K16 acetylation. The significant decrease of H3T80 phosphorylation supports a downregulation of neural progenitor cell proliferation and neurogenesis, which is consistent with previously discovered inhibition of astrocyte and neural progenitor propagation as a result of METH use. Our novel findings, showing correlation between individual demand and the abundance of post‐translational modifications, offer a novel insight into the epigenetic regulation associated with METH use and further the current understanding of the impact of substance use on epigenome remodeling, while also elucidating potential therapeutic targets for future studies.Support or Funding InformationThis project was funded by the Hamel Center for Undergraduate Research whose financial support made this research possible.

Rett syndrome-causing mutations compromise MeCP2-mediated liquid-liquid phase separation of chromatin.

Rett syndrome (RTT), a severe postnatal neurodevelopmental disorder, is caused by mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2). MeCP2 is a chromatin organizer regulating gene expression. RTT-causing mutations have been shown to affect this function. However, the mechanism by which MeCP2 organizes chromatin is unclear. In this study, we found that MeCP2 can induce compaction and liquid-liquid phase separation of nucleosomal arrays in vitro, and DNA methylation further enhances formation of chromatin condensates by MeCP2. Interestingly, RTT-causing mutations compromise MeCP2-mediated chromatin phase separation, while benign variants have little effect on this process. Moreover, MeCP2 competes with linker histone H1 to form mutually exclusive chromatin condensates in vitro and distinct heterochromatin foci in vivo. RTT-causing mutations reduce or even abolish the ability of MeCP2 to compete with histone H1 and to form chromatin condensates. Together, our results identify a novel mechanism by which phase separation underlies MeCP2-mediated heterochromatin formation and reveal the potential link between this process and the pathology of RTT.

Heterologous peptide display on chromatin nanofibers: A new strategy for peptide vaccines

Chromatin organization starts from a “beads-on-a string” 10 nm fiber, a basic nucleosomal structure consisting of DNA and core histones. Given its regular nucleosome array on DNA backbone where N-terminal tails of each histone are exposed on the surface of chromatin fiber, we hypothesized that chromatin can be utilized as a heterologous peptide carrier to elicit a peptide-specific immune response. The plasmid DNA containing the Widom’s clone 601 sequence and the recombinant chimeric histones containing the peptide derived from ras oncogene (G12V) were used to assemble the chromatin fiber in vitro. The immunogenicity of the assembled chromatin was tested in mice as a single vaccine component or formulated with adjuvants. G12V tagged-chromatin co-administered with adjuvants induced higher antibody responses against the G12V peptide than vaccination with adjuvant alone, while chimeric histones did not generate a significant antibody response. Interestingly, splenocytes from mice vaccinated with the G12V tagged-chromatin vaccine did not generate significant antigen-specific cytokine responses. Our studies suggest that chromatin can be utilized as an effective carrier of antigenic peptides for inducing specific antibody responses.

Unraveling the Nucleosome through Microscopic Simulations

The DNA of higher organisms associates with histone proteins to organize into an array of nucleosomes. The nucleosome, however, is not one monolithic structure, but rather, refers to an ensemble of assemblies that vary in the chemical modifications present, histone composition, and surrounding DNA sequence. Here, we connect computation with experimental efforts to reveal the effects of acetylation on the H4 histone tail, the impact of a centromere-specific variant on histone assembly structure and dynamics, and the influence of nucleotide sequence on nucleosome breathing. We discover increasing levels of acetylation introduce greater transient order to the H4 histone tail with the mono-acetylation of lysine 16 causing specific structural effects; replacing histone H3 with centromere-specific CENP-A confers enhanced flexibility to dimers and entire nucleosomes, but, interestingly, more rigidity to tetramers; and lastly, that DNA sequences with greater CG content form more stable nucleosomes, undergoing fewer events in which the outer stretches of DNA spontaneously detach and reattach to the histone core.

Beads on a string-nucleosome array arrangements and folding of the chromatin fiber.

Understanding how the genome is structurally organized as chromatin is essential for understanding its function. Here, we review recent developments that allowed the readdressing of old questions regarding the primary level of chromatin structure, the arrangement of nucleosomes along the DNA and the folding of the nucleosome fiber in nuclear space. In contrast to earlier views of nucleosome arrays as uniformly regular and folded, recent findings reveal heterogeneous array organization and diverse modes of folding. Local structure variations reflect a continuum of functional states characterized by differences in post-translational histone modifications, associated chromatin-interacting proteins and nucleosome-remodeling enzymes.

Solid-state NMR 13C, 15N assignments of human histone H3 in the nucleosome core particle.

Nucleosome core particle (NCP), the basic unit of chromatin in eukaryotic cells, consists of ~ 147bp DNA wrapped around a histone octamer (HO) formed by two H2A-H2B dimers and one (H3-H4)2 tetramer. Histones undergo various post-translational modifications (PTMs), which regulates genomic activities in different cellular phases. High-resolution structures have been solved for many nucleosomes primarily including NCPs. However, the atomic-resolution structures of nucleosome arrays and chromatin fiber, as well as the dynamics of nucleosomes remain poorly understood. Solid-state NMR (SSNMR) is one of the premier techniques to answer these questions. In this study, we present the13C and 15Nchemical shifts assignments for the globular domain of human histone H3 (hH3) using multidimensional SSNMR experiments. The obtained spectra are of outstanding resolution and the assignments are nearly 100% complete for the backbone 13C and 15N spins of R42-G132 and ~ 80% when taking into account the side chains. The secondary structure derived from the chemical shifts agrees with the previously reported X-ray crystal structure. The reported chemical shifts can be carried over to future SSNMR studies of structure and dynamics of hH3 in NCPs, nucleosome array, chromatin fibers and nucleosome-protein complexes.

Condensed Chromatin Behaves Like a Solid on the Mesoscale in Vitro and in Living Cells

The association of nuclear DNA with histones to form chromatin is essential to the temporal and spatial control of eukaryotic genomes. In this study, we examined the physical state of condensed chromatin in vitro and in vivo. Our in vitro studies demonstrate that self-association of nucleosomal arrays under a wide range of solution conditions produces supramolecular condensates in which the chromatin is physically constrained and solid-like. By measuring DNA mobility in living cells, we show that condensed chromatin also exhibits solid-like behavior in vivo. Representative heterochromatin proteins, however, display liquid-like behavior and coalesce around the solid chromatin scaffold. Importantly, both euchromatin and heterochromatin show solid-like behavior even under conditions that produce limited interactions between chromatin fibers. Our results reveal that condensed chromatin exists in a solid-like state whose properties resist external forces and create a matrix that supports liquid-liquid phase separation of chromatin binding proteins in the nucleus.

Architecture of the chromatin remodeler RSC and insights into its nucleosome engagement.

Eukaryotic DNA is packaged into nucleosome arrays, which are repositioned by chromatin remodeling complexes to control DNA accessibility. The Saccharomyces cerevisiae RSC (Remodeling the Structure of Chromatin) complex, a member of the SWI/SNF chromatin remodeler family, plays critical roles in genome maintenance, transcription, and DNA repair. Here, we report cryo-electron microscopy (cryo-EM) and crosslinking mass spectrometry (CLMS) studies of yeast RSC complex and show that RSC is composed of a rigid tripartite core and two flexible lobes. The core structure is scaffolded by an asymmetric Rsc8 dimer and built with the evolutionarily conserved subunits Sfh1, Rsc6, Rsc9 and Sth1. The flexible ATPase lobe, composed of helicase subunit Sth1, Arp7, Arp9 and Rtt102, is anchored to this core by the N-terminus of Sth1. Our cryo-EM analysis of RSC bound to a nucleosome core particle shows that in addition to the expected nucleosome-Sth1 interactions, RSC engages histones and nucleosomal DNA through one arm of the core structure, composed of the Rsc8 SWIRM domains, Sfh1 and Npl6. Our findings provide structural insights into the conserved assembly process for all members of the SWI/SNF family of remodelers, and illustrate how RSC selects, engages, and remodels nucleosomes.

Histone 1 Mutations Drive Lymphomagenesis By Inducing Primitive Stem Cell Functions and Epigenetic Instructions through Profound 3D Re-Organization of the B-Cell Genome

Melnick: Janssen: Research Funding; Epizyme: Consultancy; Constellation: Consultancy.

Acetylation of the histone H3 tail domain regulates base excision repair on higher-order chromatin structures

Despite recent evidence suggesting that histone lysine acetylation contributes to base excision repair (BER) in cells, their exact mechanistic role remains unclear. In order to examine the influence of histone acetylation on the initial steps of BER, we assembled nucleosome arrays consisting of homogeneously acetylated histone H3 (H3K18 and H3K27) and measured the repair of a site-specifically positioned 2′-deoxyuridine (dU) residue by uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE1). We find that H3K18ac and H3K27ac differentially influence the combined activities of UDG/APE1 on compact chromatin, suggesting that acetylated lysine residues on the H3 tail domain play distinct roles in regulating the initial steps of BER. In addition, we show that the effects of H3 tail domain acetylation on UDG/APE1 activity are at the nucleosome level and do not influence higher-order chromatin folding. Overall, these results establish a novel regulatory role for histone H3 acetylation during the initiation of BER on chromatin.