Hongxin Zhong,Jie Zhang,Yuli Lu,Gongcheng Hu,Guangjin Pan,Hongjie Yao
3D genome perspective on cell fate determination, organ regeneration, and diseases
Abstract The nucleosome is the fundamental subunit of chromatin. Nucleosome structures are formed by the combination of histone octamers and genomic DNA. Through a systematic and precise process of folding and compression, these structures form a 30‐nm chromatin fibre that is further organized within the nucleus in a hierarchical manner, known as the 3D genome. Understanding the intricacies of chromatin structure and the regulatory mode governing chromatin interactions is essential for unravelling the complexities of cellular architecture and function, particularly in relation to cell fate determination, regeneration, and the development of diseases. Here, we provide a general overview of the hierarchical structure of chromatin as well as of the evolution of chromatin conformation capture techniques. We also discuss the dynamic regulatory changes in higher‐order chromatin structure that occur during stem cell lineage differentiation and somatic cell reprogramming, potential regulatory insights at the chromatin level in organ regeneration, and aberrant chromatin regulation in diseases.
3D genome perspective on cell fate determination, organ regeneration, and diseases

The nucleosome is the fundamental subunit of chromatin. Nucleosome structures are formed by the combination of histone octamers and genomic DNA. Through a systematic and precise process of folding and compression, these structures form a 30‐nm chromatin fibre that is further organized within the nucleus in a hierarchical manner, known as the 3D genome. Understanding the intricacies of chromatin structure and the regulatory mode governing chromatin interactions is essential for unravelling the complexities of cellular architecture and function, particularly in relation to cell fate determination, regeneration, and the development of diseases. Here, we provide a general overview of the hierarchical structure of chromatin as well as of the evolution of chromatin conformation capture techniques. We also discuss the dynamic regulatory changes in higher‐order chromatin structure that occur during stem cell lineage differentiation and somatic cell reprogramming, potential regulatory insights at the chromatin level in organ regeneration, and aberrant chromatin regulation in diseases.

If stretched end‐to‐end, the diploid human genome is ~2 m long, yet it fits inside a nucleus that is only a few microns across. The chromatin is folded and assembled into a complex hierarchical structure. , , Transcriptional regulation relies on precise 3D genomic architecture. , To capture chromatin structure, numerous chromosome conformation capture (3C) techniques and their derivatives, followed by high‐throughput sequencing have been developed in the past two decades. , A fresh perspective on the 3D hierarchical organization of the genome has emerged from the analysis of genome‐wide chromatin contact maps, and this has revolutionized our knowledge of chromatin organization as well as our understanding of the transcriptional control of gene expression. Chromatin architectural proteins, such as CCCTC binding factor (CTCF) and the cohesin complex, play the essential roles in the formation of 3D genome. , Cell fate changes dynamically during normal development, cell lineage differentiation, organ injury and regeneration, and disease progression. Moreover, cell fate determination is frequently regulated by transcription factors (TFs), chromatin accessibility, and modifications, as well as higher‐order chromatin structure. , , An investigation of multidimensional chromatin architecture provides theoretical guidance for understanding the basic organization of life activities, the regeneration of organs, and the occurrence and development of diseases. In this review, we present a summary of the hierarchical organization of the 3D genome, discuss the development of 3C techniques and their derivatives in studying the 3D genome, introduce recent discoveries about higher‐order chromatin structure during stem cell lineage differentiation and somatic reprogramming, review the role of chromatin regulation in organ regeneration and diseases, and provide new insights and perspectives for future investigation of these topics.

Functional elements in eukaryotic genomes, including coding genes, non‐coding genes, cis‐regulatory elements, repetitive elements, and others, interact specifically in dynamic higher‐order structures rather than being arranged in linear order in the spatial organization. The ‘3D genome’, normally refers to the higher‐order chromatin structure, affects numerous biological processes, including gene expression, DNA replication, DNA damage repair, cell differentiation, and embryonic development. , , , The multidimensional genome organization of eukaryotes can be described in the following order, from large to small: chromosome territories (CTs), chromatin compartments, topologically associating domains (TADs), chromatin loops (Figure 1). These structures are associated with gene expression in multiple dimensions and thereby influence the progression of cell fate transitions.

During interphase, chromatin could occupy distinct regions within the nucleus known as CTs (Figure 1). It has been reported that the nuclear topological arrangement of CTs has remained stable over 30 million years of primate evolution. Several factors contribute to the creation of CTs, including chromosome size and the number of genes present on specific chromosomes. Gene‐dense CTs are enriched inside the nucleus, whereas gene‐poor CTs are located at the periphery of the nuclear membrane. The spatial arrangement of CTs determines the frequency of genomic translocations. The presence of correct CTs can shield the genome from potentially harmful translocations in the event of DNA damage.

The genome is further separated into active A compartments and inactive B compartments (Figure 1). The A compartments, which are mainly located in the interior of the nucleus, have the relatively open chromatin structure in contrast to the B compartments, are enriched with housekeeping genes, short interspersed repetitive elements (SINEs), and histone markers such as H3K27ac and H3K36me3 that indicate active transcription. However, the B compartments are primarily located at the edge of the nuclear membrane or around the nucleolus and are associated with tissue‐specific genes, long terminal repeats, long interspersed nuclear elements, and repressive histone modifications such as H3K9me3 and H3K27me3 that are involved in transcriptional silencing. , Nuclear re‐localization and A/B compartment switching are regulated by chromatin‐associated proteins such as TFs and chromatin‐modifying enzymes, as indicated by functional analysis of specific genomic loci. , The A/B compartments are spatially polarized structures that may contribute to the local concentration of transcriptional machinery and epigenetic regulators and thereby increase the efficient utilization of these biological resources.

Genomic compartments can be further subdivided into topologically associated domains (TADs) at the sub‐megabase scale (Figure 1). TADs are separated by distinct boundaries and are characterized by the presence of frequent internal chromatin contacts in the same TAD but infrequent interactions across TADs. This means that TADs frequently function as autonomous domains of gene control. , , CTCF, cohesin complexes, SINE elements, tRNAs, housekeeping genes, and active histone modifications such as H3K4me3 and H3K36me3 are abundant at TAD boundaries. These elements are typically closely related to transcriptional activity and epigenetic chromatin modification signatures. As an insulator at the boundary, CTCF prevents the interaction of regulatory elements located on two nearby TADs. Furthermore, TADs can be further divided into smaller sub‐topological domains (sub‐TADs) that are highly conserved across species and stable across diverse cell types. TADs are functional units of chromatin, and co‐regulated genes located in a given TAD exhibit comparable patterns of expression during differentiation. , Disruption of TAD boundaries could result in aberrant chromatin interactions, long‐range transcriptional dysregulation, altered gene expression, and diseases. ,

At the kilobase scale, chromatin fibres condense into loop‐like structures known as chromatin loops (Figure 1). The ‘loop extrusion model’ is currently one of the most well‐known models that explain how chromatin loops are formed. The cohesin complex movement is halted when it encounters CTCF‐occupied sites while sliding and compressing along chromatin. The chromatin loops mediated by CTCF and the cohesin complex within a specific chromatin environment are intimately associated with gene expression. Direct evidence for the loop extrusion paradigm has been provided by in vitro single‐molecule imaging technology showing that the cohesin‐NIPBL complex may compress DNA through ATP‐driven loop extrusion. At a finer scale, the cohesin‐associated loop extrusion machinery can be further helped by RNA polymerase II when the transcriptional machinery and cohesin complex clash, which may cause the development of enhancer–promoter loop or promoter–promoter loop. Chromatin loops is closely related with regulation of gene expression. Our recent study revealed that CTCF has an alternative splicing isoform that skips Exons 3 and 4, causing translation to start at Exon 5 and resulting in a shortened CTCF protein (CTCF‐s). Competition between CTCF‐s and classical CTCF for DNA binding results in disruption of the CTCF‐mediated chromatin loops and promotes apoptosis through activation of interferon‐inducible protein 6 (IFI6). CTCF, along with other potential factors, governs long‐range chromatin interactions. We screened the factors that may be involved in the regulation of chromatin loop formation and indicated that basic helix–loop–helix family member e40 (BHLHE40) regulates CTCF binding and thereby impacts the stability of chromatin loops. Additionally, Yin Yang 1 (YY1) functions as a chromatin structural protein by mediating the interaction between enhancers and promoters and thus regulates gene expression. However, current investigations have shown that acute depletion of architectural proteins (CTCF, cohesion, WAPL, and YY1) using a degradation system had relatively moderate effects on the expression of most genes though these proteins mediate chromatin loops, , indicating that the dynamic changes of higher‐order chromatin structure are not positive correlation with the gene expression. Therefore, the relationships between higher‐order chromatin structure and gene expression need us to further investigate.

The spatial conformation of chromatin is one of the essential components of transcriptional regulation. Advances in 3C and 3C‐based derivative technologies over the past two decades have made it easier for us to study 3D genome organization (Figure 2). , 3C was initially reported in 2002, which could be used to deduce the spatial architecture of chromatin based on quantitative measurement of the frequency of interactions between two loci. Based on the 3C method, circular chromosome conformation capture or chromosome conformation capture‐on‐chip (4C) technology for capturing all loci that interact with a single specific locus, , and chromatin conformation capture carbon copy (5C) technology used for detecting all interactions within a specific locus, were rapidly developed. Hi‐C technology, which makes genome‐wide chromatin interaction analysis possible, was developed in 2009. Despite the shortcomings of the initial version of the Hi‐C technique, including high expenses, complicated and time‐consuming experimental methods, lots of randomly ligated DNA noise, and a lack of simple noise evaluation, Hi‐C marks the beginning of the era of mapping genome‐wide chromatin interactions.

The most notable improvement of the Hi‐C technique is the invention of in situ Hi‐C technology. In situ Hi‐C employs a specific four‐base cutter enzyme to fragment DNA, enhancing the number and diversity of DNA fragments and boosting sequencing coverage and resolution relative to Hi‐C using a six‐base cutter enzyme. Furthermore, in situ Hi‐C executes adjacent ligation and previous steps in the nucleus, thereby capturing chromatin interactions closer to its natural state. In situ Hi‐C has overcome the shortcomings of traditional Hi‐C, and has been widely adopted and improved upon in subsequent versions. For example, sisHi‐C, an assay that captures information from a small number of cells by optimizing the procedure based on in situ Hi‐C, was used for mapping chromatin interactions of early embryonic samples. Bridge‐Linker Hi‐C, an improved method in which biotinylated linker sequences are used, makes the technique more efficient and sensitive in capturing active chromatin interactions. Micro‐C significantly improves the resolution of the Hi‐C method by using micrococcal nuclease (MNase) to cleave the genome. In addition to the methods described above for capturing whole genome chromatin interactions, the techniques for capturing local chromatin interactions are being developed. Micro‐Capture‐C (MCC) and Tiled‐MCC technologies, both of which are based on Micro‐C, were developed for high‐resolution chromatin interaction of specific loci. As needed, other derivative technologies centred on specific factors (e.g., TFs or histones) or specific elements (e.g., promoter or enhancer regions) were established, such as ChIA‐PET, HiChIP, Capture Hi‐C, ChIATAC, and others (Figure 2). Furthermore, the development of single‐cell Hi‐C technology allowed us to detect thousands of chromatin contacts in single cells. In addition, the optimized versions of single‐cell Dip‐C and Methyl‐HiC successively emerged after that.

Simultaneously, nonadjacent‐junction approaches have been developed to avoid the inherent linkage bias of standard adjacent‐junction methods; these include genome architecture mapping (GAM) and its derived immunoGAM, SPRITE and ChIA‐Drop (Figure 2). These technologies have advantages in capturing long‐range and multiway contacts at TAD scale and have been described in detail. Other ligation‐free methods such as Trac‐looping and its derived high‐resolution technology, Hi‐TrAC exploit the bridging ability of TN5 transposase to reveal fine and comprehensive interactions between regulatory elements at the chromatin loop level.

The advances in 3D genome technology have linked the regulation of chromatin hierarchy to cell fate transitions. A number of reviews have described the contribution of the 3D genome to early embryonic development, cell lineage differentiation, and cell fate transitions. , Here, we mainly focus on the role of CTCF‐mediated chromatin regulations in cell fate determination.

Embryonic stem cells can generally differentiate into intermediate‐state progenitor cells of different lineages and thence into terminally differentiated somatic cells according to established routes. Chromatin compartments experience dynamic changes as stem cells transition to other cell types during differentiation. For instance, 36% of A and B compartments in the genome switches during the differentiation of human embryonic stem cells (hESCs). However, the observed changes in gene expression are subtle, which suggests a little link between switches in compartments and changes in gene expression during hESC differentiation. TAD borders have been shown to form early in development and remain stable during differentiation. In contrast, the differentiating cells undergo a significant reorganization of the contacts inside TADs, resulting in the creation of particular interactions between enhancers and promoters. Studies of the differentiation of mouse embryonic stem cells (mESCs) into neural progenitor cells (NPCs) also revealed significant structural reorganization of the genome on the sub‐megabase scale. Loss of stem cell pluripotency is accompanied by the rearrangement of chromatin loops. CTCF, cohesin complex, and YY1, which are especially abundant in NPC‐specific chromatin loops, control how chromatin loops interact at different scales. , , V(D)J rearrangement is a key event in the development and maturation of immune cells. The dynamic process of chromatin loop extrusion driven by CTCF is an important mechanism of V(D)J rearrangement and the generation of antibody diversity in immune cells. As mentioned earlier, CTCF usually acts as an insulator at the TAD boundary and prevents the interactions between the two sides of the regulatory elements. Simultaneously, CTCF also mediates loop extrusion together with the cohesin complex, which regulates the mutual interaction of distant regulatory elements. Although acute degradation of CTCF in cell lines has little effect on gene expression, prolonged degradation of CTCF leads to morphological abnormalities and cellular differentiation. In mESCs, CTCF degradation promotes the spontaneous and asynchronous transformation of cells into 2C‐like cells, and this effect is reversible when CTCF levels are restored. This 2C‐like transition is uniquely observed in pluripotent cells in response to CTCF degradation. Similarly, degradation of CTCF in mESCs during neural differentiation delays axonogenesis, and CTCF binding at cell type‐specific gene promoters drive CTCF‐dependent EP interactions. CTCF is also necessary for the differentiation of mESCs into myocardial mesoderm and mutation of lysine 20 in CTCF suppresses mESC differentiation into early cardiac mesoderm by reducing CTCF‐mediated chromatin binding and chromatin interactions, as well as chromatin accessibility. It was recently discovered that CTCF also controls the higher‐dimensional structure of chromatin at the level of compartments. Specifically, CTCF controls the interaction of inter‐A compartments in the form of phase separation via RYBP (RING1 and YY1 Binding Protein), thereby preventing the differentiation of mESCs into neural progenitors. The critical function of the 3D genome in deciding cell fate transition has been enriched by studies of how chromatin structure is mediated by architectural proteins during cell lineage differentiation. Further investigation is needed to allow us to understand the relationships between architectural proteins and lineage‐specific specification, how these proteins coordinate in the lineage differentiation process, and whether there are distinct regulatory paradigms in chromatin organization during the differentiation of distinct lineages.

Terminally differentiated cells undergo cell fate reversal through somatic cell nuclear transfer (SCNT), overexpression of TF sets, or induction by small molecule compounds. , , Conversion of somatic cells to induced pluripotent stem cells (iPSCs) could be executed by either TFs or small molecules. , , During transdifferentiation (also known as direct reprogramming), the cells switch directly from one lineage to another without going through a pluripotent state. SCNT technology transforms cells into a totipotent form similar to the cells present in zygotes. Here, we further review how chromatin structure participates in the reprogramming of somatic cells into iPSCs. Since Takahashi and Yamanaka successfully transformed mouse embryonic fibroblasts (MEFs) into iPSCs using four TFs, OSKM (Oct4, Klf4, Sox2, and c‐Myc). This system for cell pluripotency acquisition has been widely used to investigate the biological mechanisms that underlie cell fate transition. Somatic cell reprogramming involves the suppression of somatic gene expression programmes and the activation of pluripotency programmes. During reprogramming, chromatin is dynamically reorganized, and multiple genetic and epigenetic barriers must be overcome. Previous studies used multi‐omics techniques to depict the rearrangement of TFs in enhancer regions and the remodelling of the epigenetic landscape during reprogramming. , Enhancer elements are essential for cell type‐specific gene expression as TF binding platforms. During the reprogramming process, TFs such as OCT4, SOX2, and KLF4 slowly shift from early enrichment at somatic cell‐associated enhancers to late enrichment at pluripotency enhancers, thereby altering the gene expression programme. , After effectively eliminating the tissue‐specific 3D genome structure, iPSCs derived from multiple lineage cells are able to establish an embryonic stem cell‐like 3D genome. A recent critical study revealed that the overall changes in gene expression, genome topology, and chromatin state are tightly related. But in general, this relationship occurs non‐synchronously: changes in transcription often come after changes in sub‐nuclear compartmentalization, TAD connectivity, and the strength of TAD‐border insulation. During cell reprogramming, TFs cause a series of effects to the genome structure and chromatin state, which permits the rewiring of the gene regulatory network. The interactions between long‐distance enhancers and promoters are crucial in regulating chromatin structure and gene expression. For instance, removing individual KLF4 binding sites inside pluripotent stem cell‐specific enhancers was sufficient to damage EP interactions and decrease the expression of related genes during the reprogramming of MEFs into iPSCs. During the reprogramming of mouse pre‐B cells to pluripotent cells, reorganization of chromatin compartments and TAD structures, as well as alterations in cell type‐specific chromatin loops, occur in a stage‐specific manner. The reorganization of chromatin during somatic reprogramming is also associated with TF‐driven phase separation. For example, OCT4 modulates TAD reprogramming by altering CTCF binding at the TAD boundary through phase separation. CTCF plays a vital role in 3D genome organization during the reprogramming process. Moreover, we recently discovered that CTCF represses the expression of somatic genes by acting as a chromatin insulator and functions as a chromatin remodeller to maintain the accessibility of pluripotency genes.

Organ regeneration is a major goal of regenerative medicine, which includes the regeneration and repair of damaged tissues as well as the rebuilding of organs using stem cell technology. The essence of regenerative biology at the cellular level is associated with cell fate transition. Therefore, it is crucial to examine how programmes involved in regeneration are affected by changes in the chromatin environment. The regulatory mechanisms that operate at the chromatin level during regeneration events have been extensively studied using model animals such as Hydra, Drosophila, zebrafish, and mice. To offer fresh perspectives on chromatin organization during regeneration processes, we mainly focus on the multidimensional regulation of chromatin during regeneration events; this multidimensional regulation involves chromatin remodelling, epigenetic alterations, and regulatory elements that participate in regeneration.

By changing the physical spacing of the nucleosomes, ATP‐dependent chromatin remodelling complexes (SWI/SNF, ISWI, CHD, and INO80) make DNA more accessible to particular protein factors and thereby change gene expression programmes. In studies of liver regeneration in mice, SMARCA4, a component of the SWI/SNF remodelling complex, was found to activate the Wnt/β‐catenin pathway by interacting with β‐catenin protein and thereby promote hepatocyte proliferation. Deletion of SMARCA4 inhibits liver regeneration and affects survival after hepatectomy. In contrast, deletion of the SWI/SNF complex component ARID1A significantly improves organ regeneration after liver injury in mice. Therefore, the balance of chromatin remodelling complex components may be one of the factors limiting tissue regeneration. The role of chromatin remodelling factors in maintaining differentiation‐related programmes and activating regeneration‐related programmes at the level of chromatin conformation remains to be further studied. SMARCA4 has been shown to bind the enhancers of Myc genes in leukaemia cells and is required for the regulation of distant chromatin interactions. In human mammary epithelial cells, deletion of SMARCA4 leads to significant changes in higher‐order chromatin structure. Our recent study showed that SMARCA5, the ATPase of the ISWI complex, cooperates with CTCF to maintain chromatin accessibility and promote the reprogramming of mouse fibroblasts into iPSCs. The regulatory regions of proliferation‐related genes in regenerating hepatocytes display increased chromatin accessibility and are enriched with ELK1 and CTCF, according to a study of dynamic chromatin architecture in regenerating liver. Further study should be required to understand how chromatin remodelling and higher dimensional chromatin conformations change during regenerative events and whether these changes are dependent on CTCF and cohesin complex‐mediated chromatin structure. Obtaining this information may open up new avenues to understanding how regenerative programmes are initiated and processed.

The studies related to 3D genome in regulating regeneration are really rare; however, epigenetic modifications are highly connected to 3D genomic regulation. , Epigenetic modifications such as histone modification and DNA methylation are also involved in organ regeneration after injury. Polycomb groups (PcGs) are classical epigenetic inhibitory complexes that are divided into two main types of complexes, PRC1 and PRC2. These complexes mediate the ubiquitination of histone H2A on lysine 119 (H2AK119ub) catalysed by RING1A/B and the trimethylation of histone on lysine 27 (H3K27me3) catalysed by EZH1/2, respectively. In regeneration‐related studies, EZH2‐deficient zebrafish failed to regenerate after caudal fin transection injury, suggesting that EZH2 and the histone modifications may play a role in the regeneration process. In contrast, during cardiac regeneration in mice, EZH1, but not EZH2, is required to activate cardiac regeneration‐related genes, suggesting that PcG may play a role in both transcriptional repression and transcriptional activation during regeneration events. Several recent studies have also shown that, unlike the classical repressive process, PcG can activate gene expression programmes during cell fate transition. , , Comparison of multi‐omics data from quiescent and regenerating hepatocytes in mouse liver revealed that pro‐regeneration‐related genes tended to be found in the active region of the genome and were enriched in the H3K27me3 modification in the resting state; this modification was erased during regeneration, and proliferation‐associated gene expression programmes were quickly activated. The multiple regulatory actions of PcG can be explained by the existence of higher dimensional chromatin structural network. Specifically, detailed suggestions regarding the role of PcG in 3D genome folding have been made. Thus, it may be possible to determine how the PcG complex orchestrates the regeneration programme at the level of chromatin structure in the future.

Chromatin regulatory elements (e.g., promoters and enhancers) play an essential role in gene expression. With the development of multi‐omics technologies, chromatin regulatory elements associated with regeneration events have been identified in Hydra, Drosophila, zebrafish, and other organisms by integrating omics information on chromatin state and histone modifications. , , The results show that some elements are relatively conserved among different species. For example, a class of tissue regeneration enhancer elements (TREEs) identified in regenerating tissues of zebrafish can trigger the repair programme at the injury site. TREE can also induce reporter gene expression at injury sites in mice. Recent studies have used TREE and recombinant adenoviral AAV vectors to achieve precise repair of damaged areas of the mouse heart, demonstrating the great potential of TREE in regenerative medicine applications. The spatial interaction of chromatin regulatory elements is an important part of gene expression, but the mechanism by which TREEs drive regeneration‐related gene expression remains to be studied. Is the appropriate 3D chromatin structure between TREE and regeneration‐related genes the basis for their function? We propose that a stable chromatin conformation is required for the initiation and maintenance of a regeneration programme and that this can be achieved by the coordinated action of TFs, remodelling complexes, mediators, epigenetic modification, TREE, and architectural proteins (Figure 3). Therefore, further analysis of the multidimensional chromatin environment near TREEs will help us to understand the regulatory process that occurs during regeneration events and further guide clinical application.

Recently, a number of studies have taken advantage of 3C‐derived techologies combined with high‐throughput sequencing to study the relationships between disease‐related genetic variation and chromatin conformation. Abnormal regulation of chromatin structure in diseases has become a focus of current research.

Genomic structural variants (SVs; including deletions, duplications, inversions, translocations, and insertions of sequences on the genome) and single nucleotide variants are common causes of genetically inherited developmental diseases and cancer. To a certain extent, disease‐associated SVs cause variation in the spatial conformation of the genome, and this may be due to aberrant regulation of the higher‐order structure of chromatin, which in turn leads to observable disease phenotypes. For example, genomic structural variation at the TAD boundary may cause rearrangement of EP contacts, resulting in abnormal expression of disease‐related genes. Single nucleotide polymorphisms (SNPs) in non‐coding regions may also contribute to abnormal chromatin structure, usually as a result of SNPs affecting specific TF binding motifs or specific DNA methylation sites, ultimately affecting the overall chromatin conformational environment (Figure 4). , ,

Multiple developmental disorders have been linked to specific loci in chromatin. At the EPHA4‐PAX3 locus, for instance, genomic variation has been associated with several rare limb malformations. A linear distribution of WNT6‐IHH‐EPHA4‐PAX3 genes in three TADs separated by two boundary elements characterizes this locus, at which the enhancer element and the EPHA4 gene are in the same TAD. Pathogenic structural variations at these loci in humans include fragment deletions, inversions, and duplications that may cause visible finger malformation phenotypes if the variant involves the boundary element. For example, deletion of EPHA4 at the TAD boundary results in the fusion of two TADs and aberrant contact of the enhancer with the PAX3 gene, leading to misexpression of PAX3 and a brachydactyly phenotype. Inversion of the enhancer leads to abnormal activation of WNT6 and causes syndactyly, while duplication of the enhancer and the IHH sequences leads to misexpression of the IHH gene, causing polydactyly. The EPHA4‐PAX3 locus contains tandem CTCF sites at the TAD boundary, which are conserved in mice. Recent study has used animal models to analyse in great detail how each CTCF at the boundary affects gene expression, enhancer insulation, and pathological traits. Studies have revealed that tandem CTCFs serve redundant roles and that insulation of the boundary can have a direct impact on gene expression and on the accompanying phenotype. Genomic SVs may also lead to the generation of new TAD structures, resulting in disease‐associated dysregulation of gene expression, primarily due to the duplication of boundary elements. For example, replication of the IGF2 locus has been observed in colorectal cancer patients and shown to lead to new TAD formation and aberrant overexpression of IGF2. If a duplication of the SOX9 locus involves the neighbouring KCNJ2 gene, a new TAD will be created, and the KCNJ2 gene will be in aberrant contact with the SOX9 regulatory element and misexpressed, resulting in limb deformity. The generation of aberrant chromatin structures by sequence duplication is also related to copy number and duplication area. If the duplication around the SOX9 locus does not include the KCNJ2 gene, it does not cause misexpression or a disease phenotype.

SVs in genomic DNA have been linked to abnormal chromatin structure, leading to disease‐associated dysregulation of gene expression. However, the production of abnormal regulation of gene expression usually requires certain conditions to be met, such as changes in boundary elements and ectopic chromatin contacts. One of the critical phenomena related to ectopic contacts in chromatin is ‘enhancer hijacking’, in which SVs lead to aberrant regulation of the expression of disease‐associated genes by enhancers. In several recent tumour‐related studies, it was discovered that enhancer hijacking events are the driving factors of high levels of oncogene expression. Algorithmic tools have been developed to identify SVs that may cause enhancer hijacking events. These studies enrich current mechanistic studies on how chromatin structure is related to cancer development.

There is mounting evidence that connects the occurrence of diseases to anomalies in chromatin arrangement. Mutations in the genes that encode architectural proteins are another form of connection between structural changes in chromatin and developmental disorders (Figure 4). According to the information gained from studies of several recent clinical cases and large‐scale sequencing research on developmental disorders, mutations in genes related to CTCF and cohesin complexes are frequently connected to the occurrence of neurodevelopmental disorders. , , , , , , , These genetic mutations, including truncation or functional amino acid mutations, disrupt the normal chromatin biology of these proteins, resulting in more widespread effects compared with those caused by variations in specific sequence element, such as boundary elements, in SVs. Aberrant expression of certain tissue‐specific genes influenced by chromatin structural regulation may be a comprehensive manifestation of developmental disorders featuring multi‐tissue abnormalities, as previous studies have associated certain cell and tissue‐specific chromatin conformations with gene expression and specialized functions. Chromatin insulation mediated by CTCF and cohesin complexes, as well as chromatin loop dysfunction, might be the main cause of these developmental issues. However, further study is required using relevant mutant cell models or disease animal models to elucidate in detail. Understanding the occurrence and progression of diseases from the standpoint of chromatin structural variation will thus be a future direction for improving disease diagnosis. On the one hand, more sophisticated detection methods and computational tools are required to make it possible to discover relevant chromatin structural alterations. On the other hand, gene editing approaches could be used to create equivalent cell or animal models to explore the possible regulatory processes. Disease simulation and mechanism studies could benefit from using human iPSC‐derived organoid technology, which has shown great promise in disease modelling and regenerative medicine in recent years.

This review summarizes the recent history of 3C and its derived techniques and describes their application and direction for revealing the role of chromatin architecture in stem cell differentiation, somatic cell re‐programming, organ regeneration, and diseases. Examination of the 3D structure of chromatin and the mechanisms that govern chromatin interactions is necessary to gain insight into how cells coordinate their most basic biological functions. Theoretically, understanding how chromatin organization changes dynamically during cell fate transition could help us to create in vitro cell treatment materials that are more effective and safer than currently available. Investigation of chromatin architecture in regeneration and diseases provides theoretical guidelines for in vitro simulation of organ regeneration and disease development with potentially clinical usefulness. However, there are several limitations of the current study: First, the lack of high‐resolution methods to capture 3D chromatin interactions at single cell type or even single cell level. Second, the scarcity of data on 3D genome organization in various cell types, developmental stages, and regeneration processes. Third, the difficulty in correlating and integrating multi‐omics data such as chromatin structure, epigenetic modifications, TF networks in the same biological event, and so forth. Therefore, the utilization of high‐resolution technologies in the future may be necessary to resolve chromatin conformation at the level of single cell types or even single cells. In addition, coordination of multi‐omics sources and functional analysis will be required to comprehensively delineate the rules of chromatin regulation in cell fate transition, regeneration events and disease process.

Hongxin Zhong, Jie Zhang, and Yuli Lu wrote the first draft of this paper. Gongcheng Hu and Guangjin Pan helped to prepare for this work. Hongjie Yao supervised and approved the final version.

This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16010502), National Key R&D Program of China (2021YFA1100300), and National Natural Science Foundation of China (U21A20195, 31925009).

The authors declare that they have no competing interests.