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Chromatin
Chromatin
Chromatin
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The major structures in DNA compaction: DNA, the nucleosome, the 11 nm beads on a string chromatin fibre and the metaphase chromosome.

Chromatin is a complex of DNA and protein found in eukaryotic cells.[1] The primary function is to package long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important roles in reinforcing the DNA during cell division, preventing DNA damage, and regulating gene expression and DNA replication. During mitosis and meiosis, chromatin facilitates proper segregation of the chromosomes in anaphase; the characteristic shapes of chromosomes visible during this stage are the result of DNA being coiled into highly condensed chromatin.

The primary protein components of chromatin are histones. An octamer of two sets of four histone cores (Histone H2A, Histone H2B, Histone H3, and Histone H4) bind to DNA and function as "anchors" around which the strands are wound.[2] In general, there are three levels of chromatin organization:

  1. DNA wraps around histone proteins, forming nucleosomes and the so-called beads on a string structure (euchromatin).
  2. Multiple histones wrap into a 30-nanometer fiber consisting of nucleosome arrays in their most compact form (heterochromatin).[a]
  3. Higher-level DNA supercoiling of the 30 nm fiber produces the metaphase chromosome (during mitosis and meiosis).

Many organisms, however, do not follow this organization scheme. For example, spermatozoa and avian red blood cells have more tightly packed chromatin than most eukaryotic cells, and trypanosomatid protozoa do not condense their chromatin into visible chromosomes at all. Prokaryotic cells have entirely different structures for organizing their DNA (the prokaryotic chromosome equivalent is called a genophore and is localized within the nucleoid region).

The overall structure of the chromatin network further depends on the stage of the cell cycle. During interphase, the chromatin is structurally loose to allow access to RNA and DNA polymerases that transcribe and replicate the DNA. The local structure of chromatin during interphase depends on the specific genes present in the DNA. Regions of DNA containing genes which are actively transcribed ("turned on") are less tightly compacted and closely associated with RNA polymerases in a structure known as euchromatin, while regions containing inactive genes ("turned off") are generally more condensed and associated with structural proteins in heterochromatin.[4] Epigenetic modification of the structural proteins in chromatin via methylation and acetylation also alters local chromatin structure and therefore gene expression. There is limited understanding of chromatin structure and it is active area of research in molecular biology.

Dynamic chromatin structure and hierarchy

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Basic units of chromatin structure
the structure of chromatin within a chromosome

Chromatin undergoes various structural changes during a cell cycle. Histone proteins are the basic packers and arrangers of chromatin and can be modified by various post-translational modifications to alter chromatin packing (histone modification). Most modifications occur on histone tails. The positively charged histone cores only partially counteract the negative charge of the DNA phosphate backbone resulting in a negative net charge of the overall structure. An imbalance of charge within the polymer causes electrostatic repulsion between neighboring chromatin regions that promote interactions with positively charged proteins, molecules, and cations. As these modifications occur, the electrostatic environment surrounding the chromatin will flux and the level of chromatin compaction will alter.[2] The consequences in terms of chromatin accessibility and compaction depend both on the modified amino acid and the type of modification. For example, histone acetylation results in loosening and increased accessibility of chromatin for replication and transcription. Lysine trimethylation can either lead to increased transcriptional activity (trimethylation of histone H3 lysine 4) or transcriptional repression and chromatin compaction (trimethylation of histone H3, lysine 9 or lysine 27). Several studies suggested that different modifications could occur simultaneously. For example, it was proposed that a bivalent structure (with trimethylation of both lysine 4 and 27 on histone H3) is involved in early mammalian development. Another study tested the role of acetylation of histone 4 on lysine 16 on chromatin structure and found that homogeneous acetylation inhibited 30 nm chromatin formation and blocked adenosine triphosphate remodeling. This singular modification changed the dynamics of the chromatin which shows that acetylation of H4 at K16 is vital for proper intra- and inter- functionality of chromatin structure.[5] [6]

Polycomb-group proteins play a role in regulating genes through modulation of chromatin structure.[7]

For additional information, see Chromatin variant, Histone modifications in chromatin regulation and RNA polymerase control by chromatin structure.

Structure of DNA

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The structures of A-, B-, and Z-DNA.
Main articles: Mechanical properties of DNA and Z-DNA

In nature, DNA can form three structures, A-, B-, and Z-DNA. A- and B-DNA are very similar, forming right-handed helices, whereas Z-DNA is a left-handed helix with a zig-zag phosphate backbone. Z-DNA is thought to play a specific role in chromatin structure and transcription because of the properties of the junction between B- and Z-DNA.

At the junction of B- and Z-DNA, one pair of bases is flipped out from normal bonding. These play a dual role of a site of recognition by many proteins and as a sink for torsional stress from RNA polymerase or nucleosome binding. DNA bases are stored as a code structure with four chemical bases such as "Adenine (A), Guanine (G), Cytosine (C), and Thymine (T)". The order and sequences of these chemical structures of DNA are reflected as information available for the creation and control of human organisms. "A with T and C with G" pairing up to build the DNA base pair. Sugar and phosphate molecules are also paired with these bases, making DNA nucleotides arrange 2 long spiral strands unitedly called "double helix".[8] In eukaryotes, DNA consists of a cell nucleus and the DNA is providing strength and direction to the mechanism of heredity. Moreover, between the nitrogenous bonds of the 2 DNA, homogenous bonds are forming.

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Nucleosomes and beads-on-a-string

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Main articles: Nucleosome, Chromatosome, and Histone
A cartoon representation of the nucleosome structure. From PDB: 1KX5​.

The basic repeat element of chromatin is the nucleosome, interconnected by sections of linker DNA, a far shorter arrangement than pure DNA in solution.

In addition to core histones, a linker histone H1 exists that contacts the exit/entry of the DNA strand on the nucleosome. The nucleosome core particle, together with histone H1, is known as a chromatosome. Nucleosomes, with about 20 to 60 base pairs of linker DNA, can form, under non-physiological conditions, an approximately 11 nm beads on a string fibre.

The nucleosomes bind DNA non-specifically, as required by their function in general DNA packaging. There are, however, large DNA sequence preferences that govern nucleosome positioning. This is due primarily to the varying physical properties of different DNA sequences: For instance, adenine (A), and thymine (T) is more favorably compressed into the inner minor grooves. This means nucleosomes can bind preferentially at one position approximately every 10 base pairs (the helical repeat of DNA)- where the DNA is rotated to maximise the number of A and T bases that will lie in the inner minor groove. (See nucleic acid structure.)

30-nm chromatin fiber in mitosis

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Two proposed structures of the 30 nm chromatin filament.
Left: 1 start helix "solenoid" structure.
Right: 2 start loose helix structure.
Note: the histones are omitted in this diagram - only the DNA is shown.

With addition of H1, during mitosis the beads-on-a-string structure can coil into a 30 nm-diameter helical structure known as the 30 nm fibre or filament. The precise structure of the chromatin fiber in the cell is not known in detail.[10]

This level of chromatin structure is thought to be the form of heterochromatin, which contains mostly transcriptionally silent genes. Electron microscopy studies have demonstrated that the 30 nm fiber is highly dynamic such that it unfolds into a 10 nm fiber beads-on-a-string structure when transversed by an RNA polymerase engaged in transcription.

Four proposed structures of the 30 nm chromatin filament for DNA repeat length per nucleosomes ranging from 177 to 207 bp.
Linker DNA in yellow and nucleosomal DNA in pink.

The existing models commonly accept that the nucleosomes lie perpendicular to the axis of the fibre, with linker histones arranged internally. A stable 30 nm fibre relies on the regular positioning of nucleosomes along DNA. Linker DNA is relatively resistant to bending and rotation. This makes the length of linker DNA critical to the stability of the fibre, requiring nucleosomes to be separated by lengths that permit rotation and folding into the required orientation without excessive stress to the DNA. In this view, different lengths of the linker DNA should produce different folding topologies of the chromatin fiber. Recent theoretical work, based on electron-microscopy images[11] of reconstituted fibers supports this view.[12]

DNA loops

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Animated representation of the dynamic formation of chromatin loops through CTCF (red) and condensin rings (yellow)[13]

The beads-on-a-string chromatin structure has a tendency to form loops. These loops allow interactions between different regions of DNA by bringing them closer to each other, which increases the efficiency of gene interactions. This process is dynamic, with loops forming and disappearing. The loops are regulated by two main elements:[14]

  • Cohesins, protein complexes that generate loops by extrusion of the DNA fiber through the ring-like structure of the complex itself.[13][15]
  • CTCF, a transcription factor that limits the frontier of the DNA loop. To stop the growth of a loop, two CTCF molecules must be positioned in opposite directions to block the movement of the cohesin ring (see video).[13][16]

There are many other elements involved. For example, Jpx regulates the binding sites of CTCF molecules along the DNA fiber.[17]

Spatial organization of chromatin in the cell nucleus

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The spatial arrangement of the chromatin within the nucleus is not random - specific regions of the chromatin can be found in certain territories. Territories are, for example, the lamina-associated domains (LADs), and the topologically associating domains (TADs), which are bound together by protein complexes.[18] Currently, polymer models such as the Strings & Binders Switch (SBS) model[19] and the Dynamic Loop (DL) model[20] are used to describe the folding of chromatin within the nucleus. The arrangement of chromatin within the nucleus may also play a role in nuclear stress and restoring nuclear membrane deformation by mechanical stress. When chromatin is condensed, the nucleus becomes more rigid. When chromatin is decondensed, the nucleus becomes more elastic with less force exerted on the inner nuclear membrane. This observation sheds light on other possible cellular functions of chromatin organization outside of genomic regulation.[2]

Cell-cycle dependent structural organization

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Karyogram of human male using Giemsa staining, showing the classic metaphase chromatin structure.
Condensation and resolution of human sister chromatids in early mitosis
  1. Interphase: The structure of chromatin during interphase of mitosis is optimized to allow simple access of transcription and DNA repair factors to the DNA while compacting the DNA into the nucleus. The structure varies depending on the access required to the DNA. Genes that require regular access by RNA polymerase require the looser structure provided by euchromatin.
  2. Metaphase: The metaphase structure of chromatin differs vastly to that of interphase. It is optimised for physical strength[citation needed] and manageability, forming the classic chromosome structure seen in karyotypes. The structure of the condensed chromatin is thought to be loops of 30 nm fibre to a central scaffold of proteins. It is, however, not well-characterised. Chromosome scaffolds play an important role to hold the chromatin into compact chromosomes. Loops of 30 nm structure further condense with scaffold, into higher order structures.[21] Chromosome scaffolds are made of proteins including condensin, type IIA topoisomerase and kinesin family member 4 (KIF4).[22] The physical strength of chromatin is vital for this stage of division to prevent shear damage to the DNA as the daughter chromosomes are separated. To maximise strength the composition of the chromatin changes as it approaches the centromere, primarily through alternative histone H1 analogues. During mitosis, although most of the chromatin is tightly compacted, there are small regions that are not as tightly compacted. These regions often correspond to promoter regions of genes that were active in that cell type prior to chromatin formation. The lack of compaction of these regions is called bookmarking, which is an epigenetic mechanism believed to be important for transmitting to daughter cells the "memory" of which genes were active prior to entry into mitosis.[23] This bookmarking mechanism is needed to help transmit this memory because transcription ceases during mitosis.

Chromatin and bursts of transcription

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Chromatin and its interaction with enzymes has been researched, and a conclusion being made is that it is relevant and an important factor in gene expression. Vincent G. Allfrey, a professor at Rockefeller University, stated that RNA synthesis is related to histone acetylation.[24] The lysine amino acid attached to the end of the histones is positively charged. The acetylation of these tails would make the chromatin ends neutral, allowing for DNA access.

When the chromatin decondenses, the DNA is open to entry of molecular machinery. Fluctuations between open and closed chromatin may contribute to the discontinuity of transcription, or transcriptional bursting. Other factors are probably involved, such as the association and dissociation of transcription factor complexes with chromatin. Specifically, RNA polymerase and transcriptional proteins have been shown to congregate into droplets via phase separation, and recent studies have suggested that 10 nm chromatin demonstrates liquid-like behavior increasing the targetability of genomic DNA.[25] The interactions between linker histones and disordered tail regions act as an electrostatic glue organizing large-scale chromatin into a dynamic, liquid-like domain. Decreased chromatin compaction comes with increased chromatin mobility and easier transcriptional access to DNA.[2] The phenomenon, as opposed to simple probabilistic models of transcription, can account for the high variability in gene expression occurring between cells in isogenic populations.[26]

Alternative chromatin organizations

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During metazoan spermiogenesis, the spermatid's chromatin is remodeled into a more spaced-packaged, widened, almost crystal-like structure. This process is associated with the cessation of transcription and involves nuclear protein exchange. The histones are mostly displaced, and replaced by protamines (small, arginine-rich proteins).[27] It is proposed that in yeast, regions devoid of histones become very fragile after transcription; HMO1, an HMG-box protein, helps in stabilizing nucleosomes-free chromatin.[28][29]

Chromatin and DNA repair

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A variety of internal and external agents can cause DNA damage in cells. Many factors influence how the repair route is selected, including the cell cycle phase and chromatin segment where the break occurred. In terms of initiating 5' end DNA repair, the p53 binding protein 1 (53BP1) and BRCA1 are important protein components that influence double-strand break repair pathway selection. The 53BP1 complex attaches to chromatin near DNA breaks and activates downstream factors such as Rap1-Interacting Factor 1 (RIF1) and shieldin, which protects DNA ends against nucleolytic destruction. DNA damage process occurs within the condition of chromatin, and the constantly changing chromatin environment has a large effect on it.[30] Accessing and repairing the damaged cell of DNA, the genome condenses into chromatin and repairing it through modifying the histone residues. Through altering the chromatin structure, histones residues are adding chemical groups namely phosphate, acetyl and one or more methyl groups and these control the expressions of gene building by proteins to acquire DNA.[31] Moreover, resynthesis of the delighted zone, DNA will be repaired by processing and restructuring the damaged bases. In order to maintain genomic integrity, "homologous recombination and classical non-homologous end joining process" has been followed by DNA to be repaired.[32]

The packaging of eukaryotic DNA into chromatin presents a barrier to all DNA-based processes that require recruitment of enzymes to their sites of action.[33] To allow the critical cellular process of DNA repair, the chromatin must be remodeled. In eukaryotes, ATP-dependent chromatin remodeling complexes and histone-modifying enzymes are two predominant factors employed to accomplish this remodeling process.[34]

Chromatin relaxation occurs rapidly at the site of DNA damage.[35] This process is initiated by PARP1 protein that starts to appear at DNA damage in less than a second, with half maximum accumulation within 1.6 seconds after the damage occurs.[36] Next the chromatin remodeler Alc1 quickly attaches to the product of PARP1, and completes arrival at the DNA damage within 10 seconds of the damage.[35] About half of the maximum chromatin relaxation, presumably due to action of Alc1, occurs by 10 seconds.[35] This then allows recruitment of the DNA repair enzyme MRE11, to initiate DNA repair, within 13 seconds.[36]

γH2AX, the phosphorylated form of H2AX is also involved in the early steps leading to chromatin decondensation after DNA damage occurrence. The histone variant H2AX constitutes about 10% of the H2A histones in human chromatin.[37] γH2AX (H2AX phosphorylated on serine 139) can be detected as soon as 20 seconds after irradiation of cells (with DNA double-strand break formation), and half maximum accumulation of γH2AX occurs in one minute.[37] The extent of chromatin with phosphorylated γH2AX is about two million base pairs at the site of a DNA double-strand break.[37] γH2AX does not, itself, cause chromatin decondensation, but within 30 seconds of irradiation, RNF8 protein can be detected in association with γH2AX.[38] RNF8 mediates extensive chromatin decondensation, through its subsequent interaction with CHD4,[39] a component of the nucleosome remodeling and deacetylase complex NuRD.

After undergoing relaxation subsequent to DNA damage, followed by DNA repair, chromatin recovers to a compaction state close to its pre-damage level after about 20 min.[35]

Methods to investigate chromatin

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Microscopy of heterochromatic versus euchromatic nuclei (H&E stain).
Granular "salt-and-pepper chromatin", seen on H&E, Pap stain and comparison to actual salt and pepper. Its finding on microscopy indicates mainly medullary thyroid carcinoma, neuroendocrine tumours[40] or pheochromocytoma.[41]
Schematic karyogram of a human, showing an overview of the human genome using G banding, which is a method that includes Giemsa staining, wherein the lighter staining regions are generally more euchromatic (and more transcriptionally active), whereas darker regions generally are more heterochromatic.
Further information: Karyotype
  1. ChIP-seq (Chromatin immunoprecipitation sequencing) is recognized as the vastly utilized chromatin identification method it has been using the antibodies that actively select, identify and combine with proteins including "histones, histone restructuring, transcription factors and cofactors". This has been providing data about the state of chromatin and the transcription of a gene by trimming "oligonucleotides" that are unbound.[42] Chromatin immunoprecipitation sequencing aimed against different histone modifications, can be used to identify chromatin states throughout the genome. Different modifications have been linked to various states of chromatin.[43]
  2. DNase-seq (DNase I hypersensitive sites Sequencing) uses the sensitivity of accessible regions in the genome to the DNase I enzyme to map open or accessible regions in the genome.
  3. FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements sequencing) uses the chemical properties of protein-bound DNA in a two-phase separation method to extract nucleosome depleted regions from the genome.[44]
  4. ATAC-seq (Assay for Transposable Accessible Chromatin sequencing) uses the Tn5 transposase to integrate (synthetic) transposons into accessible regions of the genome consequentially highlighting the localisation of nucleosomes and transcription factors across the genome.
  5. DNA footprinting is a method aimed at identifying protein-bound DNA. It uses labeling and fragmentation coupled to gel electrophoresis to identify areas of the genome that have been bound by proteins.[45]
  6. MNase-seq (Micrococcal Nuclease sequencing) uses the micrococcal nuclease enzyme to identify nucleosome positioning throughout the genome.[46][47]
  7. Chromosome conformation capture determines the spatial organization of chromatin in the nucleus, by inferring genomic locations that physically interact.
  8. MACC profiling (Micrococcal nuclease ACCessibility profiling) uses titration series of chromatin digests with micrococcal nuclease to identify chromatin accessibility as well as to map nucleosomes and non-histone DNA-binding proteins in both open and closed regions of the genome.[48]

Chromatin and knots

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It has been a puzzle how decondensed interphase chromosomes remain essentially unknotted. The natural expectation is that in the presence of type II DNA topoisomerases that permit passages of double-stranded DNA regions through each other, all chromosomes should reach the state of topological equilibrium. The topological equilibrium in highly crowded interphase chromosomes forming chromosome territories would result in formation of highly knotted chromatin fibres. However, Chromosome Conformation Capture (3C) methods revealed that the decay of contacts with the genomic distance in interphase chromosomes is practically the same as in the crumpled globule state that is formed when long polymers condense without formation of any knots. To remove knots from highly crowded chromatin, one would need an active process that should not only provide the energy to move the system from the state of topological equilibrium but also guide topoisomerase-mediated passages in such a way that knots would be efficiently unknotted instead of making the knots even more complex. It has been shown that the process of chromatin-loop extrusion is ideally suited to actively unknot chromatin fibres in interphase chromosomes.[49]

Chromatin: alternative definitions

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The term, introduced by Walther Flemming, has multiple meanings:

  1. Simple and concise definition: Chromatin is a macromolecular complex of a DNA macromolecule and protein macromolecules (and RNA). The proteins package and arrange the DNA and control its functions within the cell nucleus.
  2. A biochemists' operational definition: Chromatin is the DNA/protein/RNA complex extracted from eukaryotic lysed interphase nuclei. Just which of the multitudinous substances present in a nucleus will constitute a part of the extracted material partly depends on the technique each researcher uses. Furthermore, the composition and properties of chromatin vary from one cell type to another, during the development of a specific cell type, and at different stages in the cell cycle.
  3. The DNA + histone = chromatin definition: The DNA double helix in the cell nucleus is packaged by special proteins termed histones. The formed protein/DNA complex is called chromatin. The basic structural unit of chromatin is the nucleosome.

The first definition allows for "chromatins" to be defined in other domains of life like bacteria and archaea, using any DNA-binding proteins that condenses the molecule. These proteins are usually referred to nucleoid-associated proteins (NAPs); examples include AsnC/LrpC with HU. In addition, some archaea do produce nucleosomes from proteins homologous to eukaryotic histones.[50]

Chromatin Remodeling:

Chromatin remodeling can result from covalent modification of histones that physically remodel, move or remove nucleosomes.[51] Studies of Sanosaka et al. 2022, says that Chromatin remodeler CHD7 regulate cell type-specific gene expression in human neural crest cells.[52]

See also

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Notes

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References

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Revisions and contributorsEdit on WikipediaRead on Wikipedia

Chromatin

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from Grokipedia
Chromatin is a complex of DNA and proteins that packages the eukaryotic genome into chromosomes, enabling the compaction of vast lengths of genetic material—approximately 2 meters in humans—into the micron-sized nucleus of a cell. The fundamental structural unit of chromatin is the nucleosome, consisting of approximately 147 base pairs of DNA wrapped around an octamer of histone proteins (two each of H2A, H2B, H3, and H4), with linker DNA connecting adjacent nucleosomes and often stabilized by linker histone H1. This "beads-on-a-string" arrangement allows chromatin to exist in two primary states: euchromatin, which is relatively loose and transcriptionally active, and heterochromatin, which is densely packed and generally repressive for gene expression. Beyond basic packaging, chromatin dynamically regulates essential cellular processes through its multilevel organization and modifications. At higher orders, chromatin folds into loops, topologically associating domains (TADs), and nuclear compartments, influenced by proteins like and , which facilitate long-range interactions critical for gene regulation. Epigenetic modifications, such as histone , , and , alter chromatin accessibility by recruiting regulatory factors or changing stability, thereby controlling access to DNA for transcription factors and . These mechanisms ensure precise spatiotemporal control of gene expression, DNA replication, repair, and maintenance of genome stability, with disruptions linked to diseases including cancer and developmental disorders. Recent advances in techniques like sequencing and have revealed chromatin's four-dimensional dynamics, including into liquid-like condensates that compartmentalize functional genomic regions. During , chromatin condenses further into discrete chromosomes visible under a light microscope, facilitating accurate segregation to daughter cells. Overall, chromatin's structure and plasticity underpin eukaryotic life's complexity, integrating genetic and environmental cues to orchestrate cellular identity and response.

Definition and Composition

Core Definition

Chromatin is the macromolecular complex of DNA and proteins found in the nuclei of eukaryotic cells, primarily consisting of DNA wrapped around histone proteins along with various non-histone proteins that collectively package the genetic material. This assembly enables the compaction of approximately two meters of DNA in a typical human cell into a nucleus measuring just a few micrometers in diameter, achieving an overall compaction ratio of approximately 10,000-fold. The basic structural unit of chromatin is the nucleosome, formed by DNA coiled around a core of histone proteins. The term "chromatin" was coined in 1882 by German biologist , who observed thread-like structures in stained cell nuclei while studying in animal tissues. Flemming described these as basophilic (dye-affine) substances in his seminal work Zellsubstanz, Kern und Zelltheilung, distinguishing them from other nuclear components. By the early , advances in cytology clarified the distinction between chromatin as the diffuse, protein-DNA complex throughout the and chromosomes as the highly condensed forms of chromatin visible primarily during . Chromatin serves essential functions, including protecting DNA from physical damage and chemical insults through its compact organization, regulating access to the genome for processes such as and transcription, and facilitating the inheritance of epigenetic states that influence across cell generations. These roles are mediated by chromatin's dynamic structure, which can alternate between loosely packed —characterized by open configuration and association with active gene transcription—and densely packed , which is transcriptionally repressed and maintains genomic stability.

Molecular Components

Chromatin consists of DNA and associated proteins, with DNA serving as the primary scaffold in the form of a double-stranded, negatively charged polymer that electrostatically interacts with positively charged proteins. This DNA is typically organized in a linear fashion within the nucleus, comprising the genetic material that is packaged and regulated by protein components. Quantitatively, chromatin incorporates segments of DNA, with approximately 147 base pairs associating with core histone proteins to form structural units. The histone proteins are the predominant molecular components, categorized into core and linker types. Core histones include two molecules each of H2A, H2B, H3, and H4, which together form a histone octamer around which DNA wraps. The linker histone H1 binds externally to the DNA-histone complex, contributing to structural stability through interactions with linker DNA segments between core units. Non-histone proteins constitute a diverse group within chromatin, including high-mobility group (HMG) proteins that influence DNA bending and accessibility, RNA polymerases that associate with the chromatin template, and architectural factors such as CTCF that help maintain chromatin conformation. These proteins, while varying in abundance and specificity, interact with DNA and histones to modulate the overall molecular architecture without being integral to the core repeating unit. Although present in smaller quantities relative to DNA and proteins, non-coding RNAs represent an emerging class of chromatin components with roles in scaffolding and structural support. Long non-coding RNAs, for instance, can bind chromatin and contribute to its organizational framework through interactions with proteins and DNA. The nucleosome, integrating these DNA and protein elements, acts as the fundamental repeating unit of chromatin.

Hierarchical Structure

Nucleosome Assembly

The nucleosome core particle serves as the fundamental unit of chromatin packaging, consisting of approximately 147 base pairs of DNA wrapped in a left-handed superhelix around a histone octamer composed of two copies each of the core histones H2A, H2B, H3, and H4. This wrapping occurs for about 1.65 turns, forming a compact structure approximately 5.5 nm in diameter and 6 nm in height, with the DNA path following a helical trajectory of roughly 147 bp total length and a pitch of ~10 bp per turn. Adjacent nucleosomes are connected by stretches of linker DNA, typically 10–80 bp in length, resulting in the characteristic "beads-on-a-string" appearance observed in electron micrographs of decondensed chromatin. Nucleosome assembly can be recapitulated in vitro through methods such as stepwise salt dialysis, where core histones and DNA are mixed under high salt conditions (e.g., 2 M NaCl) to promote nonspecific interactions, followed by gradual reduction of salt concentration to allow sequential deposition and wrapping of DNA around the . This technique yields regularly spaced arrays mimicking physiological structures and has been widely used to study chromatin reconstitution. In vivo, assembly is a tightly regulated process mediated by histone chaperones and ATP-dependent remodelers to ensure proper deposition during , repair, or transcription. Key players include the chaperone nucleosome assembly protein 1 (NAP-1), which facilitates histone deposition by shielding basic histone tails and preventing nonspecific DNA binding, and the ATP-utilizing chromatin assembly and remodeling factor (ACF), which uses to space at regular intervals of ~190 . Insights into the atomic structure of the core particle were provided by the seminal study at 2.8 resolution, which revealed the characteristic fold—a long α-helix flanked by shorter helices and loops—that forms the dimerization interface for H2A-H2B and H3-H4 pairs within the octamer. This structure also highlighted the extensive -DNA interactions, primarily electrostatic in nature, involving and residues from the histone tails and cores forming hydrogen bonds and salt bridges with the DNA phosphate backbone at 14 distinct sites along the superhelical ramp. These interactions stabilize the wrapped DNA conformation despite the significant bending strain, with the histone octamer's saddle-shaped surface guiding the DNA's path. Histone variants contribute to structural diversity in nucleosomes while maintaining the core architecture. For instance, H2A.Z, which replaces canonical H2A in about 5–10% of nucleosomes, introduces subtle alterations in the octamer's surface that affect DNA wrapping stability and nucleosome positioning, often leading to more dynamic structures at promoter regions. Similarly, the variant H3.3, differing from canonical H3 by only four amino acids, incorporates into nucleosomes with comparable wrapping geometry but influences the rigidity of the histone core, thereby modulating overall nucleosome stability without disrupting the fundamental octamer assembly. These variants preserve the essential DNA-histone interactions but fine-tune the nucleosome's structural properties for specialized chromatin contexts.

Chromatin Fibers and Loops

Beyond the basic nucleosome structure, chromatin organizes into higher-order fibers and loops that facilitate compaction and functional compartmentalization. The classical 30-nm fiber model posits a folded structure of the 10-nm "beads-on-a-string" nucleosomal array, where nucleosomes coil into a solenoid helix with approximately six nucleosomes per turn, achieving a diameter of about 30 nm. This model, stabilized by linker histone H1 binding to linker DNA, was derived from electron microscopy observations of chromatin in low ionic strength conditions. An alternative zigzag model proposes a two-start helical arrangement, with nucleosomes alternating positions connected by straight linker DNA segments, also yielding a ~30-nm diameter but with a more irregular pitch. However, the existence of a uniform 30-nm fiber in vivo remains debated, as in situ studies often fail to detect it, suggesting chromatin maintains a more dynamic, irregular conformation rather than a stable helical fold. Recent advances in cryo-electron tomography (cryo-ET) have provided direct visualization of native chromatin fibers, revealing flexible, irregular arrays of nucleosomes without a 30-nm structure; instead, often forms straight, patterns between nucleosomes, varying with ionic conditions and variants. These findings challenge the and models, indicating that higher-order folding may involve transient interactions rather than fixed helices, with compaction driven by multivalent tails and non-histone proteins. At larger scales, chromatin forms DNA loops of 50–100 kb, anchored by the insulator protein at convergent binding sites and extruded by complexes, which act as molecular motors to pull distant genomic regions together. These loops define topologically associating domains (TADs), self-interacting regions averaging 1 Mb that insulate enhancers from non-target genes, as identified through () techniques. The loop extrusion model explains TAD formation: cohesin loads onto DNA and reels in loops until stalled by CTCF barriers, creating stable domains that enhance regulatory specificity. During mitosis, chromatin undergoes extreme condensation, transforming into highly compacted chromosomes visible as distinct entities; this involves radial loop organization around a protein scaffold enriched in topoisomerase II (topo II), which decatenates intertwined DNA strands to resolve entanglements. Topo II, along with condensin, forms the core scaffold, enabling chromosomes to achieve a ~10,000-fold compaction from extended DNA while maintaining structural integrity for segregation. Hierarchically, chromatin scaling progresses from the 10-nm nucleosomal fiber to irregular ~30-nm folds, then to looped domains, and further to 200–300-nm chromonema fibers observed in chromosomes via electron microscopy tomography. This multi-level organization, visualized , supports a model of progressive, irregular compaction without rigid intermediate fibers, integrating loops and scaffolds for mitotic readiness.

Nuclear Spatial Organization

Chromatin within the eukaryotic nucleus is organized into distinct three-dimensional structures that influence and stability. Each of the 23 pairs of occupies a discrete, non-overlapping region known as a chromosome territory (CT), a principle established through (FISH) techniques developed in the 1990s. These territories maintain spatial separation during , with gene-rich chromosomes often positioned toward the nuclear interior and gene-poor ones toward the periphery, facilitating efficient nuclear function without extensive intermingling. Genome-wide chromatin conformation capture methods, such as introduced in 2009, have revealed that chromosomes fold into globule structures, where long-range interactions form contact maps exhibiting scale-invariant patterns without knots, allowing compact yet accessible packaging. These maps further delineate the into two major compartments: the A compartment, enriched in and active, gene-dense regions, and the B compartment, dominated by repressive often associated with the . The A compartment promotes open, transcriptionally permissive environments, while the B compartment enforces silencing through spatial segregation. Interactions with the nuclear lamina, a meshwork of lamin proteins lining the inner nuclear membrane, tether specific heterochromatic regions to the periphery via lamina-associated domains (LADs), which span 0.1 to 10 Mb and correlate with low gene expression. LADs, first mapped in 2008 using DamID sequencing in human fibroblasts, exhibit enrichment for histone marks like H3K9me2/3 and contribute to stable repression by positioning chromatin away from transcription factories. Emerging models invoke liquid-liquid phase separation (LLPS) to explain heterochromatin compartmentalization, particularly in B compartments, where heterochromatin protein 1 (HP1) multivalently binds H3K9me-modified nucleosomes and, in concert with RNA molecules, drives condensate formation. This LLPS mechanism, demonstrated in Drosophila embryos in 2017, enables dynamic yet reversible clustering of heterochromatin into liquid-like droplets that exclude active factors, enhancing spatial organization without rigid scaffolding.

Dynamic Organization

Cell Cycle Variations

During the G1/S phase transition, chromatin undergoes replication-coupled assembly to duplicate its structure alongside . This process incorporates newly synthesized s, which are produced in late G1 and peak during , into nascent DNA strands via histone chaperones such as Asf1 and CAF-1. Parental histones are recycled and randomly segregated to daughter strands, helping to propagate epigenetic information. To facilitate replication fork progression, chromatin temporarily loosens, allowing access to DNA origins and reducing barriers, with rapid reassembly restoring structure post-replication. In mitosis, chromatin achieves hypercondensation to form compact essential for segregation. A key event is the of at serine 10 (H3 Ser10), which correlates temporally with starting in and peaking in , promoting higher-order folding by altering histone-DNA interactions. This modification, conserved across eukaryotes, facilitates the organization of chromatin into linear loop arrays anchored to a protein scaffold, including topoisomerase II and . (CDK1) plays a central role by phosphorylating non-histone proteins, such as the CAP-D3 subunit of condensin II at Thr1415, which recruits Polo-like kinase 1 (Plk1) to initiate axial assembly and ensure timely compaction. This mitotic condensation achieves a total compaction of approximately 10,000-fold relative to the extended length of naked DNA, with additional structural organization beyond the already compacted interphase state, though recent estimates suggest the increase in density may be only 2-3 fold or even negligible. However, recent electron microscopy studies suggest that the increase in chromatin density during mitosis may be minimal (approximately 1-fold), with compaction primarily involving structural rearrangements into cylindrical forms. Following , during post-mitotic reassembly in early G1, chromatin decondenses as the reforms, restoring organization. marks, such as H3K27ac at enhancers and promoters, act as mitotic bookmarks to retain epigenetic memory, associating with mitotic chromatin to guide rapid reactivation of and preserve cell identity. For instance, over 50% of enhancers in pluripotent stem cells retain H3K27ac through , ensuring faithful propagation of regulatory states. This bookmarking mechanism, supported by stable modifications like and , prevents stochastic loss of epigenetic information during division.

Transcription-Associated Changes

During active transcription, chromatin undergoes transient structural alterations to facilitate access by (Pol II) and associated factors. These changes are dynamic and reversible, enabling bursts of while maintaining overall genomic stability. In the bursts model, transcription occurs in stochastic pulses where chromatin domains open intermittently, allowing Pol II to access promoter regions for short periods, typically lasting a few minutes before recondensing. This stochastic opening is driven by the probabilistic nature of molecular interactions, resulting in variable mRNA output across cells, as demonstrated in early single-cell studies of eukaryotic genes.00582-4) Chromatin remodelers play a central role in these processes by altering positioning and composition. The family of ATP-dependent complexes, first identified in for their role in gene activation, slides or evicts nucleosomes to expose DNA sequences for transcriptional initiation in mammalian systems. For instance, the mammalian BAF complex variant of SWI/SNF repositions nucleosomes at promoters, facilitating Pol II recruitment and elongation. Complementing this, the FACT (facilitates chromatin transcription) complex acts as a chaperone, temporarily removing H2A-H2B dimers from nucleosomes during Pol II passage, which reduces barriers to transcription without fully disassembling the octamer. This dimer eviction and reassembly mechanism was elucidated through transcription assays, highlighting FACT's specificity for transcribing chromatin templates. Enhancer-promoter interactions further contribute to these changes by forming spatial loops that bring distant regulatory elements into proximity with genes. The complex, a large coactivator scaffold, bridges enhancers and promoters, while stabilizes these loops through DNA extrusion, enabling efficient signal transmission from activators to the basal transcription machinery. This looping is particularly evident at active loci, where depletion of or Mediator disrupts contacts and reduces transcriptional output, as shown in embryonic stem cells.01100-0) Transcriptional organization can vary between punctuated bursts and more continuous modes, influenced by . Punctuated transcription aligns with bursty kinetics at many metazoan genes, whereas continuous patterns occur at highly stable loci. Super-enhancers, clusters of enhancers marked by high and occupancy, often form phase-separated condensates that act as hubs for concentrated transcriptional machinery, enhancing burst frequency and amplitude at cell identity genes. These liquid-like hubs, observed via live imaging, promote robust activation by sequestering factors in a chromatin-tethered microenvironment. Live-cell imaging techniques, such as (FRAP), reveal the kinetics of these dynamics. FRAP experiments show that mobility increases during transcription, with recovery half-times of approximately 1-5 minutes in active regions, reflecting nucleosome disassembly and reassembly coupled to Pol II progression. In contrast, recovery is slower (tens of minutes) in repressed chromatin, underscoring transcription's role in accelerating histone exchange. These measurements, pioneered in mammalian cells, quantify how active genes exhibit heightened turnover compared to silent domains.

Functional Roles

Role in Gene Expression

Chromatin plays a pivotal role in regulating gene expression by modulating the accessibility of DNA to transcription factors (TFs) and the transcriptional machinery. In euchromatin, the relatively open and decondensed structure facilitates TF binding and subsequent gene activation. Open chromatin regions, comprising approximately 2–3% of the genome, harbor over 90% of TF binding sites, enabling the recruitment of RNA polymerase II and co-activators at promoters and enhancers. This accessibility is dynamically maintained by ATP-dependent chromatin remodeling complexes, such as SWI/SNF, which reposition nucleosomes to expose DNA sequences, thereby promoting transcriptional initiation and elongation essential for cellular processes like development and response to stimuli. Insulator elements further refine this activation by preventing the inappropriate spread of euchromatic signals into adjacent repressive domains, ensuring precise spatial control of gene expression. In contrast, enforces transcriptional repression through a more compact structure that limits TF access and promotes . Constitutive , found in gene-poor regions like centromeres and telomeres, maintains permanent repression via modifications such as and binding of (HP1), safeguarding stability by suppressing repetitive elements and recombination. Facultative , however, exhibits reversible silencing in response to developmental cues; a prime example is X-chromosome inactivation in female mammals, where the coats the inactive , recruiting silencing factors like SHARP and PRC2 to deposit marks, thereby repressing for dosage compensation. This distinction allows facultative regions to toggle between active and inactive states, contrasting with the fixed repression of constitutive . Epigenetic memory ensures the stable inheritance of chromatin states across cell divisions, preserving patterns through . Polycomb repressive complex 2 (PRC2)-mediated modifications exemplify this, as parental nucleosomes marked with are recycled during replication by histone chaperones like CAF-1, while PRC2 restores the mark on nascent chromatin through a self-propagating feedback loop, maintaining repression without continuous external signals. This mechanism underpins heritable silencing, such as in developmental lineage commitment, where diluted marks post-replication are efficiently re-established to avoid stochastic reactivation. Dysregulation of chromatin-mediated gene expression contributes to diseases, particularly cancer, where mutations in chromatin regulators disrupt these controls. For instance, loss-of-function mutations in SMARCB1 (a subunit) occur in nearly all rhabdoid tumors, leading to elevated activity and widespread deposition that aberrantly represses tumor suppressor genes, driving uncontrolled proliferation. Similarly, gain-of-function mutations in , such as Y646F, redistribute in lymphomas and melanomas, enforcing oncogenic repression of differentiation genes and promoting tumor progression. These alterations highlight chromatin regulators as key drivers of dysregulated expression, with therapeutic potential in targeting such mutants to restore balanced transcription.

Involvement in DNA Repair

Chromatin structure plays a critical role in the detection and repair of DNA damage by influencing the accessibility of repair machinery to lesion sites. Upon the occurrence of DNA double-strand breaks (DSBs), the histone variant H2AX is rapidly phosphorylated at serine 139 to form γ-H2AX, which serves as a molecular beacon that spreads along megabases of chromatin flanking the break site. This modification facilitates the recruitment of downstream repair factors, including the MRN complex (Mre11-Rad50-Nbs1) and ataxia-telangiectasia mutated (ATM) kinase, thereby initiating damage signaling and coordinating the DNA damage response (DDR). The formation of γ-H2AX foci thus marks chromatin domains requiring repair, promoting the assembly of multiprotein complexes essential for lesion resolution. Different DNA repair pathways interact distinctly with chromatin architecture to address various types of damage. (NER), which removes bulky helix-distorting lesions such as UV-induced cyclobutane , operates efficiently even in condensed chromatin regions by leveraging modifications and remodelers to transiently expose damaged sites without full decompaction. In contrast, (HR), a high-fidelity pathway for repairing DSBs, necessitates chromatin decondensation to enable strand invasion and template-directed synthesis, often involving the relaxation of arrays near the break to facilitate access to homologous sequences. This decondensation is mediated by ATP-dependent chromatin remodelers, such as the INO80 complex, which evicts at DSB sites to promote resection of DNA ends and subsequent HR progression. INO80's role is particularly vital in the early phases of repair, where it disassembles repressive barriers to allow binding of resection factors like CtIP and MRN. Links between chromatin dynamics and further ensure repair fidelity. ATM and ATR kinases, activated by DSBs and replication stress respectively, phosphorylate variants and remodelers to induce local chromatin alterations that support G2/M arrest, preventing progression through until damage is resolved. These kinases promote the opening of chromatin domains around lesions, enhancing repair factor recruitment while enforcing checkpoint activation via pathways involving Chk1 and Chk2. Overall repair efficiency varies by chromatin context, with processes in proceeding more slowly than in due to the need for extensive decompaction and higher-order structure reconfiguration.

Epigenetic Regulation

Histone Modifications

Histone modifications are covalent chemical alterations to the residues of proteins, primarily on their flexible N-terminal tails, that serve as key epigenetic mechanisms influencing chromatin structure and function. These post-translational modifications include , , , and ubiquitination, each mediated by specific enzymes known as "writers" and reversed by "erasers," with their effects often interpreted by "reader" proteins that recruit additional factors to modulate . involves the addition of an to residues, typically by histone acetyltransferases (HATs) such as p300/CBP, which neutralizes the positive charge of lysine, thereby reducing the electrostatic attraction between histones and negatively charged DNA, leading to a more open chromatin conformation that facilitates access for transcription machinery. Methylation, another prevalent modification, occurs on and residues and can result in mono-, di-, or trimethylation, with outcomes ranging from activation to repression depending on the site and degree. For instance, trimethylation at 4 (), catalyzed by histone methyltransferases (HMTs) of the Trithorax group such as MLL1, marks active promoters and is associated with transcriptional activation by recruiting reader proteins that stabilize open chromatin states. In contrast, trimethylation at H3 27 (), deposited by the Polycomb repressive complex 2 (PRC2) via its catalytic subunit , promotes gene repression by compacting chromatin and inhibiting transcription factor binding. adds a group to serine, , or residues, often in response to signaling events, while ubiquitination attaches to lysines, influencing processes like and , though these are less central to steady-state epigenetic patterning compared to and methylation. The code posits that the combinatorial patterns of these modifications form a "code" that is dynamically read by effector proteins to dictate specific chromatin responses, extending the informational content of the genome beyond DNA sequence alone. Readers such as bromodomain-containing proteins (e.g., ) specifically recognize acetylated lysines, recruiting co-activators to enhance transcription, while chromodomains in Polycomb proteins bind to propagate repressive states. Erasers, including deacetylases (HDACs) for and demethylases like UTX for , ensure reversibility, with modification turnover rates varying by context—e.g., exhibits slower dynamics at stable active loci compared to transient adjustments during development. This writer-reader-eraser triad, exemplified by the antagonistic Trithorax (activating) and Polycomb (repressive) groups, underlies the plasticity of chromatin-mediated regulation.

DNA and RNA Modifications

DNA methylation is a fundamental epigenetic modification involving the addition of a to the fifth carbon of bases, primarily at CpG dinucleotides, catalyzed by DNA methyltransferases (DNMTs) such as , DNMT3A, and DNMT3B. This modification typically occurs in promoter regions and CpG islands, where it represses by inhibiting binding and recruiting methyl-CpG-binding domain (MBD) proteins like MeCP2, which in turn facilitate chromatin compaction through interactions with deacetylases and other repressive complexes. In mammals, de novo methylation is established by DNMT3A and DNMT3B during early embryogenesis, while maintenance methylation by ensures propagation through cell divisions. Hydroxymethylation of DNA, marked by (5hmC), serves as an intermediate in active and is generated by the ten-eleven translocation (TET) family of enzymes (TET1, TET2, TET3), which oxidize (5mC). Unlike 5mC, 5hmC is enriched in gene bodies of actively transcribed genes and enhancers, particularly in post-mitotic neurons, where it promotes chromatin accessibility and neuronal differentiation by facilitating the binding of transcription factors and reducing repressive marks. TET-mediated oxidation can lead to further modifications like 5-formylcytosine and 5-carboxylcytosine, ultimately enabling and demethylation, thus dynamically regulating chromatin states in response to environmental cues. Non-coding RNAs, particularly long non-coding RNAs (lncRNAs), play crucial roles in modulating chromatin structure and function through direct coating and recruitment of modifying factors. The lncRNA , essential for X-chromosome inactivation in female mammals, spreads along the , recruiting polycomb repressive complexes and promoting into nuclear condensates that enforce silencing over large genomic domains. This RNA-mediated mechanism alters chromatin accessibility and three-dimensional organization, distinct from sequence-specific methylation, and is vital for dosage compensation. Other lncRNAs similarly influence chromatin by scaffolding protein complexes or guiding epigenetic writers to target loci, contributing to compartmentalization and gene repression. The interplay between DNA modifications and RNAs is exemplified by RNA-directed DNA methylation (RdDM) in , where small interfering RNAs (siRNAs) and lncRNAs direct DNMTs like DRM2 to homologous DNA sequences, establishing de novo methylation at transposable elements and to maintain stability. These mechanisms highlight how RNA molecules can orchestrate DNA modifications to fine-tune chromatin states across kingdoms. Evolutionarily, DNA methylation patterns are highly conserved among vertebrates, with CpG island hypermethylation repressing developmental genes and widespread gene-body marking genes, reflecting adaptations for complex gene regulation. In contrast, exhibit more variable and often sparser methylation landscapes, lacking prominent CpG islands and relying less on this modification for silencing, which correlates with simpler chromatin architectures and diverse epigenetic strategies. This divergence underscores 's role in vertebrate-specific innovations like imprinting and long-range chromatin interactions.

Methods of Study

Biochemical and Imaging Techniques

(ChIP) is a cornerstone biochemical technique for isolating and identifying specific DNA-protein interactions , particularly those involving modifications and chromatin-associated factors. The method relies on crosslinking proteins to DNA using , followed by chromatin shearing, antibody-mediated , and purification of the bound DNA fragments. Originally developed by Solomon, Larsen, and Varshavsky in 1988, ChIP demonstrated its efficacy by mapping the retention of H4 on actively transcribed genes in , revealing that core histones remain associated with DNA during transcription. This pull-down approach enables precise mapping of modifications, such as or , to specific genomic loci, providing insights into epigenetic without requiring prior knowledge of binding sites. Variations like sequential ChIP (re-ChIP) further allow interrogation of multi-protein complexes on chromatin. Micrococcal nuclease (MNase) digestion serves as an enzymatic tool to evaluate positioning, , and chromatin in vitro and in isolated nuclei. MNase preferentially cleaves between nucleosomes, generating a characteristic ladder of DNA fragments upon , with mononucleosomal cores protecting approximately 147 base pairs and dinucleosomes around 300 base pairs. Seminal work by Noll in established this repeating subunit structure of chromatin through limited digestion experiments, showing that eukaryotic genomes are organized into discrete nucleosomal units of about 200 base pairs total, including linkers. By titrating concentrations, researchers can assess regional ; for instance, open chromatin regions exhibit faster digestion rates, yielding shorter fragments, while compact domains resist cleavage. This technique has been refined for quantitative analysis, distinguishing positioned from delocalized nucleosomes based on protection patterns. Electron (EM) provides direct visualization of chromatin's higher-order architecture, from individual nucleosomes to folded fibers. Conventional transmission EM, applied to fixed and stained samples, first revealed the "beads-on-a-string" nucleofilament and its condensation into thicker structures under physiological salt conditions. and Klug's 1976 study used EM to propose the solenoidal 30-nm fiber model, where six nucleosomes coil into a helical structure with a pitch of about 11 nm, supported by images of magnesium-induced compaction. More recently, cryo-EM has advanced to near-atomic resolution without fixation artifacts, elucidating detailed nucleosome conformations and short-range folding. For example, cryo-EM reconstructions of reconstituted chromatin arrays have resolved tetranucleosomal units twisting into a double helix, confirming irregular 30-nm-like fibers with variable linker angles. These structures highlight how tails and linker s influence folding stability. Super-resolution imaging techniques surpass the diffraction limit of conventional light microscopy (~200 nm), enabling nanoscale observation of chromatin organization and dynamics in live or fixed cells. Stimulated emission depletion (STED) microscopy achieves resolutions down to 50 nm by depleting fluorescence around an excitation spot, allowing tracking of chromatin compaction states and fiber trajectories. In chromatin studies, STED has visualized dense, fiber-like domains in interphase nuclei, correlating epigenetic marks with local densities. Photoactivated localization microscopy (PALM), a stochastic optical reconstruction method, localizes individual fluorophores to ~20 nm precision over thousands of frames, revealing transient chromatin loops and domain boundaries. PALM imaging of labeled histones has shown dynamic extrusion of loops in real time, with extrusion rates varying by cell type and averaging 1-2 kb/s in mammalian cells. These approaches capture loop-mediated interactions, such as enhancer-promoter contacts, that underpin gene regulation. Despite their power, these techniques face inherent limitations that can introduce biases in interpreting chromatin structure. Fixation in ChIP and EM often stabilizes non-native conformations, potentially crosslinking distant interactions or disrupting dynamic associations, as evidenced by discrepancies between fixed and live . Pre-2010s methods were constrained by resolutions above 100 nm, obscuring fine arrays and loop scales below 50 kb, though super-resolution has mitigated this. Biochemical assays like MNase may over-digest accessible regions unevenly due to preferences, skewing occupancy maps. These methods integrate with genomic sequencing for validation, as detailed in complementary approaches.

Genomic and Computational Approaches

Genomic and computational approaches have revolutionized the study of chromatin by enabling high-throughput mapping of its structure and dynamics at genome-wide scales. techniques, such as , provide comprehensive 3D interaction maps by capturing pairwise chromatin contacts through cross-linking, digestion, and proximity ligation followed by high-throughput sequencing. The original method, introduced in 2009, provided genome-wide maps revealing compartmentalization into open (A) and closed (B) regions in mammalian genomes, with initial resolutions down to megabase scales. Subsequent higher-resolution studies identified topologically associating domains (TADs). Variants like (circular ) focus on interactions involving a single bait locus by using inverse PCR to detect genome-wide contacts, offering targeted insights into enhancer-promoter pairings. Similarly, 5C ( ) employs ligation-mediated amplification with pools of bait and prey primers to generate high-resolution interaction matrices for predefined genomic regions, typically resolving interactions at tens of kilobase precision. Assays like and ChIP-seq complement by profiling chromatin accessibility and modifications, respectively, facilitating multi-omics integration for a layered view of regulatory landscapes. uses a hyperactive Tn5 to insert sequencing adapters into accessible chromatin regions, enabling rapid, low-input mapping of open chromatin with nucleosome-level resolution in diverse cell types. ChIP-seq, which immunoprecipitates chromatin bound by specific antibodies against modifications or transcription factors, quantifies modification profiles such as H3K27ac enrichment at active enhancers, often integrated with data to annotate functional loops. Multi-omics pipelines, such as those combining , ChIP-seq, and , reveal how accessibility correlates with interaction strength, as seen in studies linking open chromatin to insulated neighborhoods in developmental contexts. Computational models grounded in polymer physics simulate chromatin folding to interpret experimental data and predict structural transitions. Loop extrusion models posit that cohesin and CTCF proteins form loops by actively reeling in DNA, modeled as Brownian ratchets on self-avoiding polymer chains to recapitulate TAD formation and contact probabilities decaying as a power law with genomic distance. These simulations, often using Monte Carlo methods on bead-spring polymers, quantify how extrusion speed and barrier strengths influence loop sizes, achieving agreement with Hi-C maps at kilobase resolutions. Machine learning approaches, such as convolutional neural networks, predict chromatin states from sequence features alone, training on epigenomic profiles to forecast accessibility or interaction probabilities with accuracies exceeding 80% in cross-validation. Post-2015 advances have enhanced resolution and scalability, particularly through single-cell methods that dissect heterogeneity in chromatin architecture. Techniques like sci- use combinatorial indexing to profile thousands of cells, revealing cell-to-cell variability in loop formation during differentiation, with contact maps at 100 kb resolution. Recent developments as of 2025 include deep-learning methods for reconstructing 3D chromatin structures from single-cell and cryo-EM studies elucidating mechanisms of enzymes like SNF2H. AI-driven models for enhancer prediction, leveraging graph neural networks on multi-omics , identify distal regulatory elements by integrating motifs with interaction graphs, improving recall by 20-30% over rule-based methods in human genomes. Despite these gains, challenges persist, including normalization for biases like restriction fragment length and , addressed by iterative correction algorithms that equalize expected contacts across replicates. Achieving 1 kb resolution requires deep (billions of reads) to overcome sparse signals in low-abundance interactions, with tools like cooler matrices mitigating computational overhead.

Advanced and Alternative Concepts

Chromatin Topology and Entanglement

Chromatin topology encompasses the spatial organization and physical entanglements of DNA within the nucleus, where supercoiling arises from the helical twisting of DNA around nucleosomes, leading to torsional stress that can propagate through chromatin fibers. In looped chromatin structures, catenanes—interlinked DNA rings—form during replication and transcription, potentially hindering processes like gene expression and chromosome segregation. Knotting becomes particularly prevalent in highly compacted mitotic chromosomes, where the dense folding of chromatin increases the likelihood of intramolecular tangles, as evidenced by analyses of three-dimensional chromosome structures from Hi-C data showing over 80% of chromosomes containing knots. Topoisomerases are essential enzymes that resolve these topological constraints in chromatin. Type I topoisomerases, such as TOP1 and TOP3A, create single-strand breaks to relax supercoils and facilitate limited decatenation, particularly in and during replication to prevent fork collapse. Type II topoisomerases, including TOP2A and TOP2B, introduce double-strand breaks to decatenate intertwined chromatids and relax both positive and negative supercoils more efficiently in nucleosomal contexts; TOP2A is indispensable for mitotic segregation, as its inhibition leads to unresolved entanglements and segregation failure. These enzymes interact with chromatin remodelers to ensure timely resolution, maintaining stability. Monte Carlo simulations of chromatin models demonstrate that knot probability escalates with increasing compaction, as denser folding confines polymer chains and promotes self-entanglements. In coarse-grained representations of double-stranded DNA within chromatin, knotting frequencies rise nonlinearly with chain length and compaction density, reflecting the entropic drive toward tangled states in confined volumes. These models, often using self-avoiding walks or Kratky-Porod chains, predict that transcriptional supercoiling further boosts knot formation during chromatin condensation. Experimental detection of chromatin knots relies on high-resolution , which separates knotted DNA species based on mobility shifts, revealing steady-state knot fractions of 2–3% in minichromosomes irrespective of replication. Recent studies highlight entropic barriers in entanglement resolution, showing that mitotic chromosomes start highly self-entangled and disentangle progressively through topoisomerase II activity during and early G1, with loop extrusion aiding in reducing residual tangles. Chromatin's architecture limits knot complexity beyond ~20 nucleosomes, minimizing severe entanglements. Unresolved topological knots and catenanes in chromatin impair chromosome segregation, leading to by causing chromatin bridges and unequal distribution during . In TOP2-deficient cells, persistent entanglements trigger DNA breaks and genomic instability, with chromatin bridges from unresolved linkages inducing lesions that propagate in daughter cells. These defects underscore the biological cost of topological failures, linking them to conditions like cancer.

Evolving Models and Definitions

The traditional model of chromatin structure, dominant from the to the early , envisioned it as a static, hierarchical assembly of nucleosomes folded into a 30-nm that further compacted into higher-order structures during and . This view, supported by electron microscopy and studies, posited a regular, fiber-like organization enabling efficient DNA packaging. However, post-2010 observations from advanced imaging techniques in living cells revealed chromatin as a highly dynamic, irregular network rather than a rigid , shifting paradigms toward models emphasizing fluidity and adaptability. By the mid-2010s, evidence mounted for liquid-like behaviors, where chromatin domains exhibit rapid rearrangements driven by molecular interactions, marking a transition from static to dynamic conceptualizations. Alternative definitions have emerged to capture chromatin's complexity beyond nucleosome-linker histone units. The fractal globule model describes chromatin as a compact, unknotted configuration that allows efficient folding of the while preserving accessibility for processes like transcription, supported by data showing scale-free contact probabilities. Similarly, the polymer melt model portrays interphase chromatin as a disordered, interdigitated assembly of 10-nm fibers akin to a viscous melt, where nucleosomes interact promiscuously without forming regular helices, consistent with from native chromosomes. These polymer-based views highlight chromatin's entropic and topological properties, contrasting with earlier rigid frameworks. Debates surrounding the 30-nm fiber intensified in the 2000s, culminating in its dismissal as a prevalent structure based on cryo-electron and showing no widespread 30-nm periodicity in eukaryotic nuclei. Instead, data favor heterogeneous, clutch-like groupings without solenoid folding. For chromatin, the bottlebrush model proposes a radial extension of loops from a central axis, creating bristle-like protrusions that enhance and organization without dense compaction, aligning with simulations of loop extrusion. Recent updates from 2020 to 2025 highlight as a primary mechanism for chromatin organization, where multivalent interactions drive liquid-liquid (LLPS) into condensates that compartmentalize the nucleus dynamically. These condensates, influenced by modifications and , enable rapid responses to cellular signals, as reviewed in studies integrating and biochemistry. Concurrently, AI-integrated models, such as graph neural networks combined with simulations, have advanced predictions of chromatin heterogeneity, capturing cell-to-cell variations in 3D conformations from single-cell data. To broaden inclusivity, prokaryotic systems offer analogs through nucleoid-associated proteins (NAPs) like HU and H-NS, which compact bacterial DNA into a nucleoid resembling eukaryotic chromatin by bending and bridging DNA segments, facilitating gene regulation without histones. These proteins enable phase-like condensates in bacteria, paralleling eukaryotic LLPS and highlighting conserved principles of genome organization across domains of life.

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