Topologically Associating Domains Research Articles

Integrating Dynamic 3D Chromatin Architecture and Gene Expression Alterations Reveal Heterosis in Brassica rapa.

Heterosis plays a significant role in enhancing variety, boosting yield, and raising economic value in crops, but the molecular mechanism is still unclear. We analyzed the transcriptomes and 3D genomes of a hybrid (F1) and its parents (w30 and 082). The analysis of the expression revealed a total of 485 specially expressed genes (SEGs), 173 differentially expressed genes (DEGs) above the parental expression level, more actively expressed genes, and up-regulated DEGs in the F1. Further study revealed that the DEGs detected in the F1 and its parents were mainly involved in the response to auxin, plant hormone signal transduction, DNA metabolic process, purine metabolism, starch, and sucrose metabolism, which suggested that these biological processes may play a crucial role in the heterosis of Brassica rapa. The analysis of 3D genome data revealed that hybrid F1 plants tend to contain more transcriptionally active A chromatin compartments after hybridization. Supplementaryly, the F1 had a smaller TAD (topologically associated domain) genome length, but the number was the highest, and the expression change in activated TAD was higher than that of repressed TAD. More specific TAD boundaries were detected between the parents and F1. Subsequently, 140 DEGs with genomic structural variants were selected as potential candidate genes. We found two DEGs with consistent expression changes in A/B compartments and TADs. Our findings suggested that genomic structural variants, such as TADs and A/B chromatin compartments, may affect gene expression and contribute to heterosis in Brassica rapa. This study provides further insight into the molecular mechanism of heterosis in Brassica rapa.

Transposable elements as scaffold/matrix attachment regions: shaping organization and functions in genomes.

The hierarchical structure of eukaryotic genomes has regulatory layers, one of them being epigenetic "indexing" of the genome that leads to cell-type-specific patterns of gene expression. By establishing loops and defining chromatin domains, cells can achieve coordinated control over multi-locus segments of the genome. This is thought to be achieved using scaffold/matrix attachment regions (S/MARs) that establish structural and functional loops and topologically associating domains (TADs) that define a self-interacting region of the genome. Large-scale genome-wide mapping of S/MARs has begun to uncover these aspects of genome organization. A recent genome-wide study showed the association of transposable elements (TEs) with a significant fraction of S/MARs, suggesting that the multitude of TE-derived repeats constitute a class of anchorage sites of chromatin loops to nuclear architecture. In this study, we provide an insight that TE-driven dispersal of S/MARs has the potential to restructure the chromosomes by creating novel loops and domains. The combination of TEs and S/MARs, as elements that can hop through the genome along with regulatory capabilities, may provide an active mechanism of genome evolution leading to the emergence of novel features in biological systems. The significance is that a genome-wide study mapping developmental S/MARs reveals an intriguing link between these elements and TEs. This article highlights the potential of the TE-S/MAR combination to drive evolution by restructuring and shaping the genome.

Giant pandas in captivity undergo short-term adaptation in nerve-related pathways.

Behaviors in captive animals, including changes in appetite, activity level, and social interaction, are often seen as adaptive responses. However, these behaviors may become progressively maladaptive, leading to stress, anxiety, depression, and other negative reactions in animals. In this study, we investigated the whole-genome sequencing data of 39 giant panda individuals, including 11 in captivity and 28 in the wild. To eliminate the mountain range effect and focus on the factor of captivity only, we first performed a principal component analysis. We then enumerated the 21,474,180 combinations of wild giant pandas (11 chosen from 28) and calculated their distances from the 11 captive individuals. The 11 wild individuals with the closest distances were used for the subsequent analysis. The linkage disequilibrium (LD) patterns demonstrated that the population was almost eliminated. We identified 505 robust selected genomic regions harboring at least one SNP, and the absolute frequency difference was greater than 0.6 between the two populations. GO analysis revealed that genes in these regions were mainly involved in nerve-related pathways. Furthermore, we identified 22 GO terms for which the selection strength significantly differed between the two populations, and there were 10 nerve-related pathways among them. Genes in the differentially abundant regions were involved in nerve-related pathways, indicating that giant pandas in captivity underwent minor genomic selection. Additionally, we investigated the relationship between genetic variation and chromatin conformation structures. We found that nucleotide diversity (θπ) in the captive population was correlated with chromatin conformation structures, which included A/B compartments, topologically associated domains (TADs) and TAD-cliques. For each GO term, we then compared the expression level of genes regulated by the above four factors (AB index, TAD intactness, TAD clique and PEI) with the corresponding genomic background. The retained 10 GO terms were all coordinately regulated by the four factors, and three of them were associated with nerve-related pathways. This study revealed that giant pandas in captivity undergo short-term adaptation in nerve-related pathways. Furthermore, it provides new insights into the molecular mechanism of gene expression regulation under short-term adaptation to environmental change.

LoopSage: An energy-based Monte Carlo approach for the loop extrusion modeling of chromatin

The connection between the patterns observed in 3C-type experiments and the modeling of polymers remains unresolved. This paper presents a simulation pipeline that generates thermodynamic ensembles of 3D structures for topologically associated domain (TAD) regions by loop extrusion model (LEM). The simulations consist of two main components: a stochastic simulation phase, employing a Monte Carlo approach to simulate the binding positions of cohesins, and a dynamical simulation phase, utilizing these cohesins' positions to create 3D structures. In this approach, the system's total energy is the combined result of the Monte Carlo energy and the molecular simulation energy, which are iteratively updated. The structural maintenance of chromosomes (SMC) protein complexes are represented as loop extruders, while the CCCTC-binding factor (CTCF) locations on DNA sequence are modeled as energy minima on the Monte Carlo energy landscape. Finally, the spatial distances between DNA segments from ChIA-PET experiments are compared with the computer simulations, and we observe significant Pearson correlations between predictions and the real data. LoopSage model offers a fresh perspective on chromatin loop dynamics, allowing us to observe phase transition between sparse and condensed states in chromatin.

Topologically associating domains define the impact of de novo promoter variants on autism spectrum disorder risk

Whole-genome sequencing (WGS) studies of autism spectrum disorder (ASD) have demonstrated the roles of rare promoter de novo variants (DNVs). However, most promoter DNVs in ASD are not located immediately upstream of known ASD genes. In this study analyzing WGS data of 5,044 ASD probands, 4,095 unaffected siblings, and their parents, we show that promoter DNVs within topologically associating domains (TADs) containing ASD genes are significantly and specifically associated with ASD. An analysis considering TADs as functional units identified specific TADs enriched for promoter DNVs in ASD and indicated that common variants in these regions also confer ASD heritability. Experimental validation using human induced pluripotent stem cells (iPSCs) showed that likely deleterious promoter DNVs in ASD can influence multiple genes within the same TAD, resulting in overall dysregulation of ASD-associated genes. These results highlight the importance of TADs and gene-regulatory mechanisms in better understanding the genetic architecture of ASD.

Cis-regulatory modes of Ultrabithorax inactivation in butterfly forewings.

Hox gene clusters encode transcription factors that drive regional specialization during animal development: for example the Hox factor Ubx is expressed in the insect metathoracic (T3) wing appendages and differentiates them from T2 mesothoracic identities. Hox transcriptional regulation requires silencing activities that prevent spurious activation and regulatory crosstalks in the wrong tissues, but this has seldom been studied in insects other than Drosophila, which shows a derived Hox dislocation into two genomic clusters that disjoined Antennapedia (Antp) and Ultrabithorax (Ubx). Here, we investigated how Ubx is restricted to the hindwing in butterflies, amidst a contiguous Hox cluster. By analysing Hi-C and ATAC-seq data in the butterfly Junonia coenia, we show that a Topologically Associated Domain (TAD) maintains a hindwing-enriched profile of chromatin opening around Ubx. This TAD is bordered by a Boundary Element (BE) that separates it from a region of joined wing activity around the Antp locus. CRISPR mutational perturbation of this BE releases ectopic Ubx expression in forewings, inducing homeotic clones with hindwing identities. Further mutational interrogation of two non-coding RNA encoding regions and one putative cis-regulatory module within the Ubx TAD cause rare homeotic transformations in both directions, indicating the presence of both activating and repressing chromatin features. We also describe a series of spontaneous forewing homeotic phenotypes obtained in Heliconius butterflies, and discuss their possible mutational basis. By leveraging the extensive wing specialization found in butterflies, our initial exploration of Ubx regulation demonstrates the existence of silencing and insulating sequences that prevent its spurious expression in forewings.

Cis-regulatory modes of Ultrabithorax inactivation in butterfly forewings

Hox gene clusters encode transcription factors that drive regional specialization during animal development: for example the Hox factor Ubx is expressed in the insect metathoracic (T3) wing appendages and differentiates them from T2 mesothoracic identities. Hox transcriptional regulation requires silencing activities that prevent spurious activation and regulatory crosstalks in the wrong tissues, but this has seldom been studied in insects other than Drosophila, which shows a derived Hox dislocation into two genomic clusters that disjoined Antennapedia (Antp) and Ultrabithorax (Ubx). Here, we investigated how Ubx is restricted to the hindwing in butterflies, amidst a contiguous Hox cluster. By analysing Hi-C and ATAC-seq data in the butterfly Junonia coenia, we show that a Topologically Associated Domain (TAD) maintains a hindwing-enriched profile of chromatin opening around Ubx. This TAD is bordered by a Boundary Element (BE) that separates it from a region of joined wing activity around the Antp locus. CRISPR mutational perturbation of this BE releases ectopic Ubx expression in forewings, inducing homeotic clones with hindwing identities. Further mutational interrogation of two non-coding RNA encoding regions and one putative cis-regulatory module within the Ubx TAD cause rare homeotic transformations in both directions, indicating the presence of both activating and repressing chromatin features. We also describe a series of spontaneous forewing homeotic phenotypes obtained in Heliconius butterflies, and discuss their possible mutational basis. By leveraging the extensive wing specialization found in butterflies, our initial exploration of Ubx regulation demonstrates the existence of silencing and insulating sequences that prevent its spurious expression in forewings.

Boundary stacking interactions enable cross-TAD enhancer-promoter communication during limb development.

Although promoters and their enhancers are frequently contained within a topologically associating domain (TAD), some developmentally important genes have their promoter and enhancers within different TADs. Hypotheses about molecular mechanisms enabling cross-TAD interactions remain to be assessed. To test these hypotheses, we used optical reconstruction of chromatin architecture to characterize the conformations of the Pitx1 locus on single chromosomes in developing mouse limbs. Our data support a model in which neighboring boundaries are stacked as a result of loop extrusion, bringing boundary-proximal cis-elements into contact. This stacking interaction also contributes to the appearance of architectural stripes in the population average maps. Through molecular dynamics simulations, we found that increasing boundary strengths facilitates the formation of the stacked boundary conformation, counter-intuitively facilitating border bypass. This work provides a revised view of the TAD borders' function, both facilitating and preventing cis-regulatory interactions, and introduces a framework to distinguish border-crossing from border-respecting enhancer-promoter pairs.

DiffDomain enables identification of structurally reorganized topologically associating domains

Topologically associating domains (TADs) are critical structural units in three-dimensional genome organization of mammalian genome. Dynamic reorganizations of TADs between health and disease states are associated with essential genome functions. However, computational methods for identifying reorganized TADs are still in the early stages of development. Here, we present DiffDomain, an algorithm leveraging high-dimensional random matrix theory to identify structurally reorganized TADs using high-throughput chromosome conformation capture (Hi–C) contact maps. Method comparison using multiple real Hi–C datasets reveals that DiffDomain outperforms alternative methods for false positive rates, true positive rates, and identifying a new subtype of reorganized TADs. Applying DiffDomain to Hi–C data from different cell types and disease states demonstrates its biological relevance. Identified reorganized TADs are associated with structural variations and epigenomic changes such as changes in CTCF binding sites. By applying to a single-cell Hi–C data from mouse neuronal development, DiffDomain can identify reorganized TADs between cell types with reasonable reproducibility using pseudo-bulk Hi–C data from as few as 100 cells per condition. Moreover, DiffDomain reveals differential cell-to-population variability and heterogeneous cell-to-cell variability in TADs. Therefore, DiffDomain is a statistically sound method for better comparative analysis of TADs using both Hi–C and single-cell Hi–C data.

Disruption of OVOL2 Distal Regulatory Elements as a Possible Mechanism Implicated in Corneal Endothelial Dystrophy

The genetic architecture of corneal endothelial dystrophies remains unknown in a substantial number of affected individuals. The proband investigated in the current study was diagnosed in the neonatal period with bilateral corneal opacification due to primary endothelial cell dysfunction. Neither his parents nor his sister had signs of corneal disease. Conventional karyotyping revealed a de novo translocation involving chromosomes 3 and 20, t(3;20)(q25;p11-12). Following genome and targeted Sanger sequencing analysis, the breakpoints were mapped at the nucleotide level. Notably, the breakpoint on chromosome 20 was identified to lie within the same topologically associated domain (TAD) as corneal endothelial dystrophy-associated gene OVOL2, and it is predicted to disrupt distal enhancers. The breakpoint at chromosome 3 is located within intron 2 of PFN2, which is currently not associated with any human disease. Further interrogation of the proband’s genome failed to identify any additional potentially pathogenic variants in corneal endothelial dystrophy-associated genes. Disruption of a candidate cis-regulatory element and/or positional effects induced by translocation of OVOL2 to a novel genomic context may lead to an aberrant OVOL2 expression, a previously characterized disease mechanism of corneal endothelial dystrophy. Further research is necessary to explore how disruption of regulatory elements may elucidate genetically unsolved corneal endothelial dystrophies.

Mapping nucleosome-resolution chromatin organization and enhancer-promoter loops in plants using Micro-C-XL

Although chromatin organizations in plants have been dissected at the scales of compartments and topologically associating domain (TAD)-like domains, there remains a gap in resolving fine-scale structures. Here, we use Micro-C-XL, a high-throughput chromosome conformation capture (Hi-C)-based technology that involves micrococcal nuclease (instead of restriction enzymes) and long cross-linkers, to dissect single nucleosome-resolution chromatin organization in Arabidopsis. Insulation analysis reveals more than 14,000 boundaries, which mostly include chromatin accessibility, epigenetic modifications, and transcription factors. Micro-C-XL reveals associations between RNA Pols and local chromatin organizations, suggesting that gene transcription substantially contributes to the establishment of local chromatin domains. By perturbing Pol II both genetically and chemically at the gene level, we confirm its function in regulating chromatin organization. Visible loops and stripes are assigned to super-enhancers and their targeted genes, thus providing direct insights for the identification and mechanistic analysis of distal CREs and their working modes in plants. We further investigate possible factors regulating these chromatin loops. Subsequently, we expand Micro-C-XL to soybean and rice. In summary, we use Micro-C-XL for analyses of plants, which reveal fine-scale chromatin organization and enhancer-promoter loops and provide insights regarding three-dimensional genomes in plants.

ZNF143 deletion alters enhancer/promoter looping and CTCF/cohesin geometry

The transcription factor ZNF143 contains a central domain of seven zinc fingers in a tandem array and is involved in 3D genome construction. However, the mechanism by which ZNF143 functions in chromatin looping remains unclear. Here, we show that ZNF143 directionally recognizes a diverse range of genomic sites directly within enhancers and promoters and is required for chromatin looping between these sites. In addition, ZNF143 is located between CTCF and cohesin at numerous CTCF sites, and ZNF143 removal narrows the space between CTCF and cohesin. Moreover, genetic deletion of ZNF143, in conjunction with acute CTCF degradation, reveals that ZNF143 and CTCF collaborate to regulate higher-order topological chromatin organization. Finally, CTCF depletion enlarges direct ZNF143 chromatin looping. Thus, ZNF143 is recruited by CTCF to the CTCF sites to regulate CTCF/cohesin configuration and TAD (topologically associating domain) formation, whereas directional recognition of genomic DNA motifs directly by ZNF143 itself regulates promoter activity via chromatin looping.

Strategies of Integrated Analysis of ATAC-seq and RNA-seq Data

The ATAC-seq and RNA-seq have been widely used in the epigenomes analysis of mammals. However, there is a lack of comprehensive integrated analysis of ATAC-seq and RNA-seq data. Here, we explored two effective strategies for analyzing ATAC-seq and RNA-seq data using proliferation and differentiation myoblast cells as model. One strategy is a joint analysis by integrating Hi-C data with ATAC-seq and RNA-seq data. The other strategy is to investigate the association between ATAC-seq and RNA-seq data only based on genome physical distance in the case of no availability of Hi-C data. Hi-C data-based integrated analysis showed a strong correlation between genes and distal enhancers, particularly between genes and promoters, within the topologically associated domains (TADs) and chromatin loops. In the absence of Hi-C data, the integrated analysis based on 500 kb physical distance between genes and associated peaks revealed a strong positive correlation between gene expression and chromatin accessibility. Moreover, the function enrichment analysis indicated that both integrated analysis results could reflect the cellular states. Our proposed two integrated analysis strategies combine ATAC-seq and RNA-seq data, which lay a foundation for subsequent epigenetics studies.

A Comprehensive Evaluation of Generalizability of Deep Learning-Based Hi-C Resolution Improvement Methods.

Hi-C is a widely used technique to study the 3D organization of the genome. Due to its high sequencing cost, most of the generated datasets are of a coarse resolution, which makes it impractical to study finer chromatin features such as Topologically Associating Domains (TADs) and chromatin loops. Multiple deep learning-based methods have recently been proposed to increase the resolution of these datasets by imputing Hi-C reads (typically called upscaling). However, the existing works evaluate these methods on either synthetically downsampled datasets, or a small subset of experimentally generated sparse Hi-C datasets, making it hard to establish their generalizability in the real-world use case. We present our framework-Hi-CY-that compares existing Hi-C resolution upscaling methods on seven experimentally generated low-resolution Hi-C datasets belonging to various levels of read sparsities originating from three cell lines on a comprehensive set of evaluation metrics. Hi-CY also includes four downstream analysis tasks, such as TAD and chromatin loops recall, to provide a thorough report on the generalizability of these methods. We observe that existing deep learning methods fail to generalize to experimentally generated sparse Hi-C datasets, showing a performance reduction of up to 57%. As a potential solution, we find that retraining deep learning-based methods with experimentally generated Hi-C datasets improves performance by up to 31%. More importantly, Hi-CY shows that even with retraining, the existing deep learning-based methods struggle to recover biological features such as chromatin loops and TADs when provided with sparse Hi-C datasets. Our study, through the Hi-CY framework, highlights the need for rigorous evaluation in the future. We identify specific avenues for improvements in the current deep learning-based Hi-C upscaling methods, including but not limited to using experimentally generated datasets for training.

A chromatin-regulated biphasic circuit coordinates IL-1β-mediated inflammation.

Inflammation is characterized by a biphasic cycle consisting initially of a proinflammatory phase that is subsequently resolved by anti-inflammatory processes. Interleukin-1β (IL-1β) is a master regulator of proinflammation and is encoded within the same topologically associating domain (TAD) as IL-37, which is an anti-inflammatory cytokine that opposes the function of IL-1β. Within this TAD, we identified a long noncoding RNA called AMANZI, which negatively regulates IL-1β expression and trained immunity through the induction of IL37 transcription. We found that the activation of IL37 occurs through the formation of a dynamic long-range chromatin contact that leads to the temporal delay of anti-inflammatory responses. The common variant rs16944 present in AMANZI augments this regulatory circuit, predisposing individuals to enhanced proinflammation or immunosuppression. Our work illuminates a chromatin-mediated biphasic circuit coordinating expression of IL-1β and IL-37, thereby regulating two functionally opposed states of inflammation from within a single TAD.

Enhancer-promoter interactions can form independently of genomic distance and be functional across TAD boundaries.

Topologically Associating Domains (TADs) have been suggested to facilitate and constrain enhancer-promoter interactions. However, the role of TAD boundaries in effectively restricting these interactions remains unclear. Here, we show that a significant proportion of enhancer-promoter interactions are established across TAD boundaries in Drosophila embryos, but that developmental genes are strikingly enriched in intra- but not inter-TAD interactions. We pursued this observation using the twist locus, a master regulator of mesoderm development, and systematically relocated one of its enhancers to various genomic locations. While this developmental gene can establish inter-TAD interactions with its enhancer, the functionality of these interactions remains limited, highlighting the existence of topological constraints. Furthermore, contrary to intra-TAD interactions, the formation of inter-TAD enhancer-promoter interactions is not solely driven by genomic distance, with distal interactions sometimes favored over proximal ones. These observations suggest that other general mechanisms must exist to establish and maintain specific enhancer-promoter interactions across large distances.

TAD evolutionary and functional characterization reveals diversity in mammalian TAD boundary properties and function

Topological associating domains (TADs) are self-interacting genomic units crucial for shaping gene regulation patterns. Despite their importance, the extent of their evolutionary conservation and its functional implications remain largely unknown. In this study, we generate Hi-C and ChIP-seq data and compare TAD organization across four primate and four rodent species and characterize the genetic and epigenetic properties of TAD boundaries in correspondence to their evolutionary conservation. We find 14% of all human TAD boundaries to be shared among all eight species (ultraconserved), while 15% are human-specific. Ultraconserved TAD boundaries have stronger insulation strength, CTCF binding, and enrichment of older retrotransposons compared to species-specific boundaries. CRISPR-Cas9 knockouts of an ultraconserved boundary in a mouse model lead to tissue-specific gene expression changes and morphological phenotypes. Deletion of a human-specific boundary near the autism-related AUTS2 gene results in the upregulation of this gene in neurons. Overall, our study provides pertinent TAD boundary evolutionary conservation annotations and showcases the functional importance of TAD evolution.

Cohesin and CTCF do not assemble TADs in Xenopus sperm and male pronuclei.

Paternal genomes are compacted during spermiogenesis and decompacted following fertilization. These processes are fundamental for inheritance but incompletely understood. We analyzed these processes in the frog Xenopus laevis, whose sperm can be assembled into functional pronuclei in egg extracts in vitro. In such extracts, cohesin extrudes DNA into loops, but in vivo cohesin only assembles topologically associating domains (TADs) at the mid-blastula transition (MBT). Why cohesin assembles TADs only at this stage is unknown. We first analyzed genome architecture in frog sperm and compared it to human and mouse. Our results indicate that sperm genome organization is conserved between frogs and humans and occurs without formation of TADs. TADs can be detected in mouse sperm samples, as reported, but these structures might originate from somatic chromatin contaminations. We therefore discuss the possibility that the absence of TADs might be a general feature of vertebrate sperm. To analyze sperm genome remodeling upon fertilization, we reconstituted male pronuclei in Xenopus egg extracts. In pronuclei, chromatin compartmentalization increases, but cohesin does not accumulate at CTCF sites and assemble TADs. However, if pronuclei are formed in the presence of exogenous CTCF, CTCF binds to its consensus sites, and cohesin accumulates at these and forms short-range chromatin loops, which are preferentially anchored at CTCF's N terminus. These results indicate that TADs are only assembled at MBT because before this stage CTCF sites are not occupied and cohesin only forms short-range chromatin loops.

Stage-specific dynamic reorganization of genome topology shapes transcriptional neighborhoods in developing human retinal organoids

We have generated a high-resolution Hi-C map of developing human retinal organoids to elucidate spatiotemporal dynamics of genomic architecture and its relationship with gene expression patterns. We demonstrate progressive stage-specific alterations in DNA topology and correlate these changes with transcription of cell-type-restricted gene markers during retinal differentiation. Temporal Hi-C reveals a shift toward A compartment for protein-coding genes and B compartment for non-coding RNAs, displaying high and low expression, respectively. Notably, retina-enriched genes are clustered near lost boundaries of topologically associated domains (TADs), and higher-order assemblages (i.e., TAD cliques) localize in active chromatin regions with binding sites for eye-field transcription factors. These genes gain chromatin contacts at their transcription start site as organoid differentiation proceeds. Our study provides a global view of chromatin architecture dynamics associated with diversification of cell types during retinal development and serves as a foundational resource for in-depth functional investigations of retinal developmental traits.

Hi-C sequencing unravels dynamic three-dimensional chromatin interactions in muntjac lineage: insights from chromosome fusions in Fea's muntjac genome.

Eukaryotes have varying numbers and structures of characteristic chromosomes across lineages or species. The evolutionary trajectory of species may have been affected by spontaneous genome rearrangements. Chromosome fusion drastically alters karyotypes. However, the mechanisms and consequences of chromosome fusions, particularly in muntjac species, are poorly understood. Recent research-based advancements in three-dimensional (3D) genomics, particularly high-throughput chromatin conformation capture (Hi-C) sequencing, have allowed for the identification of chromosome fusions and provided mechanistic insights into three muntjac species: Muntiacus muntjak, M. reevesi, and M. crinifrons. This study aimed to uncover potential genome rearrangement patterns in the threatened species Fea's muntjac (Muntiacus feae), which have not been previously examined for such characteristics. Deep Hi-C sequencing (31.42 × coverage) was performed to reveal the 3D chromatin architecture of the Fea's muntjac genome. Patterns of repeated chromosome fusions that were potentially mediated by high-abundance transposable elements were identified. Comparative Hi-C maps demonstrated linkage homology between the sex chromosomes in Fea's muntjac and autosomes in M. reevesi, indicating that fusions may have played a crucial role in the evolution of the sex chromosomes of the lineage. The species-level dynamics of topologically associated domains (TADs) suggest that TAD organization could be altered by differential chromosome interactions owing to repeated chromosome fusions. However, research on the effect of TADs on muntjac genome evolution is insufficient. This study generated Hi-C data for the Fea's muntjac, providing a genomic resource for future investigations of the evolutionary patterns of chromatin conformation at the chromosomal level.