DNA Compaction Research Articles

Understanding and optimising the transfection of lipopolyplexes formulated in saline: the effects of peptide and serum.

Lipopolyplexes (LPDs) are of considerable interest for use as gene delivery vehicles. Here LPDs have been prepared from cationic vesicles (composed of a 1 : 1 molar ratio of DOTMA with the neutral helper lipid, DOPE), singly branched cationic peptides and plasmid DNA. All peptides contained a linker sequence (cleaved by endosomal furin) attached to a targeting sequence selected to bind human airway epithelial cells and mediate gene delivery. The current study investigates the effects of novel Arg-containing cationic peptide sequences on the biophysical and transfection properties of LPDs. Mixed His/Arg cationic peptides were of particular interest, as these sequences have not been previously used in LPD formulations. Lengthening the number of cationic residues in a homopolymer from 6 to 12 in each branch reduced transfection using LPDs, most likely due to increased DNA compaction hindering the release of pDNA within the target cell. Furthermore, LPDs containing mixed Arg-containing peptides, particularly an alternating Arg/His sequence exhibited an increase in transfection, probably because of their optimal ability to complex and subsequently release pDNA. To confer stability in serum, LPDs were prepared in 0.12 M sodium chloride solution (as opposed to the more commonly used water) yielding multilamellar LPDs with very high levels of size reproducibility and DNA protection, especially when compared to the (unilamellar) LPDs formed in water. Significantly for the clinical applications of the LPDs, those prepared in the presence of sodium chloride retained high levels of transfection in the presence of media supplemented with fetal bovine serum. This work therefore represents a significant advance for the optimisation of LPD formulation for gene delivery, under physiologically relevant conditions, in vivo.

Multi-phase separation in mitochondrial nucleoids and eukaryotic nuclei.

In mammalian cells, besides nuclei, mitochondria are the only semi-autonomous organelles possessing own DNA organized in the form of nucleoids. While eukaryotic nuclear DNA compaction, chromatin compartmentalization and transcription are regulated by phase separation, our recent work proposed a model of mitochondrial nucleoid self-assembly and transcriptional regulation by multi-phase separation. Herein, we summarized the phase separation both in the nucleus and mitochondrial nucleoids, and did a comparison of the organization and activity regulating, which would provide new insight into the understanding of both architecture and genetics of nucleus and mitochondrial nucleoids.

DNA compaction enhances the sensitivity of fluorescence-based nucleic acid assays: a game changer in point of care sensors?

Fluorescence-based nucleic acid assays frequently exhibit a feeble signal at low analyte concentrations, necessitating complex, expensive methods such as the development of sequence-specific oligo tags, molecular beacons, and chemical modifications to maintain high detection sensitivity. Hence, there is growing interest in accomplishing fluorescence enhancement in nucleic acid assays using robust and cost-effective strategies. The study exploits the use of two compaction agents, PEG 8000 and CTAB, to compact the ITS-2 amplicon of the fungus Candida albicans and evaluates the effect of both of these agents on the fluorescence intensity of SYTO-9 labelled nucleic acids. Conventional fluorometric measurements showed that both CTAB and PEG 8000 enhanced the emission intensity by ∼1.2- and 2-fold, respectively. Furthermore, we leveraged paper-based spot tests and distance-based assays to validate the effect of DNA compaction for enhancing sensitivity in the point-of-care context. The spot assay performed on paper with compacted samples showed an increase in the emission intensity of SYTO-9 and this was manifested by an elevated G channel intensity in the order of PEG 8000 compacted > CTAB compacted > amplified. Moreover, in the distance-based assay, the PEG 8000 compacted sample was found to migrate farther compared to CTAB compacted and amplified DNA samples at amplicon concentrations, 15 μg ml-1 and 39.65 μg ml-1. The limit of detection (LOD) for PEG 8000 and CTAB compacted samples on both paper-spot and distance-based assays were found to be 0.4 μg ml-1 and 0.5 μg ml-1, respectively. Hence our work provides an overview of employing DNA compaction as an approach for enhancing the sensitivity of fluorescence-based point-of-care nucleic acid assays without the need for cumbersome sensitivity enhancement methods.

Previously Unidentified Histone H1-Like Protein Is Involved in Cell Division and Ribosome Biosynthesis in Toxoplasma gondii.

Chromatin dynamics can regulate all DNA-dependent processes. Access to DNA within chromatin is orchestrated mainly by histones and their posttranslational modifications (PTMs). Like other eukaryotes, the apicomplexan parasite Toxoplasma gondii encodes four canonical histones and five histone variants. In contrast, the linker histone (H1) has never been identified in apicomplexan parasites. In other eukaryotes, histone H1 compacts the chromatin by linking the nucleosome and increasing the DNA compaction. H1 is a multifunctional protein and can be involved in different steps of DNA metabolism or associated with protein complexes related to distinct biological processes. We have identified a novel protein in T. gondii ("TgH1-like") that, although lacking the globular domain of mammalian H1, is remarkably like the H1-like proteins of bacteria and trypanosomatids. Our results demonstrate that TgH1-like is a nuclear protein associated with chromatin and other histones. Curiously, TgH1-like is also in the nucleolus and associated with ribosomal proteins, indicating a versatile function in this parasite. Although knockout of the tgh1-like gene does not affect the cell cycle, it causes endopolygeny and asynchronous division. Interestingly, mutation of posttranslationally modified amino acids results in defects in cell division like those in the Δtgh1-like mutant, showing that these sites are important for protein function. Furthermore, in the bradyzoite stage, this protein is expressed only in dividing parasites, reinforcing its importance in cell division. Indeed, the absence of TgH1-like decreases compaction of peripheral chromatin, confirming its role in the chromatin modulation in T. gondii. IMPORTANCE Histone H1, or linker histone, is an important protein that binds to the nucleosome, aiding chromatin compaction. Here, we characterize for the first time a linker histone in T. gondii, named TgH1-like. It is a small and basic protein that corresponds only to the C-terminal portion of the human H1 but is similar to histone H1 from trypanosomatids and bacteria. TgH1-like is located in the nucleus, interacts with nucleosome histones, and acts in chromatin structure and cell division. Our findings show for the first time the presence of a histone H1 protein in an apicomplexan parasite and will provide new insights into cell division and chromatin dynamics in T. gondii and related parasites.

Extracting and characterizing protein-free megabase-pair DNA for in vitro experiments

Chromosome structure and function is studied using various cell-based methods as well as with a range of invitro single-molecule techniques on short DNA substrates. Here, we present a method to obtain megabase-pair-length deproteinated DNA for invitro studies. We isolated chromosomes from bacterial cells and enzymatically digested the native proteins. Mass spectrometry indicated that 97%-100% of DNA-binding proteins are removed from the sample. Fluorescence microscopy analysis showed an increase in the radius of gyration of the DNA polymers, while the DNA length remained megabase-pair sized. In proof-of-concept experiments using these deproteinated long DNA molecules, we observed DNA compaction upon adding the DNA-binding protein Fis or PEG crowding agents and showed that it is possible to track the motion of a fluorescently labeled DNA locus. These results indicate the practical feasibility of a "genome-in-a-box" approach to study chromosome organization from the bottom up.

Stabilization of DNA fork junctions by Smc5/6 complexes revealed by single-molecule imaging.

SMC complexes play key roles in genome maintenance, where they ensure efficient genome replication and segregation. The SMC complex Smc5/6 is a crucial player in DNA replication and repair, yet many molecular features that determine its roles are unclear. Here, we use single-molecule microscopy to investigate Smc5/6's interaction with DNA. We find that Smc5/6 forms oligomers that dynamically redistribute on dsDNA by 1D diffusion and statically bind to ssDNA. Using combined force manipulation and single-molecule microscopy, we generate ssDNA-dsDNA junctions that mimic structures present in DNA repair intermediates or replication forks. We show that Smc5/6 accumulates at these junction sites, stabilizes the fork, and promotes the retention of RPA. Our observations provide a model for the complex's enrichment at sites of replication stress and DNA lesions from where it coordinates the recruitment and activation of downstream repair proteins.

Calix[4]arene Polyamine Triazoles: Synthesis, Aggregation and DNA Binding

Artificial gene delivery systems are in great demand from both scientific and practical biomedical points of view. In this paper, we present the synthesis of a new click chemistry calix[4]arene precursor with free lower rim and new water-soluble calixarene triazoles with 12 amino-groups on the upper rim (one with free phenol hydroxyl groups and two another containing four butyl or tetradecyl fragments). Aggregation in the series of amino-triazole calixarenes of different lipophilicity (calixarene with free phenol hydroxyl groups or butyl and tetradecyl fragments on the lower rim) was studied using dynamic light scattering and fluorescent pyrene probe. It was found that calix[4]arene with a free lower rim, like alkyl-substituted butyl calix[4]arene, forms stable submicron aggregates 150-200 nm in size, while the more lipophilic tetradecyl -substituted calix[4]arene forms micellar aggregates19 nm in size. Using UV-Vis spectroscopy, fluorimetry and CD, it was shown that amino-triazole calix[4]arenes bind to calf thymus DNA by classical intercalation. According to DLS and TEM data, all studied macrocycles cause significant DNA compaction, forming stable nanoparticles 50-20 nm in size. Among all studied calix[4]arenes the most lipophilic tetradecyl one proved to be the best for both binding and compaction of DNA.

Nanopore Sensing of DNA-Histone Complexes on Nucleosome Arrays.

The location of nucleosomes in DNA and their structural stability are critical in regulating DNA compaction, site accessibility, and epigenetic gene regulation. Here, we combine the nanopore platform-based fast and label-free single-molecule detection technique with a voltage-dependent force rupture assay to detect distinct structures on nucleosomal arrays and then to induce breakdown of individual nucleosome complexes. Specifically, we demonstrate direct measurement of distinct nucleosome structures present on individual 12-mer arrays. A detailed event analysis showed that nucleosomes are present as a combination of complete and partial structures, during translocation through the pore. By comparing with the voltage-dependent translocation of the mononucleosomes, we find that the partial nucleosomes result from voltage-dependent structural disintegration of nucleosomes. High signal-to-noise detection of heterogeneous levels in translocation of 12-mer array molecules quantifies the heterogeneity and nucleosomal substructure sizes on the arrays. These results facilitate the understanding of electrostatic interactions responsible for the integrity of the nucleosome structure and possible mechanisms of its unraveling by chromatin remodeling enzymes. This study also has potential applications in chromatin profiling.

Rapid Single-Pot Assembly of Modular Chromatin Proteins for Epigenetic Engineering.

Chromatin is the nucleoprotein complex that organizes genomic DNA in the nuclei of eukaryotic cells. Chromatin-modifying enzymes and chromatin-binding regulators generate chromatin states that affect DNA compaction, repair, gene expression, and ultimately cell phenotype. Many natural chromatin mediators contain subdomains that can be isolated and recombined to build synthetic regulators and probes. Engineered chromatin proteins make up a growing collection of new tools for cell engineering and can help deepen our understanding of the mechanism by which chromatin features, such as modifications of histones and DNA, contribute to the epigenetic states that govern DNA-templated processes. To support efficient exploration of the large combinatorial design space of synthetic chromatin proteins, we have developed a Golden Gate assembly method for one-step construction of protein-encoding recombinant DNA. A set of standard 2-amino acid linkers allows facile assembly of any combination of up to four protein modules, obviating the need to design different compatible overhangs to ligate different modules. Beginning with the identification of protein modules of interest, a synthetic chromatin protein can be built and expressed in vitro or in cells in under 2weeks.

Determination of Ram (Ovis aries) Sperm DNA Damage Due to Oxidative Stress: 8-OHdG Immunodetection Assay vs. SCSA®.

Conventional DNA analysis techniques can hardly detect DNA damage in ruminant spermatozoa due to high DNA compaction in these cells. Furthermore, these techniques cannot discriminate whether the damage is due to oxidative stress. The main purpose of this study was to evaluate the efficacy of two techniques for determining DNA damage in ovine sperm when the source of that damage is oxidative stress. Semen samples from twenty Manchega rams (Ovis aries) were collected and cryopreserved. After thawing, the samples were subjected to different levels of oxidative stress, and DNA oxidation was quantified using an 8-hydroxy-2′-deoxyguanosine (8-OHdG) immunodetection assay and Sperm Chromatin Structure Assay (SCSA®). For this purpose, we evaluated five different concentrations of an oxidation solution (H2O2/FeSO4•7H2O) on ram sperm DNA. Our study with the 8-OHdG immunodetection assay shows that there are higher values for DNA oxidation in samples that were subjected to the highest oxidative stress (8 M H2O2/800 µM FeSO4•7H2O) and those that were not exposed to high oxidative stress, but these differences were not significant (p ≥ 0.05). The two SCSA® parameters considered, DNA fragmentation index (DFI %) and high DNA stainability (HDS %), showed significant differences between samples that were subjected to high concentrations of the oxidation agent and those that were not (p < 0.05). We can conclude that the 8-OHdG immunodetection assay and SCSA® detect DNA damage caused by oxidative stress in ovine sperm under high oxidative conditions; SCSA® is a more straightforward method with more accurate results. For these reasons, an oxidative-stress-specific assay such as 8-OHdG immunodetection is not needed to measure DNA damage caused by oxidative stress in ram sperm samples.

The Domino Effect: Nucleosome Dynamics and the Regulation of Base Excision Repair Enzymes

DNA damage is induced by exogenous and endogenous sources, creating a variety of lesions. However, the cellular repair machinery that addresses and corrects this damage must contend with the fact that genomic DNA is sequestered in the nucleoprotein complex of chromatin. As the minimal unit of DNA compaction, the nucleosome core particle (NCP) is a major determinant of repair and poses unique barriers to DNA accessibility. This review outlines how the base excision repair (BER) pathway is modulated by the NCP and describes the structural and dynamic factors that influence the ability of BER enzymes to find and repair damage. Structural characteristics of the NCP such as nucleobase positioning and occupancy will be explored along with factors that impact the dynamic nature of NCPs to increase mobilization of nucleosomal DNA. We will discuss how altering the dynamics of NCPs initiates a domino effect that results in the regulation of BER enzymes.

Condensin DC loads and spreads from recruitment sites to create loop-anchored TADs in C. elegans.

Condensins are molecular motors that compact DNA via linear translocation. In Caenorhabditis elegans, the X-chromosome harbors a specialized condensin that participates in dosage compensation (DC). Condensin DC is recruited to and spreads from a small number of recruitment elements on the X-chromosome (rex) and is required for the formation of topologically associating domains (TADs). We take advantage of autosomes that are largely devoid of condensin DC and TADs to address how rex sites and condensin DC give rise to the formation of TADs. When an autosome and X-chromosome are physically fused, despite the spreading of condensin DC into the autosome, no TAD was created. Insertion of a strong rex on the X-chromosome results in the TAD boundary formation regardless of sequence orientation. When the same rex is inserted on an autosome, despite condensin DC recruitment, there was no spreading or features of a TAD. On the other hand, when a 'super rex' composed of six rex sites or three separate rex sites are inserted on an autosome, recruitment and spreading of condensin DC led to the formation of TADs. Therefore, recruitment to and spreading from rex sites are necessary and sufficient for recapitulating loop-anchored TADs observed on the X-chromosome. Together our data suggest a model in which rex sites are both loading sites and bidirectional barriers for condensin DC, a one-sided loop-extruder with movable inactive anchor.

Evaluation of cryodamage in small abalone, Haliotis diversicolor, spermatozoa by using a flow cytometer

AbstractWe used flow cytometry to determine the quality of small abalone, Haliotis diversicolor, sperm before freezing and after thawing. We investigated the effects of cryopreservation on the characteristics of small abalone semen and determined the motility and fertilization capacity of the pre‐freezing and post‐thawed sperm. The percentages of motility and fertility were 61 ± 2% and 67 ± 1%, respectively, for the post‐thawed sperm and 90 ± 4% and 92 ± 0%, respectively, for the pre‐freezing sperm. Sperm cells were stained with specific fluorescent dyes to measure plasma and acrosomal membrane integrity, mitochondrial status, oxidation level, DNA compaction, and viability through flow cytometry. The frozen–thawed sperm exhibited significantly higher mitopotential activity (p &lt; 0.05, damaged mitochondria; 25.01 ± 1.18%) and oxidation value (p &lt; 0.01, free radicals; 63.79 ± 3.93%) compared with the pre‐freezing sperm. The oxidation level was the most sensitive signal of the cryopreservation‐induced small abalone sperm damage. Flow cytometry is valuable for the objective, accurate, and rapid assessment of pre‐freezing and post‐thaw small abalone sperm quality.

Single-nucleus RNA-seq reveals that MBD5, MBD6, and SILENZIO maintain silencing in the vegetative cell of developing pollen.

Silencing of transposable elements (TEs) drives the evolution of numerous redundant mechanisms of transcriptional regulation. Arabidopsis MBD5, MBD6, and SILENZIO act as TE repressors downstream of DNA methylation. Here, we show, via single-nucleus RNA-seq of developing male gametophytes, that these repressors are critical for TE silencing in the pollen vegetative cell, a companion cell important for fertilization that undergoes chromatin decompaction. Instead, other silencing mutants (met1, ddm1, mom1, morc) show loss of silencing in all pollen nucleus types and somatic cells. We show that TEs repressed by MBD5/6 gain chromatin accessibility in wild-type vegetative nuclei despite remaining silent, suggesting that loss of DNA compaction makes them sensitive to loss of MBD5/6. Consistently, crossing mbd5/6 to histone 1 mutants, which have decondensed chromatin in leaves, reveals derepression of MBD5/6-dependent TEs in leaves. MBD5/6 and SILENZIO thus act as a silencing system particularly important when chromatin compaction is compromised.

Analysis of Cell Cycle and DNA Compaction Dependent Subnuclear Distribution of Histone Marks.

In eukaryotes, the organization of DNA wrapped around histones regulates DNA-dependent processes. Changes in epigenetic modifications modulate the compaction of DNA into chromatin and, thus, regulate DNA metabolism in time and space. Hence, to catalog the spatiotemporal epigenetic information and its relation to the dynamic nuclear landscape is of paramount importance. Here, we present a method, based on FiJi and the statistical image analysis tool nucim(R), to classify in 3D the nuclear DNA compaction in single interphase cells. We, furthermore, mapped the distribution of (epi)genetic marks and nuclear proteins/processes to the compaction classes along with their dynamics over the cell cycle. These techniques allow to catalog and quantify the dynamic changes in the epigenome in space and time and in single cells.

Single-Molecule Imaging of the Phase Separation-Modulated DNA Compaction to Study Transcriptional Repression.

Liquid-liquid phase separation (LLPS) often induces the formation of biomolecule condensates at the cellular level. The importance of this phenomenon has been demonstrated in many important biological functions, such as in transcription. However, the biophysical nature of LLPS containing transcriptional machinery has not yet been carefully examined. Here, we give an overview of a novel high-throughput single-molecule technique, termed as DNA Curtains. It was established recently to dissect the DNA compaction process in real time. The experimental procedures are further discussed in detail in the context of the biomolecular condensates of a transcription repressor.

Phosphorylation Regulation of a Histone-like HU Protein from Deinococcus radiodurans.

Histone-like proteins are small molecular weight DNA-binding proteins that are widely distributed in prokaryotes. These proteins have multiple functions in cellular structures and processes, including the morphological stability of the nucleoid, DNA compactness, DNA replication, and DNA repair. Deinococcus radiodurans, an extremophilic microorganism, has extraordinary DNA repair capability and encodes an essential histone-like protein, DrHU. We aim to investigate the phosphorylation regulation role of a histone-like HU protein from Deinococcus radiodurans. LC-MS/MS analysis was used to determine the phosphorylation site of endogenous DrHU. The predicted structure of DrHU-DNA was obtained from homology modeling (Swissmodel) using Staphylococcus aureus HU-DNA structure (PDB ID: 4QJU) as the starting model. Two types of mutant proteins T37E and T37A were generated to explore their DNA binding affinity. Complemented-knockout strategy was used to generate the ΔDrHU/pk-T37A and ΔDrHU/pk-T37E strains for growth curves and phenotypical analyses. The phosphorylation site Thr37, which is present in most bacterial HU proteins, is located at the putative protein-DNA interaction interface of DrHU. Compared to the wild-type protein, one in which this threonine is replaced by glutamate to mimic a permanent state of phosphorylation (T37E) showed enhanced double-stranded DNA binding but a weakened protective effect against hydroxyl radical cleavage. Complementation of T37E in a DrHU-knockout strain caused growth defects and sensitized the cells to UV radiation and oxidative stress. Phosphorylation modulates the DNA-binding capabilities of the histone-like HU protein from D. radiodurans, which contributes to the environmental adaptation of this organism.

Mechanism of endosomal escape by pH-responsive nucleic-acid vectors.

Successful intracellular delivery of nucleic acids (NAs) hinges on many factors, one of them being NAs' efficacious escape from endosomes. As competent NA vectors, pH-responsive gemini surfactants (GSs) might achieve high efficacy by facilitating endosomal escape. However, how the GSs assist the escape remains debated as many proposed mechanisms still lack experimental support, which hinders replication and further improvement of the efficient delivery. Here, via UV, fluorescence spectroscopy, and small-angle neutron scattering (SANS), we examined a pH-responsive GS's and a pH-unresponsive GS's capabilities to compact DNA and withstand binding competition, and their interactions with model endosomal and lysosomal membranes, at varied pHs. Acidification-driven enhancement of DNA-compaction capability and of stability against binding competition were found specific to the pH-responsive GS. Alongside the pH-responsive GS's structural perturbation to the membranes as observed with SANS, the features suggest that pH-responsive GSs facilitate endosomal escape by releasing excess GS molecules from DNA-GS complexes upon acidification in endosome maturation, with the released GS molecules disrupting endosomal and lysosomal membranes and thereby assisting the escape. A general design principle for NA vectors is proposed on the basis of this experimental finding.

PSV-A-7 Quality Assessments of Sperm from Gnrh-II Receptor Knockdown (KD) Boars

Abstract Pigs are the only livestock species encoding functional proteins for both the second form of gonadotropin-releasing hormone (GnRH-II) and its receptor (GnRHR-II). Previously, we determined that GnRHR-II localizes to the connecting piece of ejaculated sperm, whereas GnRH-II is present within seminal plasma, suggesting an autocrine/paracrine interaction influencing porcine sperm function. To examine the GnRH-II/GnRHR-II system further, we produced a swine line with reduced endogenous expression of GnRHR-II (GnRHR-II KD). Therefore, our objective was to assess sperm quality of ejaculates collected from GnRHR-II KD (n = 3) and littermate control (n = 3) boars. Semen was extended (37.5 x 106 sperm/mL) in Androstar Plus and subjected to flow cytometry (Kuster Research and Consulting, Inc.; Atkinson, IL). Assays measured DNA compaction, membrane fluidity and viability, acrosome integrity, mitochondrial depolarization, and morphology. Data were analyzed using the MIXED procedure of SAS; the model included ejaculate as the experimental unit and line as a fixed effect. DNA compaction, membrane fluidity, acrosome integrity, mitochondrial depolarization, and morphology were not statistically different between lines (P &amp;gt; 0.05). However, the percentage of sperm with a viable (intact) plasma membrane was reduced in GnRHR-II KD boars (87.4 ± 0.8%) compared with controls (89.6 ± 0.8%; P &amp;lt; 0.05). Although differences did not reach significance, DNA compaction (a measure of sperm chromatin integrity when undergoing acid stress) was doubled and mitochondrial depolarization (reflective of mitochondrial function) was 11% greater in sperm from transgenic males relative to littermate controls, indicating that GnRHR-II KD sperm are more susceptible to environmental stressors and have altered energy production. Thus, these initial data suggest disruptions in the function and fertilizing capacity of GnRHR-II KD sperm, implying that GnRHR-II may represent a potential biomarker for boar fertility. Supported by USDA/NIFA AFRI (2017-67015-26508) and Hatch Multistate (NEB-26-244) funds. USDA is an equal opportunity provider and employer.

The Effects of Histone H2B Ubiquitylations on the Nucleosome Structure and Internucleosomal Interactions.

Eukaryotic gene compaction takes place at multiple levels to package DNA to chromatin and chromosomes. Two of the most fundamental levels of DNA packaging are at the nucleosome and dinucleosome stacks. The nucleosome is the basic gene-packing unit and is composed of DNA wrapped around a histone core. Nucleosomes stack with one another for further compaction of DNA. The first stacking step leads to dinucleosome formation, which is driven by internucleosomal interactions between various parts of two nucleosomes. Histone proteins are rich targets for post-translational modifications, some of which affect the structure of the nucleosome and the interactions between nucleosomes. These effects are often implicated in the regulation of various genomic transactions. In particular, histone H2B ubiquitylation has been associated with facilitated transcription and hexasome formation. Here, we employed semi-synthetically ubiquitylated histone H2B and single-molecule FRET to investigate the effects of H2B ubiquitylations at lysine 34 (H2BK34) and lysine 120 (H2BK120) on the structure of the nucleosome and the interactions between two nucleosomes. Our results suggest that H2BK34 ubiquitylation widens the DNA gyre gap in the nucleosome and stabilizes long- and short-range internucleosomal interactions while H2BK120 ubiquitylation does not affect the nucleosome structure or internucleosomal interactions. These results suggest potential roles for H2B ubiquitylations in facilitated transcription and hexasome formation while maintaining the structural integrity of chromatin.