Chromatin In Eukaryotic Cells Research Articles

Combining molecular dynamics simulations and scoring method to computationally model ubiquitylated linker histones in chromatosomes.

The chromatin in eukaryotic cells plays a fundamental role in all processes during a cell's life cycle. This nucleoprotein is normally tightly packed but needs to be unpacked for expression and division. The linker histones are critical for such packaging processes and while most experimental and simulation works recognize their crucial importance, the focus is nearly always set on the nucleosome as the basic chromatin building block. Linker histones can undergo several modifications, but only few studies on their ubiquitylation have been conducted. Mono-ubiquitylated linker histones (HUb), while poorly understood, are expected to influence DNA compaction. The size of ubiquitin and the globular domain of the linker histone are comparable and one would expect an increased disorder upon ubiquitylation of the linker histone. However, the formation of higher order chromatin is not hindered and ubiquitylation of the linker histone may even promote gene expression. Structural data on chromatosomes is rare and HUb has never been modeled in a chromatosome so far. Descriptions of the chromatin complex with HUb would greatly benefit from computational structural data. In this study we generate molecular dynamics simulation data for six differently linked HUb variants with the help of a sampling scheme tailored to drive the exploration of phase space. We identify conformational sub-states of the six HUb variants using the sketch-map algorithm for dimensionality reduction and iterative HDBSCAN for clustering on the excessively sampled, shallow free energy landscapes. We present a highly efficient geometric scoring method to identify sub-states of HUb that fit into the nucleosome. We predict HUb conformations inside a nucleosome using on-dyad and off-dyad chromatosome structures as reference and show that unbiased simulations of HUb produce significantly more fitting than non-fitting HUb conformations. A tetranucleosome array is used to show that ubiquitylation can even occur in chromatin without too much steric clashes.

Nucleus size and its effect on nucleosome stability in living cells.

Spatial conformation of chromatin in eukaryotic cells is mainly determined by an intricate interplay between the DNA-binding properties of architectural proteins and physical constraints imposed on chromosomal DNA by a tight nuclear space. Yet, the exact effect of the nucleus size on DNA-protein interactions and chromatin structure currently remains obscure. Furthermore, there is even no clear understanding of molecular mechanisms responsible for the nucleus size regulation in living cells. In our study, we analyse the relative contribution of several key components of living cells to the regulation of the nucleus size through direct application of pressure on the nuclear envelope. It is shown that the nucleus size in higher eukaryotes is mainly defined by the difference between the surface tensions of the nuclear envelope and the endoplasmic reticulum membrane as well as the osmotic pressure exerted by cytosolic macromolecules on the nucleus. In addition, calculations demonstrate that the cell nucleus functions as a piezoelectric element, changing its electrostatic potential in a size-dependent manner. This effect has been found to have a profound impact on stability of nucleosomes, revealing a previously unknown link between the nucleus size and chromatin structure. Overall, our study provides new insights into the molecular mechanisms responsible for regulation of the nucleus size, as well as the potential role of nuclear organization in shaping the cell response to environmental cues.

Bottom-up proteomics analysis for adduction of the broad-spectrum herbicide atrazine to histone

Histones are the major proteinaceous components of chromatin in eukaryotic cells and an important part of the epigenome. The broad-spectrum herbicide atrazine (2-chloro-4-[ethylamino]-6-[isopropylamino]-1, 3, 5-triazine) and its metabolites are known to form protein adducts, but the formation of atrazine–histone adducts has not been studied. In this study, a bottom-up proteomics analysis method was optimized and applied to identify histone adduction by atrazine in vitro. Whole histones of calf thymus or human histone H3.3 were incubated with atrazine. After solvent-based protein precipitation, the protein was digested by trypsin/Glu-C and the resulting peptides were analyzed by high-resolution mass spectrometry using an ultra-high-performance liquid chromatograph interfaced with a quadrupole Exactive-Orbitrap mass spectrometer. The resulting tryptic/Glu-C peptide of DTNLCAIHAK from calf thymus histone H3.1 or human histone H3.3 was identified with an accurate mass shift of  +179.117 Da in atrazine incubated samples. It is deduced that a chemical group with an elemental composition of C8H13N5 (179.1171 Da) from atrazine adducted with calf thymus histone H3.1 or human histone H3.3. It was confirmed by MS/MS analysis that the adduction position was at its cysteine 110 residue. Time- and concentration-dependent assays also confirmed the non-enzymatic covalent modification of histone H3.3 by atrazine in vitro. Thus, the potential exists that atrazine adduction may lead to the alteration of histones that subsequently disturbs their normal function.Graphical abstract

Profiling and characterization of constitutive chromatin-enriched RNAs

RNA species act as architectural scaffolds for nuclear structures including chromatin in eukaryotic cells. However, the composition and dynamics of tightly bound chromatin-associated RNAs during mitosis remains elusive. Here we report the identification of chromatin-enriched RNA (cheRNAs) by biochemical nuclear fractionation coupled with RNA sequencing in both interphase and mitotic phase of A549 and HeLa-S3 cell lines. We show that highly abundant cheRNAs, mostly small noncoding RNAs, are largely maintained in mitotic chromatin, and constitute a substantial part of chromatin RNA throughout cell cycle. We also show that the mitotic retained cheRNAs tend to be cell type nonspecific and might be involved in chromatin accessibility and epigenetic memory of gene expression control. Therefore, we reveal an unexpected set of cell type-nonspecific mitotic retained chromatin-enriched RNAs. We anticipate that the landscape of RNA composition of chromatin both in interphase and mitotic phase would help understanding structure and function of chromatin.

Recent Advances in Investigating Functional Dynamics of Chromatin.

Dynamics spanning the picosecond-minute time domain and the atomic-subcellular spatial window have been observed for chromatin in vitro and in vivo. The condensed organization of chromatin in eukaryotic cells prevents regulatory factors from accessing genomic DNA, which requires dynamic stabilization and destabilization of structure to initiate downstream DNA activities. Those processes are achieved through altering conformational and dynamic properties of nucleosomes and nucleosome–protein complexes, of which delineating the atomistic pictures is essential to understand the mechanisms of chromatin regulation. In this review, we summarize recent progress in determining chromatin dynamics and their modulations by a number of factors including post-translational modifications (PTMs), incorporation of histone variants, and binding of effector proteins. We focus on experimental observations obtained using high-resolution techniques, primarily including nuclear magnetic resonance (NMR) spectroscopy, Förster (or fluorescence) resonance energy transfer (FRET) microscopy, and molecular dynamics (MD) simulations, and discuss the elucidated dynamics in the context of functional response and relevance.

Tetranucleosome Interactions Drive Chromatin Folding.

The multiscale organizational structure of chromatin in eukaryotic cells is instrumental to DNA transcription, replication, and repair. At mesoscopic length scales, nucleosomes pack in a manner that serves to regulate gene expression through condensation and expansion of the genome. The particular structures that arise and their respective thermodynamic stabilities, however, have yet to be fully resolved. In this study, we combine molecular modeling using the 1CPN mesoscale model of chromatin with nonlinear manifold learning to identify and characterize the structure and free energy of metastable states of short chromatin segments comprising between 4- and 16-nucleosomes. Our results reveal the formation of two previously characterized tetranucleosomal conformations, the “α-tetrahedron” and the “β-rhombus”, which have been suggested to play an important role in the accessibility of DNA and, respectively, induce local chromatin compaction or elongation. The spontaneous formation of these motifs is potentially responsible for the slow nucleosome dynamics observed in experimental studies. Increases of the nucleosome repeat length are accompanied by more pronounced structural irregularity and flexibility and, ultimately, a dynamic liquid-like behavior that allows for frequent structural reorganization. Our findings indicate that tetranucleosome motifs are intrinsically stable structural states, driven by local internucleosomal interactions, and support a mechanistic picture of chromatin packing, dynamics, and accessibility that is strongly influenced by emergent local mesoscale structure.

Combinatorial Histone H3 Modifications Are Dynamically Altered in Distinct Cell Cycle Phases.

The cell cycle is a highly regulated and evolutionary conserved process that results in the duplication of cell content and the equal distribution of the duplicated chromosomes into a pair of daughter cells. Histones are fundamental structural components of chromatin in eukaryotic cells, and their post-translational modifications (PTMs) benchmark DNA readout and chromosome condensation. Aberrant regulation of the cell cycle associated with dysregulation of histone PTMs is the cause of critical diseases such as cancer. Monitoring changes of histone PTMs could pave the way to understanding the molecular mechanisms associated with epigenetic regulation of cell proliferation. Previously, our lab established a novel middle-down workflow using porous graphitic carbon (PGC) as a stationary phase to analyze histone PTMs, which utilizes the same reversed-phase chromatography for gradient separation as canonical proteomics coupled with online mass spectrometry (MS). Here, we applied this novel workflow for high-throughput analysis of histone modifications of H3.1 and H3.2 during the cell cycle. Collectively, we identified 1133 uniquely modified canonical histone H3 N-terminal tails. Consistent with previous findings, histone H3 phosphorylation increased significantly during the mitosis (M) phase. Histone H3 variant-specific and cell-cycle-dependent expressions of PTMs were observed, underlining the need to not combine H3.1 and H3.2 together as H3. We confirmed previously known H3 PTM crosstalk (e.g., K9me-S10ph) and revealed new information in this area as well. These findings imply that the combinatorial PTMs play a role in cell cycle control, and they may serve as markers for proliferation.

Reflections on the organization and the physical state of chromatin in eukaryotic cells.

In recent years, our perception of chromatin structure and organization in the cell nucleus has changed in fundamental ways. The 30 nm chromatin fiber has lost its status as an essential in vivo structure. Hi-C and related biochemical methods, advanced electron and super-resolved fluorescence microscopy, together with concepts from soft matter physics, have revolutionized the field. A comprehensive understanding of the structural and functional interactions that regulate cell cycle and cell type specific nuclear functions appears within reach, but it requires the integration of top-down and bottom-up approachs. In this review, I present an update on nuclear architecture studies with an emphasis on organization and the controversy regarding the physical state of chromatin in cells.

How Human H1 Histone Recognizes DNA.

Linker H1 histone is one of the five main histone proteins (H1, H2A, H2B, H3, and H4), which are components of chromatin in eukaryotic cells. Here we have analyzed the patterns of DNA recognition by free H1 histone using a stepwise increase of the ligand complexity method; the affinity of H1 histone for various single- and double-stranded oligonucleotides (d(pN)n; n = 1–20) was evaluated using their competition with 12-mer [32P]labeled oligonucleotide and protein–oligonucleotide complex delaying on nitrocellulose membrane filters. It was shown that minimal ligands of H1 histone (like other DNA-dependent proteins and enzymes) are different mononucleotides (dNMPs; Kd = (1.30 ± 0.2) × 10−2 M). An increase in the length of single-stranded (ss) homo- and hetero-oligonucleotides (d(pA)n, d(pT)n, d(pC)n, and d(pN)n with different bases) by one nucleotide link regardless of their bases, leads to a monotonic increase in their affinity by a factor of f = 3.0 ± 0.2. This factor f corresponds to the Kd value = 1/f characterizing the affinity of one nucleotide of different ss d(pN)n for H1 at n = 2–6 (which are covered by this protein globule) is approximately 0.33 ± 0.02 M. The affinity of five out of six DNA nucleotide units is approximately 25 times lower than for one of the links. The affinity of duplexes of complementary homo- and hetero-d(pN)20 is only 1.3–3.3-fold higher in comparison with corresponding ss oligonucleotides. H1 histone forms mainly weak additive contacts with internucleoside phosphate groups of ssDNAs and one chain of double-stranded DNAs, but not with the bases.

Histone Purification Combined with High-Resolution Mass Spectrometry to Examine Histone Post-Translational Modifications and Histone Variants in Caenorhabditis elegans.

Histones are the major proteinaceous component of chromatin in eukaryotic cells and an important part of the epigenome, affecting most DNA‐related events, including transcription, DNA replication, and chromosome segregation. The properties of histones are greatly influenced by their post‐translational modifications (PTMs), over 200 of which are known today. Given this large number, researchers need sophisticated methods to study histone PTMs comprehensively. In particular, mass spectrometry (MS)−based approaches have gained popularity, allowing for the quantification of dozens of histone PTMs at once. Using these approaches, even the study of co‐occurring PTMs and the discovery of novel PTMs become feasible. The success of MS‐based approaches relies substantially on obtaining pure and well‐preserved histones for analysis, which can be difficult depending on the source material. Caenorhabditis elegans has been a popular model organism to study the epigenome, but isolation of pure histones from these animals has been challenging. Here, we address this issue, presenting a method for efficient isolation of pure histone proteins from C. elegans at good yield. Further, we describe an MS pipeline optimized for accurate relative quantification of histone PTMs from C. elegans. We alkylate and tryptically digest the histones, analyze them by bottom‐up MS, and then evaluate the resulting data by a C. elegans−adapted version of the software EpiProfile 2.0. Finally, we show the utility of this pipeline by determining differences in histone PTMs between C. elegans strains that age at different rates and thereby achieve very different lifespans. © 2020 The Authors. Basic Protocol 1: Large‐scale growth and harvesting of synchronized C. elegans Basic Protocol 2: Nuclear preparation, histone extraction, and histone purification Basic Protocol 3: Bottom‐up mass spectrometry analysis of histone PTMs and histone variants

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.

Statistical properties of a tangentially driven active filament

Active polymers play a central role in many biological systems, from bacterial flagella to cellular cytoskeletons. Minimal models of semiflexible active filaments have been used to study a variety of interesting phenomena in active systems, such as defect dynamics in active nematics, clustering and laning in motility assays, and conformational properties of chromatin in eukaryotic cells. In this paper, we map a semiflexible polymer to an exactly solvable active Rouse chain, which enables us to analytically compute configurational and dynamical properties of active polymers with arbitrary rigidity. Upon mapping back to the semiflexible filament, we see that the center of mass diffusion coefficient grows linearly with an activity parameter that is renormalized by the polymer persistence length. These results closely agree with numerical data obtained from microscopic simulations.

DIG-seq: a genome-wide CRISPR off-target profiling method using chromatin DNA.

To investigate whether and how CRISPR-Cas9 on-target and off-target activities are affected by chromatin in eukaryotic cells, we first identified a series of identical endogenous DNA sequences present in both open and closed chromatin regions and then measured mutation frequencies at these sites in human cells using Cas9 complexed with matched or mismatched sgRNAs. Unlike matched sgRNAs, mismatched sgRNAs were highly sensitive to chromatin states, suggesting that off-target but not on-target DNA cleavage is hindered by chromatin. We next performed Digenome-seq using cell-free chromatin DNA (now termed DIG-seq) and histone-free genomic DNA in parallel and found that only a subset of sites, cleaved in histone-free DNA, were cut in chromatin DNA, suggesting that chromatin can inhibit Cas9 off-target effects in favor of its genome-wide specificity in cells.

The Arabidopsis Histone Chaperone FACT: Role of the HMG-Box Domain of SSRP1

Histone chaperones play critical roles in regulated structural transitions of chromatin in eukaryotic cells that involve nucleosome disassembly and reassembly. The histone chaperone FACT is a heterodimeric complex consisting in plants and metazoa of SSRP1/SPT16 and is involved in dynamic nucleosome reorganization during various DNA-dependent processes including transcription, replication and repair. The C-terminal HMG-box domain of the SSRP1 subunit mediates interactions with DNA and nucleosomes in vitro, but its relevance in vivo is unclear. Here, we demonstrate that Arabidopsis ssrp1–2 mutant plants express a C-terminally truncated SSRP1 protein. Although the structure of the truncated HMG-box domain is distinctly disturbed, it still exhibits residual DNA-binding activity, but has lost DNA-bending activity. Since ssrp1–2 plants are phenotypically affected but viable, the HMG-box domain may be functionally non-essential. To examine this possibility, SSRP1∆HMG completely lacking the HMG-box domain was studied. SSRP1∆HMG in vitro did not bind to DNA and its interactions with nucleosomes were severely reduced. Nevertheless, the protein showed a nuclear mobility and protein interactions similar to SSRP1. Interestingly, expression of SSRP1∆HMG is almost as efficient as that of full-length SSRP1 in supporting normal growth and development of the otherwise non-viable Arabidopsis ssrp1–1 mutant. SSRP1∆HMG is structurally similar to the fungal ortholog termed Pob3 that shares clear similarity with SSRP1, but it lacks the C-terminal HMG-box. Therefore, our findings indicate that the HMG-box domain conserved among SSRP1 proteins is not critical in Arabidopsis, and thus, the functionality of SSRP1/SPT16 in plants/metazoa and Pob3/Spt16 in fungi is perhaps more similar than anticipated.

NucPosPred: Predicting species-specific genomic nucleosome positioning via four different modes of general PseKNC

The nucleosome is the basic structure of chromatin in eukaryotic cells, with essential roles in the regulation of many biological processes, such as DNA transcription, replication and repair, and RNA splicing. Because of the importance of nucleosomes, the factors that determine their positioning within genomes should be investigated. High-resolution nucleosome-positioning maps are now available for organisms including Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans, enabling the identification of nucleosome positioning by application of computational tools. Here, we describe a novel predictor called NucPosPred, which was specifically designed for large-scale identification of nucleosome positioning in C. elegans and D. melanogaster genomes. NucPosPred was separately optimized for each species for four types of DNA sequence feature extraction, with consideration of two classification algorithms (gradient-boosting decision tree and support vector machine). The overall accuracy obtained with NucPosPred was 92.29% for C. elegans and 88.26% for D. melanogaster, outperforming previous methods and demonstrating the potential for species-specific prediction of nucleosome positioning. For the convenience of most experimental scientists, a web-server for the predictor NucPosPred is available at http://121.42.167.206/NucPosPred/index.jsp.

Emerging roles of linker histones in regulating chromatin structure and function.

Together with core histones, which make up the nucleosome, the linker histone (H1) is one of the five main histone protein families present in chromatin in eukaryotic cells. H1 binds to the nucleosome to form the next structural unit of metazoan chromatin, the chromatosome, which may help chromatin to fold into higher-order structures. Despite their important roles in regulating the structure and function of chromatin, linker histones have not been studied as extensively as core histones. Nevertheless, substantial progress has been made recently. The first near-atomic resolution crystal structure of a chromatosome core particle and an 11 Å resolution cryo-electron microscopy-derived structure of the 30 nm nucleosome array have been determined, revealing unprecedented details about how linker histones interact with the nucleosome and organize higher-order chromatin structures. Moreover, several new functions of linker histones have been discovered, including their roles in epigenetic regulation and the regulation of DNA replication, DNA repair and genome stability. Studies of the molecular mechanisms of H1 action in these processes suggest a new paradigm for linker histone function beyond its architectural roles in chromatin.

Analysis of chromatin fibers in Hela cells with electron tomography.

The presence and folding pattern of chromatin in eukaryotic cells remain elusive and controversial. In this study, we prepared ultra-thin sections of Hela cells with three different fixation and sectioning methods, i.e., chemical fixation, high pressure freezing with freeze substitution, and cryo-ultramicrotomy with SEM-FIB (focused ion beam), and analyzed in vivo architecture of chromatin fibers in Hela nuclei with electron tomography technology. The results suggest that the chromatin fibers in eukaryotic Hela cells are likely organized in an architecture with a diameter of about 30 nm.

Quantitative proteomic analysis of histone modifications.

1.1. Nucleosome and Chromatin In eukaryotic cells, chromosomal DNA is packaged into a compact structure, chromatin, with the use of four core histones (H2A, H2B, H3, and H4). The fundamental repeating unit of chromatin is the nucleosome, which is composed of an octamer of the core histones, around which ~147 base pairs of DNA are wrapped. Nucleosomes are in turn folded into progressively higher-order structures. Dynamic chromatin remodeling plays a critical role in regulating diverse DNA-based biological processes, such as transcription of RNA, DNA replication, and DNA repair, as well as chromosome condensation and segregation.1 The core histone proteins (not histone octamer) are small (10–20 kDa) and highly basic. They are predominantly globular except for their N-terminal “tails”, which are unstructured and protrude from the surface of the chromatin polymer. Amino acid sequence analysis shows that histone proteins are highly conserved in eukaryotic cells from yeast to human, implying that most amino acid residues, if not all, are likely to be important for structure or function. Indeed, studies among histone variants as well as mutational evidence in cancers suggest that a change of a single amino acid residue can lead to very different biological output and even disease, such as cancer.2 Histone post-translational modification (PTM), or histone mark, in combination with DNA modifications, histone variants, and ATP-dependent protein complex formation, is used by cells to dynamically modulate chromatin structure and function. Because PTMs alter the properties of the substrate amino acid residue, typically more significant than a mutation, they are likely to affect histone structure and therefore function.3 Indeed, PTMs are abundant in histones, especially at their N-terminal tails, and have roles in modulating chromatin dynamics and diverse DNA-templated biological processes (Figure 1).1 Dysregulation of these processes has been intimately associated with the development of diseases such as cancer.4 Figure 1 Structures of histone post-translational modifications. 1.2. Biological Mechanism of Histone PTMs As of this writing, 20 types of histone PTMs had been reported: phosphorylation, acetylation, monomethylation, dimethylation, trimethylation, propionylation, butyrylation, crotonylation, 2-hydroxylisobutyrylation, malonylation, succinylation, glutarylation, formylation, hydroxylation, ubiquitination, SUMOylation, O-GlcNAcylation, ADP-ribosylation, proline isomerization, and citrullination (Figure 1).5 In more recent times, known PTM sites on histones have been identified either by sequence-specific antibodies or by mass spectrometry (MS) methods in an unbiased manner.6 The function and dynamic regulation of these PTMs have been the subject of extensive investigations over the past decade. Histone PTMs are thought to regulate chromatin structure and function by two mechanisms.1a,b First, histone PTMs can directly modulate the packaging of chromatin by either altering the charge state of histones or through inter nucleosomal interactions, thereby regulating chromatin higher-order structure and the access of DNA-binding proteins, such as transcription factors. Additionally, histone PTMs can modify chromatin structure and function either by recruiting PTM-specific binding proteins (also called “readers”) and their associated binding partners (“effector proteins”) or by inhibiting the binding of a protein to the chromatin. PTM-induced changes in protein interactions between chromatin and its binding proteins are in turn translated into biological outcomes.7 Proteins are recruited to histone PTMs through direct binding to specific domains. For example, chromo, Tudor, PHD, MBT, PWWP, WD, ADD, zf-CW, BAH, and CHD domains are all known to bind methyllysine,8 while the bromodomain binds acetyllysine.9 Proteins containing these PTM-specific binding domains may recruit additional protein factors to execute their functions. Alternatively, they may carry enzymatic activities that can further modify chromatin structure and function. Histone marks are known to be critical in regulation of diverse DNA-templated biological processes.1 Interestingly, some of these histone PTMs correlate with transcriptional activation or repression, depending on the types and the locations of the PTMs.1b,10 To execute DNA-templated processes, histone PTMs coordinate the unraveling of chromatin to carry out specific functions. For example, histone lysine acetylation (Kac) typically correlates with transcriptional activation, while lysine deacetylation correlates with transcriptional repression.1b,11 Lysine methylation (Kme) is implicated in both gene activation (H3K4, H3K36, and H3K79) and transcriptional repression (H3K9, H3K27, and H4K20).12 As examples, some monomethylation (e.g., H3K9me1 and H3K27me1) is involved in transcriptional activation, while trimethylation at the same sites (H3K9me3 and H3K27me3) is linked to repression.13 Likewise, some other histone PTMs also correlate with DNA repair (e.g., H2AS129 phosphorylation and H4S1 phosphorylation)14 and replication (e.g., acetylation).15 Dysregulation of each step of histone PTMs, including adding the histone marks by a “writer”, removing the histone mark by an “eraser”, and misinterpretation by a “reader” protein, has shown to be associated with disease, such as cancer.4a,e These histone PTMs are proposed to contribute a “histone code” or “histone language” that dictates the functions of the proteins in gene expression and chromatin dynamics.1a,c,d Addition and removal of histone PTMs are regulated by diverse groups of enzymes that were initially identified in the past decade, but still are being discovered in recent times. These enzymes are responsible for adding (“writing”) or removing (“erasing”) the histone PTM “code”. The resulting histone marks are in turn translated into biological outputs by different mechanisms. Chromatin dynamics are mainly controlled by ATP-dependent chromatin remodeling enzymes/complexes and histone PTMs.16 The “histone code” can facilitate the recruitment of diverse chromatin remodeling enzymes to regulate chromatin dynamics. Conversely, chromatin remodeling enzymes can also influence the histone PTMs.17 For example, an ATP-dependent nucleosome remodeling complex, nucleosome remodelling and deacetylation complex (NuRD), can facilitate the deacetylation of the target histones.18 Some histone PTMs, if not all, are inheritable during cell division and correlate with gene expression. Therefore, histone PTMs are linked with epigenetic phenomena and are generally considered to be a major type of epigenetic marks.19

How Do Nucleosomes Bundle DNA into Chromatin?

The binding of histones and the formation of nucleosomes play major roles in bundling DNA into chromatin in eukaryotic cells. This process not only facilitates the packaging of DNA in the cell nucleus but also influences the communication between distant genomic sites, such as enhancers and promoters found at the ends of protein-mediated loops. The details of chromatin organization, however, remain unclear. In particular, the effects of nucleosome positioning (i.e., the sequential locations of nucleosomes along DNA) on chromatin architecture are still poorly understood. With a view toward understanding the interplay between nucleosomes and DNA in chromatin, we have developed a mesoscale model of nucleosome-decorated DNA at a resolution of a single base pair. We use Monte Carlo numerical strategies to unravel how nucleosomes shape DNA into chromatin and we validate our model by successfully reproducing various experimental measurements. With the help of a cloud computing approach we have been able to perform simulations for a wide range of nucleosome positionings, that is, for a wide range of DNA linker lengths. Our results show a broad variety of chromatin organization. This structural diversity, which extends beyond the conventional solenoid and zigzag models of chromatin, comes along with very different physical and mechanical properties, such as apparent bending and twisting stiffnesses and degrees of compaction. We have also studied how fluctuations in nucleosome positioning can affect chromatin. We have found that minor changes of the order of a few base pairs in the locations of nucleosomes along DNA can greatly alter the local and global properties of chromatin. Our work shows that the synergy between DNA and nucleosomes in chromatin gives rise to a highly versatile biomolecule, which is able to accommodate tight packing while maintaining the accessibility necessary for specific expression.

Chromatin structure: The role of “linker” proteins

Structural organization of chromatin in eukaryotic cells plays an important role in the functioning of the genetic ap- paratus of the cell. The nucleoprotein complex is formed due to the interactions of DNA with histones and non-histone proteins. Here we describe the main levels of the structural organization of the chromatin. Special attention is paid to the spectroscopic techniques such as infrared absorption spectroscopy, electronic and vibrational circular dichroism and their impact to the in- vestigation of the chromatin structure. The linker proteins: histone H1 and non-histone protein HMGB1 and their role in the formation of the super compact chromatin is discussed. The chromatin of the eukaryotic cells comprises of DNA and variety of nuclear proteins. The linear dimensions of eukaryotic DNA molecule is much larger than the diameter of the cell (11). That is why one of the important functions of the chromatin is DNA compaction. The subsequent levels of DNA compaction are associated with the changes of chromatin structure resulting in a high density of DNA packing in the cell nucleus. The structural organization of the DNA in complexes with the nuclear pro- teins plays an important role, as it determines not only the structure of chromatin, but also the functioning of the genetic apparatus of the cell (3,32). The nucleoprotein complex is formed due to the interactions of DNA with histones and non-histone proteins (11). The proteins functioning in the linker regions of the chromatin (linker proteins) are the key elements of the DNA compaction (26). The most abundant linker proteins are histone H1 and HMGB non-histone proteins. The DNA-binding properties of these proteins determine the differences in the structural organization of the chromatin in the cells of different organisms, modulating the function- ing of their genomes (11,12,15,53). The HMGB1 protein is considered to be an architectural factor of transcription (9,35,63,80), and participates in transcription (1,8,23,63), recombination (66,79) and replication (67).