Nucleosome Core Research Articles

The TAFs of TFIID Bind and Rearrange the Topology of the TATA-Less RPS5 Promoter

The general transcription factor TFIID is a core promoter selectivity factor that recognizes DNA sequence elements and nucleates the assembly of a pre-initiation complex (PIC). The mechanism by which TFIID recognizes the promoter is poorly understood. The TATA-box binding protein (TBP) is a subunit of the multi-protein TFIID complex believed to be key in this process. We reconstituted transcription from highly purified components on a ribosomal protein gene (RPS5) and discovered that TFIIDΔTBP binds and rearranges the promoter DNA topology independent of TBP. TFIIDΔTBP binds ~200 bp of the promoter and changes the DNA topology to a larger extent than the nucleosome core particle. We show that TBP inhibits the DNA binding activities of TFIIDΔTBP and conclude that the complete TFIID complex may represent an auto-inhibited state. Furthermore, we show that the DNA binding activities of TFIIDΔTBP are required for assembly of a PIC poised to select the correct transcription start site (TSS).

Error-prone replication of a 5-formylcytosine-mediated DNA-peptide cross-link in human cells

DNA-protein cross-links can interfere with chromatin architecture, block DNA replication and transcription, and interfere with DNA repair. Here we synthesized a DNA 23-mer containing a site-specific DNA-peptide cross-link (DpC) by cross-linking an 11-mer peptide to the DNA epigenetic mark 5-formylcytosine in synthetic DNA and used it to generate a DpC-containing plasmid construct. Upon replication of the DpC-containing plasmid in HEK 293T cells, approximately 9% of progeny plasmids contained targeted mutations and 5% semitargeted mutations. Targeted mutations included C→T transitions and C deletions, whereas semitargeted mutations included several base substitutions and deletions near the DpC lesion. To identify DNA polymerases involved in DpC bypass, we comparatively studied translesion synthesis (TLS) efficiency and mutagenesis of the DpC in a series of cell lines with TLS polymerase knockouts or knockdowns. Knockdown of either hPol ι or hPol ζ reduced the mutation frequency by nearly 50%. However, the most significant reduction in mutation frequency (50%-70%) was observed upon simultaneous knockout of hPol η and hPol κ with knockdown of hPol ζ, suggesting that these TLS polymerases play a critical role in error-prone DpC bypass. Because TLS efficiency of the DpC construct was not significantly affected in TLS polymerase-deficient cells, we examined a possible role of replicative DNA polymerases in their bypass and determined that hPol δ and hPol ϵ can accurately bypass the DpC. We conclude that both replicative and TLS polymerases can bypass this DpC lesion in human cells but that mutations are induced mainly by TLS polymerases.

Structure and function of the Orc1 BAH-nucleosome complex

The Origin Recognition Complex (ORC) is essential for replication, heterochromatin formation, telomere maintenance and genome stability in eukaryotes. Here we present the structure of the yeast Orc1 BAH domain bound to the nucleosome core particle. Our data reveal that Orc1, unlike its close homolog Sir3 involved in gene silencing, does not appear to discriminate between acetylated and non-acetylated lysine 16, modification states of the histone H4 tail that specify open and closed chromatin respectively. We elucidate the mechanism for this unique feature of Orc1 and hypothesize that its ability to interact with nucleosomes regardless of K16 modification state enables it to perform critical functions in both hetero- and euchromatin. We also show that direct interactions with nucleosomes are essential for Orc1 to maintain the integrity of rDNA borders during meiosis, a process distinct and independent from its known roles in silencing and replication.

Positional Dependence of DNA Hole Transfer Efficiency in Nucleosome Core Particles.

Electron deficient "holes" migrate over long distances through the π-system in free DNA. Hole transfer efficiency (HTE) is strongly dependent on sequence and π-stacking. However, there is no consensus regarding the effects of nucleosome core particle (NCP) environment on hole migration. We quantitatively determined HTE in free DNA and NCPs by independently generating holes at specific positions in DNA. The relative HTE varied widely with respect to position within the NCP and proximity to tyrosine, which suppresses hole transfer. These data indicate that hole transfer in chromatin will be affected by the DNA sequence and its position with respect to histone proteins within NCPs.

The nucleosome acidic patch directly interacts with subunits of the Paf1 and FACT complexes and controls chromatin architecture in vivo.

The nucleosome core regulates DNA-templated processes through the highly conserved nucleosome acidic patch. While structural and biochemical studies have shown that the acidic patch controls chromatin factor binding and activity, few studies have elucidated its functions in vivo. We employed site-specific crosslinking to identify proteins that directly bind the acidic patch in Saccharomyces cerevisiae and demonstrated crosslinking of histone H2A to Paf1 complex subunit Rtf1 and FACT subunit Spt16. Rtf1 bound to nucleosomes through its histone modification domain, supporting its role as a cofactor in H2B K123 ubiquitylation. An acidic patch mutant showed defects in nucleosome positioning and occupancy genome-wide. Our results provide new information on the chromatin engagement of two central players in transcription elongation and emphasize the importance of the nucleosome core as a hub for proteins that regulate chromatin during transcription.

Disordered region of H3K9 methyltransferase Clr4 binds the nucleosome and contributes to its activity.

Heterochromatin is a distinctive chromatin structure that is essential for chromosome segregation, genome stability and regulation of gene expression. H3K9 methylation (H3K9me), a hallmark of heterochromatin, is deposited by the Su(var)3-9 family of proteins; however, the mechanism by which H3K9 methyltransferases bind and methylate the nucleosome is poorly understood. In this work we determined the interaction of Clr4, the fission yeast H3K9 methyltransferase, with nucleosomes using nuclear magnetic resonance, biochemical and genetic assays. Our study shows that the Clr4 chromodomain binds the H3K9me3 tail and that both, the chromodomain and the disordered region connecting the chromodomain and the SET domain, bind the nucleosome core. We show that interaction of the disordered region with the nucleosome core is independent of H3K9me and contributes to H3K9me in vitro and in vivo. Moreover, we show that those interactions with the nucleosome core are contributing to de novo deposition of H3K9me and to establishment of heterochromatin.

Reaction of Histone H1 with trans-Platinum Complexes and the Effect on DNA Platination.

Transplatin is an inactive platinum drug; however, a number of analogues, such as trans-EE and trans-PtTz, demonstrate promising antitumor activity in vitro and in vivo. Although the ultimate target is nuclear DNA, increasing evidence indicate that proteins also play important roles in the display of antitumor activity. The linker histone H1 is situated by the portal between the unwrapped DNA and the nucleosome core. Our recent study revealed that H1 can readily react with cisplatin, and the adducts tend to form ternary complexes with DNA. In this work, we have investigated the reaction of histone H1 with two antitumor-active trans-oriented complexes, trans-EE and trans-PtTz, and the effect of H1 upon the platination of DNA. The results show that trans-platinum drugs are much more reactive than cisplatin toward H1. Interestingly, in addition to the expected bidentate adducts (by displacement of the two labile chlorido ligands), also a tridentate adduct can be formed by displacement of one nonlabile carrier ligand of trans-EE or trans-PtTz. The trans-Pt/H1 adducts can then react with DNA and generate protein-Pt-DNA ternary complexes. Additionally, platinum can be transferred from trans-Pt/H1 adducts to DNA, generating binary trans-Pt/DNA complexes. Such a transfer of the platinum agent to DNA was not observed in the reaction of cisplatin. Furthermore, the detailed investigation carried out on a model peptide indicates that H1 promotes the DNA platination by trans- EE, while it reduces that of trans-PtTz and cisplatin. These results suggest that H1 can play a key role in the DNA platination and modulate the efficacy of different platinum agents.

Histone H2A Variants Enhance the Initiation of Base Excision Repair in Nucleosomes.

Substituting histone variants for their canonical counterparts can profoundly alter chromatin structure, thereby impacting multiple biological processes. Here, we investigate the influence of histone variants from the H2A family on the excision of uracil (U) by the base excision repair (BER) enzymes uracil DNA glycosylase (UDG) and single-strand selective monofunctional uracil DNA glycosylase. Using a DNA population with globally distributed U:G base pairs, enhanced excision is observed in H2A.Z and macroH2A-containing nucleosome core particles (NCPs). The U with reduced solution accessibility exhibit limited UDG activity in canonical NCPs but are more readily excised in variant NCPs, reflecting the ability of these variants to facilitate excision at sites that are otherwise poorly repaired. We also find that U with the largest increase in the level of excision in variant NCPs are clustered in regions with differential structural features between the variants and canonical H2A. Within 35-40 bp of the DNA terminus in macroH2A NCPs, the activities of both glycosylases are comparable to that on the free duplex. We show that this high level of activity results from two distinct species within the macroH2A NCP ensemble: octasomes and hexasomes. These observations reveal potential functions for H2A variants in promoting BER and preventing mutagenesis within the context of chromatin.

013 NETs generate structured antimicrobial peptide-nucleosome immune complexes with inter-DNA spacings optimal for TLR9 activation

The molecular pathogenesis of psoriasis is characterized in part by breakdown of immune tolerance to self-DNA. Recent work demonstrates that the human antimicrobial peptide (AMP) LL37 overexpressed in psoriasis organizes naked DNA into periodic nanocrystals to potently hyperactivate Toll-like receptor 9 (TLR9) in plasmacytoid dendritic cells (pDCs). Interestingly, a subset of self-DNA in psoriatic lesions remain bound to histones as histone-DNA nucleosome core particles (NCPs) released from neutrophil extracellular traps (NETs). At present, it is unknown how NET components like AMPs interact with NCPs, and whether AMP-NCP complexes form structures compatible with TLR9 activation in psoriasis. Here, we combine synchrotron X-ray scattering, cryo-electron microscopy, and computer simulations to demonstrate that under a broad range of conditions, NCPs stack into columns that present periodically arranged dsDNA ligands like threads on a screw, allowing for optimal interdigitation with clusters of TLR9. Remarkably, simulations and electron microscopy indicate that the superhelical pitch of DNA wrapped around the NCP column relaxes to a value that is well-matched with the steric size of TLR9, which predicts strong immune activation. Taken together, our results suggest that AMPs can remodel the structural organization of nucleosomes from NETs into potent amplifiers of inflammation. Preliminary immune activation experiments will be presented.

Transesterification Reaction and the Repair of Embedded Ribonucleotides in DNA Are Suppressed upon the Assembly of DNA into Nucleosome Core Particles †.

Ribonucleotides can be incorporated into DNA through many different cellular processes, and abundant amounts of ribonucleotides are detected in genomic DNA. Embedded ribonucleotides lead to genomic instability through either spontaneous ribonucleotide cleavage via internal transesterification or by inducing mutagenesis, recombination, and chromosome rearrangements. Ribonucleotides misincorporated in genomic DNA can be removed by the ribonucleotide excision repair (RER) pathway in which RNase HII initiates the repair by cleaving the 5'-phosphate of the ribonucleotide. Herein, based on in vitro reconstituted nucleosome core particles (NCPs) containing a single ribonucleotide at different positions, we studied the kinetics of ribonucleotide cleavage via the internal transesterification reaction and repair of the ribonucleotides by RNase HII in NCPs. Our results show that ribonucleotide cleavage via the internal transesterification in NCPs is suppressed compared to that in free DNA. DNA bending and structural rigidity account for the suppressed ribonucleotide cleavage in NCPs. Ribonucleotide repair by RNase HII in NCPs exhibits a strong correlation between the translational and rotational positions of the ribonucleotides. An embedded ribonucleotide located at the entry site while facing outward in NCP is repaired as efficiently as that in free DNA. However, the repair of those located in the central part of NCPs and facing inward are inhibited by up to 273-fold relative to those in free dsDNA. The difference in repair efficiency appears to arise from their different accessibility to repair enzymes in NCPs. This study reveals that a ribonucleotide misincorporated in DNA assembled into NCPs is protected against cleavage. Hence, the spontaneous cleavage of the misincorporated ribonucleotides under physiological conditions is not an essential threat to the stability of chromatin DNA. Instead, their decreased repair efficiency in NCPs may result in numerous and persistent ribonucleotides in genomic DNA, which could exert other deleterious effects on DNA such as mutagenesis and recombination.

Alterations in Nuclesome Core Particle Dynamics in the Context of Paraganglioma

Paraganglioma (PGL) is a rare form of neuroendocrine cancer for which the underlying molecular basis is poorly understood. A mysterious hallmark of many PGL tumors is the inactivation of succinate dehydrogenase (SDH), a mitochondrial enzyme of the TCA cycle. Why does SDH loss induce cancer in certain cells? As a result of SDH loss, PGL tumor cells accumulate succinate, which may have epigenetic consequences. For example, certain lysine residues on histone proteins in nucleosomes are subject to succinylation by succinyl‐CoA as a post translational modification (PTM) that converts the lysine cation to an anion. We hypothesize that the structure and stability of the nucleosome core particle (NCP) is affected by succinylation PTMs through weakening of electrostatic histone‐DNA and histone‐histone interactions. Proteomic analysis of an SDH‐loss cell line model of PGL previously demonstrated that the greatest increase in nucleosomal lysine succinylation maps to H4K79 when compared to a control cell line. The mammalian H4K79 residue lies within the nucleosome core near wrapped DNA. We specifically hypothesize that the charge inversion conferred by H4K79 succinylation, destabilizes the NCP. Point mutations at H4K79 have been generated as lysine succinylation mimics at this site. Biophysical and in vitro transcription assays are being utilized to validate the assembly of NCPs incorporating lysine succinylation mimics at this site and to interrogate their resulting stabilities. Analyses of alterations in NCP dynamics in variant NCP will help to elucidate the role of lysine succinylation of histones in the altered chromatin landscape observed during PGL oncogenesis.Support or Funding InformationGM75148‐09‐10 PREPThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of Histone Lysine Methylation on DNA Lesion Reactivity in Nucleosome Core Particles.

Lysine methylation is a common post-translational histone modification that regulates transcription and gene expression. The lysine residues in the histone tail also react with damaged nucleotides in chromatin, including abasic sites and N7-methyl-2'-deoxyguanosine, the major product of DNA methylating agents. Lysine monomethylation transforms the ε-amine into a secondary amine, which could be more nucleophilic and/or basic than the ε-amine in lysine, and therefore more reactive with damaged DNA. The effect of lysine methylation on the reactivity with abasic sites and N7-methyl-2'-deoxyguanosine was examined in nucleosome core particles using a methylated lysine analogue derived from cysteine. ε-Amine methylation increases the rate constant for abasic site reaction within nucleosome core particles. Reactivity at the two positions examined increased less than twofold. Mechanistic experiments indicate that faster β-elimination from an intermediate iminium ion accounts for accelerated abasic reactivity. The rate constants for nucleophilic attack (Schiff base/iminium ion formation) by the lysine and methylated lysine analogues are indistinguishable. Similarly, the rate constants describing nucleophilic attack by the lysine and methylated lysine analogues on β-2'-fluoro-N7-methyl-2'-deoxyguanosine to form DNA-protein cross-links are also within experimental error of one another. These data indicate that abasic site containing DNA will be destabilized by lysine methylation. However, these experiments do not indicate that DNA-protein cross-link formation, a recently discovered form of damage resulting from N7-guanine methylation, will be affected by this post-translational modification.

Backmapping from Multiresolution Coarse-Grained Models to Atomic Structures of Large Biomolecules by Restrained Molecular Dynamics Simulations Using Bayesian Inference.

Coarse-grained (CG) simulations have allowed access to larger length scales and longer time scales in the study of the dynamic processes of large biomolecules than all-atom (AA) molecular dynamics (MD) simulations. Backmapping from CG models to AA structures has long been studied because it enables us to gain detailed structure insights from CG simulations. Many methods first construct an AA structure from the CG model by fragments, random placement, or geometrical rules and subsequently optimize the solution via energy minimization, simulated annealing or position-restrained simulations. However, such methods may only work well on residue-level CG models and cannot consider the deviations of CG models. In this work, we describe, to the best of our knowledge, a new backmapping method based on Bayesian inference and restrained MD simulations. Restraints with log harmonic energy terms are defined according to the target CG model using the Bayesian inference in which the CG deviations can be estimated. From an initial AA structure obtained from either high-resolution experiments or homology modeling, a MD simulation with the aforementioned restraints is performed to obtain a final AA structure that is a backmapping of the target CG model. The method was validated using multiresolution CG models of the soluble extracellular region of the human epidermal growth factor receptor and was further applied to construct AA structures from CG simulations of the nucleosome core particle. The results demonstrate that our method can generate accurate AA structures of different types of biomolecules from multiple CG models with either residue-level resolution or much lower resolution than one-site-per-residue.

Effect of Nucleosome Assembly on Alkylation by a Dynamic Electrophile.

Quinone methides are reactive electrophiles that are generated during metabolism of various drugs, natural products, and food additives. Their chemical properties and cellular effects have been described previously, and now their response to packaging DNA in a nucleosome core is described. A model bisquinone methide precursor (bisQMP) was selected based on its ability to form reversible adducts with guanine N7 that allow for their redistribution and transfer after quinone methide regeneration. Assembly of Widom's 601 DNA with the histone octamer of H2A, H2B, H3, and H4 from Xenopus laevis significantly suppressed alkylation of the DNA. This result is a function of DNA packaging since addition of the octamer without nucleosome reconstitution only mildly protected DNA from alkylation. The lack of competition between nucleophiles of DNA and the histones was consistent with the limited number of adducts formed by the histones as detected by tryptic digestion and ultraperformance liquid chromatography-mass spectrometry. Only three peptide adducts were observed after reaction with a monofunctional analogue of bisQMP, and only two peptide adducts were observed after reaction with bisQMP. Histone reaction was also suppressed when reconstituted into the nucleosome core particle. However, bisQMP was capable of cross-linking the DNA and histones in moderate yields (∼20%) that exceeded expectations derived from reaction of cisplatin, nitrogen mustards, and diepoxybutane. The core histones also demonstrated a protective function against dynamic alkylation by trapping the reactive quinone methide after its spontaneous regeneration from DNA adducts.

ATXR5/6 Forms Alternative Protein Complexes with PCNA and the Nucleosome Core Particle

The proliferating cell nuclear antigen (PCNA) is a sliding clamp associated with DNA polymerases and serves as a binding platform for the recruitment of regulatory proteins linked to DNA damage repair, cell cycle regulation, and epigenetic signaling. The histone H3 lysine-27 (H3K27) mono-methyltransferase Arabidopsis trithorax-related protein 5/6 (ATXR5/6) associates with PCNA, and this interaction has been proposed to act as a key determinant controlling the reestablishment of H3K27 mono-methylation following replication. In this study, we provide biochemical evidence showing that PCNA inhibits ATXR6 enzymatic activity. The structure of the ATXR6 PCNA-interacting peptide (PIP) in complex with PCNA indicates that a trio of hydrophobic residues contributes to the binding of the enzyme to the sliding clamp. Finally, despite the presence of three PIP binding clefts, only two molecules of ATXR6 bind to PCNA likely enabling the recruitment of a third protein to the sliding clamp. Collectively, these results rule out the model wherein PCNA-bound ATXR6 actively reestablishes H3K27 mono-methylation following DNA replication and provides insights into the role of ATXR6 PIP motif in its interaction with PCNA.

Moderate nutrient restriction of beef heifers alters expression of genes associated with tissue metabolism, accretion, and function in fetal liver, muscle, and cerebrum by day 50 of gestation.

We hypothesized that a moderate maternal nutrient restriction during the first 50 d of gestation in beef heifers would affect transcript abundance of genes associated with tissue metabolism, accretion, and function in fetal liver, muscle, and cerebrum. Angus-cross heifers were estrus synchronized and assigned at breeding to one of two dietary treatments (CON- 100% of nutrient requirements to gain 0.45 kg/d; RES- 60% of CON). At day 50 of gestation, 14 heifers were ovariohysterectomized, and fetal liver, muscle, and cerebrum were collected. Transcriptome analysis via RNA-seq was conducted on the Illumina HiSeq 2500 platform using 50-bp paired-end reads at a depth of 2 × 10.4M reads/sample. Bioinformatic analysis was performed using the Tuxedo Suite and ontological analysis with DAVID 6.8. For fetal liver, muscle, and cerebrum, a total of 548, 317, and 151 genes, respectively (P < 0.01) were differentially expressed, of which 201, 144, and 28 genes, respectively were false discovery rate protected (FDR; q < 0.10). Differentially expressed genes were screened for fit into functional categories of pathways or ontologies associated with known impacts on tissue metabolism, accretion, and function. In fetal liver, five functional categories of interest (n = 125 genes) were affected by nutritional treatment: metabolic pathways, protein kinase, nucleosome core, mRNA splicing, and complement/coagulation cascades, of which 105 genes were upregulated in RES. In fetal muscle, three functional categories of interest (n = 106 genes) were affected by nutritional treatment: skeletal muscle, embryogenesis, and signaling cascades, of which 64 genes were upregulated in RES. In fetal cerebrum, three functional categories of interest (n = 60 genes) were affected by nutritional treatment: hippocampus and neurogenesis, metal-binding, and cytoskeleton, of which 58 genes were upregulated in RES. These results demonstrate that a moderate maternal nutrient restriction during the first 50 d of gestation in beef heifers alters transcript abundance of genes potentially impacting tissue metabolism, accretion, and function in fetal liver, muscle, and cerebrum. Furthermore, these results indicate that affected categories are tissue-specific and moderate maternal nutrient restriction generally increases expression of genes in fetuses from RES fed dams. Finally, these data lay the foundation upon which further research that identifies phenotypic responses to changes in these pathways may be elucidated.

Mechanism of Cross-talk between H2B Ubiquitination and H3 Methylation by Dot1L

Methylation of histone H3 K79 by Dot1L is a hallmark of actively transcribed genes that depends on monoubiquitination of H2B K120 (H2B-Ub) and is an example of histone modification cross-talk that is conserved from yeast to humans. We report here cryo-EM structures of Dot1L bound to ubiquitinated nucleosome that show how H2B-Ub stimulates Dot1L activity and reveal a role for the histone H4 tail in positioning Dot1L. We find that contacts mediated by Dot1L and the H4 tail induce a conformational change in the globular core of histone H3 thatreorients K79 from an inaccessible position, thus enabling this side chain to insert into the active site in a position primed for catalysis. Ourstudy provides a comprehensive mechanism ofcross-talk between histone ubiquitination and methylation and reveals structural plasticity in histones that makes it possible for histone-modifying enzymes to access residues within the nucleosome core.

Histidine Targeting Heterobimetallic Ruthenium(II)-Gold(I) Complexes.

Inspired by the preferential, allosteric binding of RAPTA-T and auranofin to the nucleosome core particle , we describe the design and synthesis of a series of heterobimetallic ruthenium(II)-gold(I) complexes with varying spacer lengths ranging from four to eight polyethylene glycol units. Evaluation of their cytotoxicity reveals IC50 values in the low micromolar range against cisplatin sensitive and resistant human ovarian carcinoma (A2780, A2780cisR) and nontumoral human embryonic kidney (HEK293) cell lines. Binding studies monitored via mass spectrometry revealed an affinity for histidine residues on a fragment of the amyloid β-protein (residues 1-16, employed as a model system), which is in accordance with the binding sites of parent drugs, RAPTA-C and auranofin, to the nucleosome core particle.

Structure and Dynamics of the Telomeric Nucleosome and Chromatin

Eukaryotic genomic DNA is condensed into chromatin: 146bp DNA wraps around the histone octamer (HO) comprising two copies each of the core histones H2A, H2B, H3 and H4 forming the nucleosome core particle (NCP). A string of nucleosomes folds into the so-called called “30nm” chromatin fibre, which compacts into metaphase chromosomes. Human telomeres constitute the protective structure at the end of the chromosomes with repetitive TTAGGG sequences that are about 10kbp. Little is known about the consequences of this unique sequence for chromatin structural and dynamic properties. Although it is established that telomeric DNA is packaged in chromatin with an unusually short (157 bp) nucleosome repeat length (NRL) and can form nucleosomes and chromatin, almost nothing is known about the telomeric nucleosome core and telomeric chromatin structure at the detailed molecular level. We have characterised the telomeric human HO 145 bp DNA NCP (Telo-NCP), using biophysical methods and determined its structure at 2.1 Å resolution with X-ray crystallography. Although the structure is largely similar to the well-known published atomic resolution NCP structures, several differences can be identified, e.g. DNA stretching on the Telo-NCP is different from that of the “601” (high-affinity nucleosome positioning sequence) and alpha satellite NCPs of the same length. We have designed and prepared 20×157 bp telomeric DNA template repeats, reconstituted these with human HO and performed characterisation of the structural and dynamic properties of the telomeric chromatin fibres with EM and single molecule magnetic tweezer experiments. Telomeric chromatin fibres are considerably more homogeneous than corresponding 157 bp repeat fibres reconstituted from 601-DNA array templates. However, the structural and dynamic properties of individual telomeric arrays saturated with 20 nucleosomes, appear similar to equivalent 601 chromatin fibres under the influence of force in single molecule pulling experiment.

Chromatin Folding Heterogeneity Inferred from High-Resolution Nucleosome Structures

DNA in eukaryotic cell nuclei occurs in a highly compact chromatin structure where the basic packaging unit is the nucleosome. This dynamic structure displays a wide range in compaction and provides a regulatory mechanism for many biologically relevant processes, such as gene expression. Since the first description of nucleosome-decorated DNA as beads on a string condensing into a 30 nm fiber in vitro, there have been many attempts to understand the nature of chromatin compaction and to create models at this scale. However, the proposed models for the 3D organization of the nucleosomes and intervening DNA vary quite significantly, and the very existence and relevance of a 30 nm structure in vivo has been highly debated. An analysis of the available high-resolution nucleosome core particle (NCP) structures shows subtle, yet significant differences in DNA wrapping around the histone octamer core, potentially due to DNA and histone modifications, presence of NCP-binding proteins, experimental crystallization/cryoEM conditions, intrinsic flexibility, etc. Monte Carlo simulations of regular nucleosomal arrays using a meso-scale representation of chromatin suggest that these local differences can result in large changes in global structure, comparable to the effect of changes in nucleosome spacing by about 2-3 base pairs. Our results suggest that a regular nucleosome array with a 177-base pair repeat can display either a loose three-stack or a more compact two-stack arrangement, depending on the mode of DNA wrapping in the nucleosome. Insights from our work may help bridge the significant divide in the proposed chromatin models based on various experimental results.