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\b[<chromatin> has organization across overlapping length scales, and each assay measures a particular aspect rather than a universal “openness”.] Local <DNA> exposure, <histone> occupancy, chemical state, long-range proximity and position within the nucleus are related but distinct. A strong <experimental design> chooses the relevant scale and combines a genomic assay with an independent structural or functional measurement.
At the molecular scale, <DNA> wraps around a <histone octamer> to form a <nucleosome> core, with roughly 147 base pairs protected; <linker DNA> connects neighbouring units, and linker <histones> can affect their organization. The core contains two copies each of H2A, H2B, H3 and H4. Crystallography established the detailed DNA–histone arrangement using a defined <nucleosome> core particle.<Crystal structure of the nucleosome core particle at 2.8 Å resolution by Karolin Luger and others>[Atomic structure of the reconstituted core particle]{ref} Such structures explain local contacts, but a purified core does not specify the arrangement of an entire chromosome in a living nucleus. <histone> variants, tail modifications and ATP-dependent remodelling change stability and access without requiring DNA-sequence changes.
For local accessibility, <ATAC-seq> uses transposase-mediated insertion of sequencing adapters into accessible <DNA>. It maps accessible <promoters> and regulatory elements, and its fragment-length distribution can contain signatures of nucleosome-free regions and neighbouring <nucleosomes>. The original assay combined rapid accessibility mapping with information about <nucleosome> organization.<Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position by Jason D. Buenrostro and others>[ATAC-seq development and accessible-region profiling]{ref} DNase I hypersensitivity provides a related cleavage-based measure. Both have sequence bias and depend on preparation and enzyme exposure; a footprint can reflect enzyme preferences or protected binding, and is not a direct identification of the occupying <transcription factor>. Compare <biological replicates>, enzyme-dose controls, appropriate background and <biological cell> composition. Accessibility does not prove that an element regulates a particular <gene>.
<nucleosome mapping by MNase-seq> addresses occupancy and spacing more directly: partial micrococcal-nuclease digestion preferentially removes exposed <linker DNA>, and sequencing protected fragments maps nucleosome-associated regions. Digestion titration is essential because over-digestion can preferentially lose fragile <nucleosomes> and create apparent depletion; MNase also has sequence preferences and can recover other protected particles. Compare fragment sizes, <histone> enrichment and an independent accessibility assay. Chemical mapping or defined in vitro reconstitution can refine particular <nucleosome> positions, while single-molecule measurements can expose heterogeneity hidden in a population average.
Chemical states require different assays. <histone acetylation> and methylation at particular residues can correlate with distinct regulatory states; they are not interchangeable with physical accessibility. <chromatin immunoprecipitation> followed by sequencing, or targeted <CUT&RUN>, maps association of a <histone modification> or <protein> with genomic regions. <antibody> specificity, <histone> abundance, input/background and calibrated normalization matter, especially when the modification changes globally. In <CUT&RUN>, an <antibody> recruits a nuclease to release nearby <DNA>, reducing some backgrounds associated with bulk <chromatin> fragmentation.<An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites by Peter J. Skene and Steven Henikoff>[Principle and validation of targeted chromatin cleavage]{ref} <DNA methylation> can be measured by appropriately controlled bisulfite-based or direct-read approaches; standard bisulfite measurements do not automatically distinguish every modified cytosine species. These assays locate a chemical mark, not the geometric folding of the fibre.
At the level of <nucleosome> arrays, regular compact fibres can form in some reconstituted or specialized conditions. A universal hierarchy in which every interphase chromosome passes through a uniform 30-nm fibre is not supported. <electron tomography> and related imaging can examine the distribution and packing of <chromatin> directly. <ChromEMT> combined DNA-selective staining with <electron tomography> and revealed irregular <chromatin> chains and variable compaction in <biological cells>.<ChromEMT: Visualizing 3D chromatin structure and compaction in interphase and mitotic cells by Horng D. Ou and others>[In situ chromatin ultrastructure and compaction]{ref} Fixation, staining, sectioning and limited sampled volumes must be considered; cryogenic and super-resolution approaches provide complementary views. Dense staining also need not imply a distinct biochemical <chromatin> state.
Longer-range folding produces loops and domains of enhanced contact. <chromosome conformation capture> crosslinks, digests and ligates spatially associated fragments; targeted variants test selected loci, whereas <Hi-C> samples contacts genome-wide. Contact matrices reveal local domains and broad A/B compartmentalization. High-resolution in situ <Hi-C> mapped <chromatin loops> and their association with <CTCF> orientation and <cohesin>.<A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping by Suhas S. P. Rao and others>[In situ contact maps, loop anchors and domain structure]{ref} A <topologically associating domain> is a region with preferential internal contacts under a stated analysis, not necessarily a stable, sharply bounded object in every <biological cell>. Contact frequency is neither an absolute distance nor proof that two sites directly interact. Restriction-site distribution, crosslinking, ligation, coverage and bin size affect resolution and interpretation.
Perturbation can distinguish mechanisms underlying similar-looking domains. Acute <cohesin> loss eliminated loop domains while compartment organization persisted, showing that local loop organization and large-scale compartmental segregation are separable.<Cohesin loss eliminates all loop domains by Suhas S. P. Rao and others>[Acute depletion, loss of loops and persistence of compartments]{ref} Imaging by <DNA fluorescence in situ hybridization> can measure distance distributions between chosen loci and test heterogeneity; live labels track dynamics but can perturb loci. Agreement between contact enrichment and a shift in distance distributions is stronger than either assay alone, although the observables still differ.
At nuclear scale, chromosomes occupy territories and regions associate preferentially with the <nuclear lamina> or <nucleolus>. <DNA> imaging maps territories and radial positions, while lamina-association assays such as <DamID> map sequences near a labelled nuclear compartment over an integration interval. The original <DamID> approach tethered a <DNA> adenine methyltransferase to a <chromatin> <protein> to record nearby genomic regions.<Identification of in vivo DNA targets of chromatin proteins using tethered Dam methyltransferase by Bas van Steensel and Steven Henikoff>[Targeted methylation as a proximity record]{ref} Such maps record proximity and history rather than a single fixed structure. Mitotic condensation is a distinct organization with additional constraints and chromosome-shaping machinery, not simply maximally closed interphase <chromatin>. \b[The appropriate pairing is exposure assays for local access, protected-fragment assays for <nucleosomes>, <protein>/mark profiling for composition, contact assays for genomic proximity and imaging for spatial organization, with perturbations to connect organization to function.]
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