Three-Dimensional Chromatin Architecture Remodeling and Mechanisms of Physiological Aging

Physiological aging is not merely the result of slow drift in transcriptional output, but also involves multilevel reorganization of chromatin in nuclear space. What changes during aging is not a single epigenetic mark or an individual gene locus, but the coordinated remodeling of three-dimensional structural units, including A/B compartment allocation, topologically associating domains, enhancer-promoter loops, lamina-associated domains, and heterochromatin clustering states. On the one hand, these changes alter the stability and noise level of gene expression programs; on the other hand, they influence DNA replication, damage responses, nuclear lamina anchoring, and maintenance of cell lineage identity, thereby extending local epigenetic drift into tissue-level functional decline.

Keywords: three-dimensional chromatin; physiological aging; A/B compartments; topologically associating domains; chromatin loops; lamina-associated domains; heterochromatin

1. Hierarchical Basis of Three-Dimensional Chromatin Structure

1.1 Spatial organizational units

(1) Compartment structure

The first layer of three-dimensional genome organization is generally represented by A/B compartments. A compartments are more frequently associated with open chromatin, active transcription, and early replication, whereas B compartments are more closely associated with compact chromatin, late replication, and transcriptional repression. Many transcriptional changes in physiological aging are therefore not determined solely by individual gene promoters, but are closely related to whether the genomic region containing a given gene undergoes compartmental switching.

(2) Domain and loop structures

Below the compartment level, topologically associating domains and enhancer-promoter loops jointly determine whether local regulatory elements can establish efficient contacts. CTCF and cohesin maintain boundary insulation and local folding stability, and thus their binding strength, boundary integrity, and loop-contact efficiency directly affect the temporal expression of lineage-related genes.

(3) Peripheral nuclear anchoring structures

Lamina-associated domains are another key layer of three-dimensional chromatin architecture. Large numbers of heterochromatic regions are anchored to the nuclear periphery through nuclear-envelope-associated factors such as lamin, LBR, and LAP2, thereby maintaining a repressive transcriptional environment and relatively low chromatin mobility. Peripheral anchoring is not merely a spatial positioning phenomenon, but an important structural foundation for preservation of cell identity and restriction of aberrant transcription.

1.2 Analytical objects in physiological aging

(1) Physiological aging versus stress-induced senescence

Physiological aging in the context of natural chronological progression more often manifests as progressive, locus-selective, and lineage-associated structural drift, whereas senescence induced by replicative exhaustion, DNA damage, or oncogenic stress more readily presents as rapid and more pronounced structural destabilization. The two may overlap in heterochromatin loss, nuclear lamina decoupling, and transcriptional imbalance, but their mechanistic hierarchy and limits of extrapolation are not identical.

(2) Mechanistic attributes of three-dimensional structure

In aging research, three-dimensional chromatin is not a background descriptor, but a mechanistic object. The key issue to explain is which structural layer changes first, which classes of genes destabilize earliest, and how these changes propagate into replication, repair, and lineage function, rather than attributing all phenomena in a broad way to "chromatin relaxation."

2. Types of Three-Dimensional Structural Remodeling in Physiological Aging

2.1 Compartment switching

(1) Redistribution of A/B compartments

Physiological aging does not necessarily manifest as globally random folding disorder, but more often as drift in compartment identity in specific genomic regions. Studies of aged bone marrow pro-B cells have shown that aging is accompanied by enhanced compartment-level interactions, while regions containing certain key developmental genes shift from A compartments into B compartments.

(2) Downward repositioning of lineage genes

In that study, the A-to-B conversion of the Ebf1 locus was associated with reduced B-cell developmental capacity, indicating that the functional significance of compartment switching lies not in spatial relocation itself, but in resetting the regulatory environment surrounding developmental genes, thereby making them more likely to undergo weakened enhancer contact and transcriptional downregulation.

2.2 Reduced insulation within domains

(1) Decreased intradomain contacts

In physiological aging, a more common feature is not simultaneous collapse of all boundaries, but reduced intradomain contact strength and weakened local regulatory coordination. In aged pro-B cells, aging is accompanied by weakening of interactions within TADs, particularly in regions related to immunoglobulin rearrangement and B-cell fate determination.

(2) Destabilization of local loop structures

Weakening of enhancer-promoter loops usually does not produce a simple on-off change in single genes, but more often manifests as reduced expression amplitude, increased transcriptional noise, and delayed responsiveness. For long-term aging, this kind of subtle mismatch is more consistent with progressive functional decline than extreme structural collapse.

2.3 Nuclear lamina decoupling

(1) Reduced LAD anchoring

During aging, coupling between heterochromatin and the nuclear lamina may gradually weaken. LADs are usually enriched in repressive chromatin and depend on lamin and related proteins to maintain peripheral positioning. When lamin or anchoring factors decline in function, LADs may detach from the nuclear periphery and undergo changes in compaction state.

(2) Consequences of peripheral structural drift

Weakening of peripheral anchoring implies not only positional change, but also destabilization of repeat-sequence repression, increased local accessibility, and aberrant transcriptional release. In long-term physiological aging, such changes are more likely to accumulate into low-level but persistent genomic instability and an inflammation-like transcriptional background.

Table 1. Major types of three-dimensional chromatin remodeling and their aging-related outputs

Structural level	Representative change	Direct consequence	Physiological aging-related output
A/B compartments	Compartment identity switching, redistribution of active/repressive compartments	Resetting of gene environment	Downregulation of developmental genes, weakening of tissue-specific function
TADs	Reduced intradomain contacts, weakened boundary insulation	Reduced enhancer coupling efficiency	Increased expression noise, drift in lineage programs
Chromatin loops	Weakening or reorganization of key enhancer-promoter loops	Destabilization of local transcriptional programs	Delayed inducible responses, reduced regenerative programs
LADs	Reduced peripheral anchoring, inward displacement of heterochromatin	Destabilization of transcriptional repression	Repeat-sequence activation, increased inflammation-like signaling
Heterochromatin clustering	Dispersal of clusters, reduced compaction	Reduced genome-protective capacity	Increased replication stress, accumulation of DNA damage

3. Molecular Linkage Mechanisms Between Three-Dimensional Structural Remodeling and Physiological Aging

3.1 Heterochromatin maintenance axis

(1) Heterochromatin compaction system

Maintenance of heterochromatin depends on H3K9me3, HP1, and associated methyltransferases that collectively establish a compacted environment. During aging, progressive loss of heterochromatin weakens the stability of large-scale repressive domains, making originally restricted regions more susceptible to aberrant transcription and structural drift.

(2) Polycomb repression layer

H3K27me3 and Polycomb-related complexes maintain repressive boundaries around many developmental and fate-determining genes. Once this layer loosens, local regulatory domains become more prone to blurred boundaries, abnormal enhancer coupling, and leakage of lineage programs.

3.2 Replication-transcription conflict and DNA damage

(1) Replication timing reprogramming

Three-dimensional chromatin structure is highly coupled to replication timing. After compartment drift and weakening of peripheral anchoring, the originally controlled early-late replication program becomes more vulnerable to disorder, causing certain fragile regions to experience replication stress earlier.

(2) Chronic accumulation of damage

Loss of heterochromatin, LAD detachment, and loosening of local structure all increase the risk of replication fork stalling, transcription-replication conflicts, and insufficient organization of repair. Physiological aging more commonly involves low-intensity but long-lasting genomic instability rather than single catastrophic injury events.

3.3 Nuclear membrane-chromatin interface

(1) Dependence on anchoring factors

LBR, LAP2, and lamin-related factors are key interface molecules for maintaining peripheral heterochromatin positioning. The nuclear membrane-chromatin connection they form is not a single-point structure, but a repressive scaffold maintained cooperatively by multiple factors.

(2) Coupling of mechanics and structure

Connections between the nuclear lamina and chromatin also provide mechanical support. Once this interface loosens, changes in intranuclear mechanical tension distribution further affect chromatin-folding stability and DNA repair efficiency. Therefore, three-dimensional structural remodeling is not merely a transcriptional issue, but also involves alteration of intranuclear mechanical homeostasis.

4. Transmission Pathways From Three-Dimensional Structural Remodeling to Aging Phenotypes

4.1 Destabilization of transcriptional programs

(1) Damage to lineage-maintenance genes

In physiological aging, the genes affected earliest are often not general survival genes, but lineage-regulatory genes that determine cell identity and tissue renewal capacity. Because these genes are more sensitive to compartment environment, local insulation, and enhancer coupling, they are often among the first to destabilize during three-dimensional structural remodeling.

(2) Increased expression noise

Even when the average expression level changes little, greater fluctuation in loop-contact efficiency and reduced coordination within TADs can still cause cell populations to exhibit greater transcriptional dispersion. This increase in noise weakens the precision of tissue responses to stimulation and repair demands.

4.2 Decline in replication and genomic stability

(1) Expansion of replication-fragile regions

Three-dimensional structural abnormalities can expose regions that were previously protected to replication stress, resulting in expansion of fragile sites and reduced completeness of replication. These changes are particularly important in highly renewing tissues and stem-cell populations.

(2) Insufficient organization of repair

DNA repair is determined not only by repair proteins, but also by the spatial chromatin environment. After loosening of domain boundaries and weakening of peripheral anchoring, repair organization at damaged loci becomes less efficient, making chronic damage accumulation more likely.

4.3 Decline in stem cells and tissue regeneration

(1) Weakening of fate control

Stem cells must maintain tightly controlled transcriptional switching among quiescence, self-renewal, and differentiation. Once compartments, LADs, or local loop contacts become unstable, stem cells become more prone to fate drift, abnormal lineage bias, and decline in regenerative potential.

(2) Tissue-level outputs

At the tissue level, such structural instability ultimately manifests as reduced immune production capacity, delayed repair, and weaker functional recovery. Tissue functional decline is not an incidental phenomenon downstream of three-dimensional structural remodeling, but a direct output of its long-term accumulation.

Table 2. Major transmission pathways from three-dimensional structural remodeling to aging phenotypes

Structural change	Molecular consequence	Cellular consequence	Tissue-level consequence
Compartment drift	Resetting of lineage-gene environment	Instability of cell identity	Reduced differentiation efficiency
Weakened TADs/loops	Reduced enhancer coupling	Increased transcriptional noise	Delayed response and insufficient regeneration
LAD detachment	Weakened repressive state	Repeat-sequence activation, inflammation-like transcription	Chronic functional decline
Heterochromatin loss	Increased vulnerability in replication and repair	Accumulation of DNA damage	Disruption of tissue homeostasis

5. Scientific Questions and Experimental Readouts

5.1 Model stratification

(1) Age-gradient models

Research on three-dimensional chromatin remodeling and physiological aging first depends on appropriate age-gradient models. Compared with acute stress models, natural aging models are more suitable for identifying progressive structural changes such as compartment drift, LAD detachment, and weakening of local loop contacts. Technically, a continuous age stratification of young-middle-aged-old is more informative than a simple young-versus-old comparison for judging cumulative trends and inflection points of structural change.

(2) Tissue and lineage selection

Different tissues do not show equal sensitivity to three-dimensional structural remodeling. Highly renewing tissues, immune systems, and stem-cell populations are usually more likely to display structural abnormalities, because these systems are more strongly dependent on lineage maintenance, replication timing, and enhancer coupling. Accordingly, the hematopoietic system, immune-cell subsets, neural stem cells, intestinal epithelial stem cells, and muscle stem cells are generally more suitable entry points for study than terminally differentiated, slowly renewing cell types.

(3) Distinguishing physiological aging from stress-induced senescence

In experimental design, naturally age-related models should be treated separately from replicative exhaustion, DNA damage induction, or oncogenic stress models. The former is better suited for studying the relationship between progressive structural drift and tissue functional decline, whereas the latter is more appropriate for amplifying extreme phenotypes such as nuclear lamina disruption, heterochromatin loss, and destabilization of domain boundaries. The two model types can serve as references for each other, but should not be directly equated.

5.2 Technical pathways

(1) Three-dimensional structural mapping

Hi-C, Micro-C, and Capture-C are core technologies for three-dimensional chromatin research. Hi-C is suitable for analysis of A/B compartment and TAD-level changes, Micro-C is more sensitive to local contacts and fine-scale structures, and Capture-C is more suitable for focusing on specific lineage genes or enhancer networks. If the purpose is to resolve which structural layers change first during physiological aging, genome-wide structural mapping generally needs to be combined with targeted mapping of key loci.

(2) Mapping of peripheral nuclear structure

Because LAD-related changes are a key layer in physiological aging research, Hi-C alone is insufficient to fully explain abnormalities in peripheral nuclear anchoring. DamID, Lamin ChIP-seq, LBR/LAP2-related localization analyses, and imaging of nuclear-envelope proteins can be used to determine whether lamina-associated domains undergo detachment, relocalization, or weakening of repressive state. In aging research, this layer of data is especially important for interpreting heterochromatin loss and nuclear-periphery decoupling.

(3) Mapping of accessibility and modification status

ATAC-seq, CUT&Tag, CUT&RUN, and ChIP-seq can be used to determine whether three-dimensional structural changes have already been translated into altered chromatin accessibility and drift in histone modifications. If compartment drift, LAD detachment, or weakened loop contact are accompanied by decreased H3K9me3, redistribution of H3K27me3, or increased accessibility, this provides stronger support for the conclusion that structural remodeling has been converted into functional output.

(4) Transcriptional and replication readouts

RNA-seq and single-cell transcriptomics can be used to determine the expression consequences of structural changes, whereas Repli-seq, EdU temporal labeling, and replication-stress-related assays can be used to analyze whether replication timing has been reprogrammed and whether fragile loci have increased. In physiological aging, mechanistic interpretation becomes substantially stronger when structural changes correspond simultaneously to increased transcriptional noise, elevated replication stress, and destabilization of lineage-gene expression.

5.3 Key readouts

(1) Readouts of compartment drift

The key readout at the compartment level is not merely the number of A/B changes, but whether regions containing specific developmental genes, tissue-homeostasis genes, and lineage-determining genes undergo compartment identity switching. Technically, the most informative result is which functional modules undergo A-to-B or B-to-A drift, rather than a simple report of the average change across the genome.

(2) Readouts of local structural instability

Key readouts at the TAD and loop levels include reduced intradomain contacts, weakened boundary strength, reduced enhancer-promoter contact frequency, and weakened local insulation around critical regulatory genes. For physiological aging research, such local structural abnormalities are often more explanatory than global structural collapse, because they more closely match the progressive nature of lineage functional decline.

(3) Readouts of peripheral decoupling

Key LAD-layer readouts include weakened peripheral localization, reduced lamin-associated contacts, inward displacement of heterochromatin, and destabilized repeat-sequence repression. If these changes are accompanied by abnormal nuclear morphology, increased inflammation-like transcription, and accumulation of DNA damage markers, this further supports the mechanistic importance of abnormalities at the nuclear membrane-chromatin interface in physiological aging.

(4) Readouts of functional output

Structural remodeling ultimately needs to be validated at the functional level. More informative outputs include decline in stem-cell self-renewal, abnormal differentiation bias, reduced immune-cell production, delayed tissue repair, and loss of regenerative capacity. If three-dimensional structural abnormalities remain only at the omics level without corresponding functional readouts, their mechanistic significance in physiological aging remains limited.

5.4 Causal validation

(1) Intervention on structural factors

CTCF, cohesin, lamin, LBR, LAP2, and heterochromatin-maintenance factors are important entry points for causal validation. Through knockdown, conditional deletion, or functional rescue experiments, one can determine whether a given structural change is a cause, consequence, or amplification layer of aging phenotypes. For a technical article, this level is more valuable than a simple description of correlational change.

(2) Reversibility analysis

If restoration of a given structural layer simultaneously improves transcriptional stability, replication stress, and tissue function, this indicates that the layer is not merely an accompanying change, but a functionally driving mechanism. Therefore, whether intervention produces reversible improvement is an important criterion for ranking mechanistic priority in research on structural aging.

(3) Multilayer validation

More convincing study designs usually do not restore just one molecule, but instead assess whether structural, transcriptional, and functional layers improve coordinately. Only when restoration of compartments, LADs, or local loop structures occurs together with stabilization of lineage-gene expression and improvement of tissue function can three-dimensional structural remodeling be more clearly defined as a mechanistic component of physiological aging.

6. Related Research Products

Table 3. Product table related to three-dimensional chromatin architecture remodeling and mechanisms of physiological aging

Name	CAS No.	Experimental stage	Key use	Use notes
5-Azacytidine	320-67-2	DNA methylation layer	Inhibits DNA methylation and is used to analyze the influence of methylation drift on compartment and LAD stability	Suitable for long-term treatment and structure-transcription coupled analysis
Decitabine	2353-33-5	DNA methylation layer	Maintenance methylation inhibitor used to study the relationship between DNA methylation and chromatin remodeling in aging	Suitable for replication-related models
Zebularine	3690-10-6	DNA methylation layer	Mild demethylation intervention used to compare three-dimensional structural changes under different methylation burdens	Suitable for continuous culture models
RG108	48208-26-0	DNMT functional validation layer	Non-nucleoside DNMT inhibitor used to validate the contribution of DNA methylation to structural remodeling	Suitable for parallel validation with 5-Azacytidine
Trichostatin A	58880-19-6	Broad-spectrum HDAC layer	HDAC inhibitor used to analyze dependence of heterochromatin compaction and peripheral anchoring on deacetylation	Suitable for short-term strong-intervention models
Vorinostat	149647-78-9	Broad-spectrum HDAC layer	Used to study the effects of altered deacetylation on compartment activity and transcriptional noise	Suitable for epigenetic-transcription coupled analysis
Panobinostat	404950-80-7	Broad-spectrum HDAC layer	Potent HDAC inhibitor used to amplify chromatin opening and boundary-vulnerability phenotypes	Suitable for structure-sensitive models
Entinostat	209783-80-2	Class I HDAC layer	Used to distinguish structural outputs of class I HDAC inhibition from those of broad-spectrum HDAC inhibition	Suitable for stratified mechanistic validation
Valproic acid	99-66-1	HDAC regulatory layer	Used for milder intervention in acetylation state and observation of nuclear structural changes	Suitable for long-term physiological treatment settings
Sodium butyrate	156-54-7	HDAC inhibition/metabolic layer	Possesses both metabolite and HDAC-inhibitory properties and can be used to analyze the effects of short-chain fatty acids on heterochromatin homeostasis	Suitable for metabolism-aging models
EX-527	49843-98-3	Sirtuin layer	Inhibits SIRT1 and is used to analyze the relationship between deacetylation, nuclear lamina stability, and transcriptional noise	Suitable for chromatin-aging models
Sirtinol	410536-97-9	Sirtuin layer	Used to assess the contribution of NAD+-dependent deacetylation to heterochromatin maintenance	Suitable for parallel design with NAD+ supplementation
Nicotinamide	98-92-0	Sirtuin comparison layer	Metabolic intervention molecule related to deacetylation, used to study the NAD+ axis and age-related structural phenotypes	Suitable for metabolism-epigenetics coupling analysis
Resveratrol	501-36-0	Sirtuin/aging layer	Commonly used in studies of the SIRT axis and aging-related chromatin homeostasis	Suitable for long-term comparative intervention
BIX-01294	935693-62-2	G9a/EHMT2 layer	Inhibits H3K9 methylation and is used to analyze compartment and LAD changes after weakened heterochromatin compaction	Suitable for heterochromatin studies
UNC0638	1255580-76-7	G9a/EHMT2 layer	Highly selective G9a inhibitor used to validate the role of H3K9me2/3 in structural maintenance	Suitable for refined mechanistic studies
Chaetocin	28097-03-2	SUV39H-related layer	Used to interfere with the H3K9me3-related heterochromatin maintenance axis	Suitable for HP1-heterochromatin studies
GSK126	1346574-57-9	EZH2/H3K27me3 layer	Inhibits EZH2 activity and is used to analyze Polycomb domain stability and destabilization of lineage genes	Suitable for developmental-gene regulation models
Tazemetostat	1403254-99-8	EZH2/H3K27me3 layer	Used to study the effects of H3K27me3 reduction on three-dimensional structural boundaries and aging phenotypes	Suitable for long-term low-intensity treatment
DZNep	120964-45-6	Polycomb intervention layer	Affects the Polycomb system by perturbing methylation-related homeostasis	Suitable for models of repressive-domain loosening
GSK-J4 HCl	1797983-09-5	KDM6 layer	Used to regulate H3K27 demethylation status and observe changes in domain plasticity	Suitable for inhibition-restoration comparisons
JQ1	1268524-70-4	BET/enhancer layer	Interferes with enhancer readout and transcriptional elongation to analyze changes at the loop-output layer	Suitable for enhancer-promoter coupling studies
1,6-Hexanediol	629-11-8	Phase separation/chromatin condensation layer	Used to disrupt weak interactions and analyze the relationship between nuclear condensates and structural stability	Suitable for short-term treatment and imaging experiments
Actinomycin D	50-76-0	Transcription inhibition layer	Used to distinguish the causal direction between transcriptional activity changes and structural changes	Suitable for loop structure-transcription dependence analysis
alpha-Amanitin	23109-05-9	Transcription inhibition layer	RNA Pol II inhibitor used to evaluate the effect of transcriptional suppression on maintenance of three-dimensional structure	Suitable for intranuclear organization studies
Aphidicolin	38966-21-1	Replication stress layer	DNA polymerase inhibitor used to induce replication stress and observe structurally vulnerable regions	Suitable for replication-structure coupling studies
Hydroxyurea	127-07-1	Replication stress layer	Induces replication fork stalling and is used to analyze the relationship between aging-related replication stress and structural remodeling	Suitable for long-term low-dose models
VE-821	1232410-49-9	ATR layer	ATR inhibitor used to validate the protective role of replication stress response in maintaining three-dimensional structural stability	Suitable for fragile-site studies
AZD6738	1352226-88-0	ATR layer	Common ATR inhibitor used to amplify replication-stress-related structural abnormalities	Suitable for combination with Aphidicolin
KU-55933	587871-26-9	ATM layer	ATM inhibitor used to analyze the influence of DNA damage response on intranuclear structural homeostasis	Suitable for damage-repair models
Olaparib	763113-22-0	PARP/repair layer	PARP inhibitor used to study cumulative effects of repair defects and chromatin remodeling	Suitable for analysis of replication-fragile regions
BIBR1532	321674-73-1	Telomere layer	Telomerase inhibitor used to construct telomere-related aging and nuclear structural change models	Suitable for long-term culture systems
2-Deoxy-D-glucose	154-17-6	Metabolic stress layer	Used to construct an energy-limited environment and observe its effects on intranuclear structural homeostasis	Suitable for metabolism-chromatin coupling studies
Rotenone	83-79-4	Mitochondrial stress layer	Induces mitochondrial ROS and metabolic stress, and is used to analyze the driving effects of metabolic stress on three-dimensional structural drift	Suitable for aging-induction backgrounds
N-acetyl-L-cysteine	616-91-1	Oxidative stress buffering layer	Used as a ROS-buffering molecule to evaluate the contribution of oxidative stress to nuclear structural abnormalities	Suitable for rescue experiments

Three-dimensional chromatin architecture remodeling is not merely an accompanying phenomenon in physiological aging, but an important organizational-level mechanism connecting heterochromatin loss, weakened peripheral anchoring, increased replication stress, and destabilization of lineage programs. Its technical significance lies not simply in showing that chromatin changes with age, but in revealing why maintenance of cell identity, tissue regeneration, and immune function decline synchronously during long-term aging.