Three-dimensional Genome Research Articles

3D-GNOME 3.0: a three-dimensional genome modelling engine for analysing changes of promoter-enhancer contacts in the human genome.

In the current update, we added a feature for analysing changes in spatial distances between promoters and enhancers in chromatin 3D model ensembles. We updated our datasets by the novel in situ CTCF and RNAPII ChIA-PET chromatin loops obtained from the GM12878 cell line mapped to the GRCh38 genome assembly and extended the 1000 Genomes SVs dataset. To handle the new datasets, we applied GPU acceleration for the modelling engine, which gives a speed-up of 30× versus the previous versions. To improve visualisation and data analysis, we embedded the IGV tool for viewing ChIA-PET arcs with additional genes and SVs annotations. For 3D model visualisation, we added a new viewer: NGL, where we provided colouring by gene and enhancer location. The models are downloadable in mmcif and xyz format. The web server is hosted and performs calculations on DGX A100 GPU servers that provide optimal performance with multitasking. 3D-GNOME 3.0 web server provides unique insights into the topological mechanism of human variations at the population scale with high speed-up and is freely available at https://3dgnome.mini.pw.edu.pl/.

Region Capture Micro-C reveals coalescence of enhancers and promoters into nested microcompartments.

Although enhancers are central regulators of mammalian gene expression, the mechanisms underlying enhancer-promoter (E-P) interactions remain unclear. Chromosome conformation capture (3C) methods effectively capture large-scale three-dimensional (3D) genome structure but struggle to achieve the depth necessary to resolve fine-scale E-P interactions. Here, we develop Region Capture Micro-C (RCMC) by combining micrococcal nuclease (MNase)-based 3C with a tiling region-capture approach and generate the deepest 3D genome maps reported with only modest sequencing. By applying RCMC in mouse embryonic stem cells and reaching the genome-wide equivalent of ~317 billion unique contacts, RCMC reveals previously unresolvable patterns of highly nested and focal 3D interactions, which we term microcompartments. Microcompartments frequently connect enhancers and promoters, and although loss of loop extrusion and inhibition of transcription disrupts some microcompartments, most are largely unaffected. We therefore propose that many E-P interactions form through a compartmentalization mechanism, which may partially explain why acute cohesin depletion only modestly affects global gene expression.

Three-dimensional genome rewiring in loci with human accelerated regions.

Human accelerated regions (HARs) are conserved genomic loci that evolved at an accelerated rate in the human lineage and may underlie human-specific traits. We generated HARs and chimpanzee accelerated regions with an automated pipeline and an alignment of 241 mammalian genomes. Combining deep learning with chromatin capture experiments in human and chimpanzee neural progenitor cells, we discovered a significant enrichment of HARs in topologically associating domains containing human-specific genomic variants that change three-dimensional (3D) genome organization. Differential gene expression between humans and chimpanzees at these loci suggests rewiring of regulatory interactions between HARs and neurodevelopmental genes. Thus, comparative genomics together with models of 3D genome folding revealed enhancer hijacking as an explanation for the rapid evolution of HARs.

Comparative three-dimensional genome architectures of adipose tissues provide insight into human-specific regulation of metabolic homeostasis

Elucidating the regulatory mechanisms of human adipose tissues (ATs) evolution is essential for understanding human-specific metabolic regulation, but the functional importance and evolutionary dynamics of three-dimensional (3D) genome organizations of ATs are not well defined. Here, we compared the 3D genome architectures of anatomically distinct ATs from humans and six representative mammalian models. We recognized evolutionarily conserved and human-specific chromatin conformation in ATs at multiple scales, including compartmentalization, topologically associating domain (TAD), and promoter-enhancer interactions (PEI), which have not been described previously. We found PEI are much more evolutionarily dynamic with respect to compartmentalization and topologically associating domain. Compared to conserved PEIs, human-specific PEIs are enriched for human-specific sequence, and the binding motifs of their potential mediators (transcription factors) are less conserved. Our data also demonstrated that genes involved in the evolutionary dynamics of chromatin organization have weaker transcriptional conservation than those associated with conserved chromatin organization. Furthermore, the genes involved in energy metabolism and the maintenance of metabolic homeostasis are enriched in human-specific chromatin organization, while housekeeping genes, health-related genes, and genetic variations are enriched in evolutionarily conserved compared to human-specific chromatin organization. Finally, we showed extensively divergent human-specific 3D genome organizations among one subcutaneous and three visceral ATs. Together, these findings provide a global overview of 3D genome architecture dynamics between ATs from human and mammalian models and new insights into understanding the regulatory evolution of human ATs.

Abstract 1486: The regulatory mechanism of LncRNA CCAT1 via three-dimensional genome structure in colorectal cancer cells

Abstract Kruppel-like factor 5 (KLF5) belongs to zinc finger transcription factors and its expression is up-regulated in specific cancer types, such as gastrointestinal cancer and squamous carcinoma. It is reported that KLF5 is associated with the maintenance of stemness and essential for the integrity of intestinal epithelium and oncogenesis. Colon Cancer Associated Transcript 1 (CCAT1) is a long noncoding RNA that was initially found to be up-regulated in colorectal cancer (CRC), and studies have reported that high CCAT1 expression is associated with poor prognosis in CRC patients. In this study, we performed in vitro enChIP (engineered DNA-binding molecule-mediated chromatin immunoprecipitation)-sequencing to identify the KLF5 enhancer region which interacts with KLF5 promoter region. We successfully identified the KLF5 enhancer region in colorectal cancer cells and further found that KLF5 promoter region interacted with the transcription start site (TSS) region of CCAT1 gene, which is located at the different chromosome. Our results indicated that KLF5 protein participated in the core regulatory circuitry together with co-factors (BRD4, MED1, and RAD21), which contributes to construction of the three-dimensional genome structures consisting of KLF5 promoter, enhancer and CCAT1 TSS region. We also found that CCAT1 expression was highly correlated with KLF5 expression in CRC clinical samples and that KLF5 regulated CCAT1 expression. In conclusion, our data propose the mechanistic insight that KLF5 protein constructs the core regulatory circuitry with co-factors in the three-dimensional genome structure and coordinately regulates KLF5 and CCAT1 expression in CRC. Citation Format: Yuhki Yokoyama, Hirofumi Yamamoto. The regulatory mechanism of LncRNA CCAT1 via three-dimensional genome structure in colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1486.

Prediction of CTCF loop anchor based on machine learning.

Introduction: Various activities in biological cells are affected by three-dimensional genome structure. The insulators play an important role in the organization of higher-order structure. CTCF is a representative of mammalian insulators, which can produce barriers to prevent the continuous extrusion of chromatin loop. As a multifunctional protein, CTCF has tens of thousands of binding sites in the genome, but only a portion of them can be used as anchors of chromatin loops. It is still unclear how cells select the anchor in the process of chromatin looping. Methods: In this paper, a comparative analysis is performed to investigate the sequence preference and binding strength of anchor and non-anchor CTCF binding sites. Furthermore, a machine learning model based on the CTCF binding intensity and DNA sequence is proposed to predict which CTCF sites can form chromatin loop anchors. Results: The accuracy of the machine learning model that we constructed for predicting the anchor of the chromatin loop mediated by CTCF reached 0.8646. And we find that the formation of loop anchor is mainly influenced by the CTCF binding strength and binding pattern (which can be interpreted as the binding of different zinc fingers). Discussion: In conclusion, our results suggest that The CTCF core motif and it's flanking sequence may be responsible for the binding specificity. This work contributes to understanding the mechanism of loop anchor selection and provides a reference for the prediction of CTCF-mediated chromatin loops.

The Principles and Applications of High-Throughput Sequencing Technologies

The advancement in high-throughput sequencing (HTS) technology has revolutionized the field of biology, including genomics, epigenomics, transcriptomics, and metagenomics. This technology has become a crucial tool in many areas of research, allowing scientists to generate vast amounts of genetic data at a much faster pace than traditional methods. With this increased speed and scale of data generation, researchers can now address critical questions and gain new insights into the inner workings of living organisms, as well as the underlying causes of various diseases. Although the first HTS technology have been introduced about two decades ago, it can still be challenging for those new to the field to understand and use effectively. This review aims to provide a comprehensive overview of commonly used HTS technologies these days and their applications in terms of genome sequencing, transcriptome, DNA methylation, DNA-protein interaction, chromatin accessibility, three-dimensional genome organization, and microbiome.

MYC reshapes CTCF-mediated chromatin architecture in prostate cancer

MYC is a well characterized oncogenic transcription factor in prostate cancer, and CTCF is the main architectural protein of three-dimensional genome organization. However, the functional link between the two master regulators has not been reported. In this study, we find that MYC rewires prostate cancer chromatin architecture by interacting with CTCF protein. Through combining the H3K27ac, AR and CTCF HiChIP profiles with CRISPR deletion of a CTCF site upstream of MYC gene, we show that MYC activation leads to profound changes of CTCF-mediated chromatin looping. Mechanistically, MYC colocalizes with CTCF at a subset of genomic sites, and enhances CTCF occupancy at these loci. Consequently, the CTCF-mediated chromatin looping is potentiated by MYC activation, resulting in the disruption of enhancer-promoter looping at neuroendocrine lineage plasticity genes. Collectively, our findings define the function of MYC as a CTCF co-factor in three-dimensional genome organization.

The 3D genome and its impacts on human health and disease

Abstract Eukaryotic genomes are highly compacted in the cell nucleus. Two loci separated by a long linear distance can be brought into proximity in space through DNA-binding proteins and RNAs, which contributes profoundly to the regulation of gene expression. Recent technology advances have enabled the development and application of the chromosome conformation capture (3C) technique and a host of 3C-based methods that enable genome-scale investigations into changes in chromatin high-order structures during diverse physiological processes and diseases. In this review, we introduce 3C-based technologies and discuss how they can be utilized to glean insights into the impacts of three-dimensional (3D) genome organization in normal physiological and disease processes.

High-throughput Pore-C reveals the single-allele topology and cell type-specificity of 3D genome folding

Canonical three-dimensional (3D) genome structures represent the ensemble average of pairwise chromatin interactions but not the single-allele topologies in populations of cells. Recently developed Pore-C can capture multiway chromatin contacts that reflect regional topologies of single chromosomes. By carrying out high-throughput Pore-C, we reveal extensive but regionally restricted clusters of single-allele topologies that aggregate into canonical 3D genome structures in two human cell types. We show that fragments in multi-contact reads generally coexist in the same TAD. In contrast, a concurrent significant proportion of multi-contact reads span multiple compartments of the same chromatin type over megabase distances. Synergistic chromatin looping between multiple sites in multi-contact reads is rare compared to pairwise interactions. Interestingly, the single-allele topology clusters are cell type-specific even inside highly conserved TADs in different types of cells. In summary, HiPore-C enables global characterization of single-allele topologies at an unprecedented depth to reveal elusive genome folding principles.

High-Resolution 3D Genome Map of Brucella Chromosomes in Exponential and Stationary Phases.

The three-dimensional (3D) genome structure of an organism or cell is highly relevant to its biological activities, but the availability of 3D genome information for bacteria, especially intracellular pathogens, is still limited. Here, we used Hi-C (high-throughput chromosome conformation capture) technology to determine the 3D chromosome structures of exponential- and stationary-phase Brucella melitensis at a 1-kb resolution. We observed that the contact heat maps of the two B. melitensis chromosomes contain a prominent diagonal and a secondary diagonal. Then, 79 chromatin interaction domains (CIDs) were detected at an optical density at 600 nm (OD600) of 0.4 (exponential phase), with the longest CID being 106 kb and the shortest being 12 kb. Moreover, we obtained 49,363 significant cis-interaction loci and 59,953 significant trans-interaction loci. Meanwhile, 82 CIDs of B. melitensis at an OD600 of 1.5 (stationary phase) were detected, with the longest CID being 94 kb and the shortest being 16 kb. In addition, 25,965 significant cis-interaction loci and 35,938 significant trans-interaction loci were obtained in this phase. Furthermore, we found that as the B. melitensis cells grew from the logarithmic to the plateau phase, the frequency of short-range interactions increased, while that of long-range interactions decreased. Finally, combined analysis of 3D genome and whole-genome transcriptome (RNA-seq) data revealed that the strength of short-range interactions in Chr1 is specifically and strongly correlated with gene expression. Overall, our study provides a global view of the chromatin interactions in the B. melitensis chromosomes, which will serve as a resource for further study of the spatial regulation of gene expression in Brucella. IMPORTANCE The spatial structure of chromatin plays important roles in normal cell functions and in the regulation of gene expression. Three-dimensional genome sequencing has been performed in many mammals and plants, but the availability of such data for bacteria, especially intracellular pathogens, is still limited. Approximately 10% of sequenced bacterial genomes contain more than one replicon. However, how multiple replicons are organized within bacterial cells, how they interact, and whether these interactions help to maintain or segregate these multipartite genomes are unresolved issues. Brucella is a Gram-negative, facultative intracellular, and zoonotic bacterium. Except for Brucella suis biovar 3, Brucella species have two chromosomes. Here, we applied Hi-C technology to determine the 3D genome structures of exponential- and stationary-phase Brucella melitensis chromosomes at a 1-kb resolution. Combined analysis of the 3D genome and RNA-seq data indicated that the strength of short-range interactions in B. melitensis Chr1 is specifically and strongly correlated with gene expression. Our study provides a resource to achieve a deeper understanding of the spatial regulation of gene expression in Brucella.

Multiscale 3D genome reorganization during skeletal muscle stem cell lineage progression and aging.

Little is known about three-dimensional (3D) genome organization in skeletal muscle stem cells [also called satellite cells (SCs)]. Here, we comprehensively map the 3D genome topology reorganization during mouse SC lineage progression. Specifically, rewiring at the compartment level is most pronounced when SCs become activated. Marked loss in topologically associating domain (TAD) border insulation and chromatin looping also occurs during early activation process. Meanwhile, TADs can form TAD clusters and super-enhancer-containing TAD clusters orchestrate stage-specific gene expression. Furthermore, we uncover that transcription factor PAX7 is pivotal in enhancer-promoter (E-P) loop formation. We also identify cis-regulatory elements that are crucial for local chromatin organization at Pax7 locus and Pax7 expression. Lastly, we unveil that geriatric SC displays a prominent gain in long-range contacts and loss of TAD border insulation. Together, our results uncover that 3D chromatin extensively reorganizes at multiple architectural levels and underpins the transcriptome remodeling during SC lineage development and SC aging.

Inducible disruption of Tet genes results in myeloid malignancy, readthrough transcription, and a heterochromatin-to-euchromatin switch

The three mammalian TET dioxygenases oxidize the methyl group of 5-methylcytosine in DNA, and the oxidized methylcytosines are essential intermediates in all known pathways of DNA demethylation. To define the invivo consequences of complete TET deficiency, we inducibly deleted all three Tet genes in the mouse genome. Tet1/2/3-inducible TKO (iTKO) mice succumbed to acute myeloid leukemia (AML) by 4 to 5 wk. Single-cell RNA sequencing of Tet iTKO bone marrow cells revealed the appearance of new myeloid cell populations characterized by a striking increase in expression of all members of the stefin/cystatin gene cluster on mouse chromosome 16. In patients with AML, high stefin/cystatin gene expression correlates with poor clinical outcomes. Increased expression of the clustered stefin/cystatin genes was associated with a heterochromatin-to-euchromatin compartment switch with readthrough transcription downstream of the clustered stefin/cystatin genes as well as other highly expressed genes, but only minor changes in DNA methylation. Our data highlight roles for TET enzymes that are distinct from their established function in DNA demethylation and instead involve increased transcriptional readthrough and changes in three-dimensional genome organization.

Polymer modelling accurately predicts three-dimensional chromosome reorganization with a seasonal coronavirus infection.

Many vital processes, such as DNA transcription, replication, and repair, depending on the cell's structural organization of chromosomes. Three-dimensional genome spatial information is essential for understanding gene regulation and epigenetic effects, which can regulate the 3D architecture of the whole genome. Various advances in experimental techniques such as chromosome conformation capture (Hi-C), Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) and other related methods have led to the finding of topologically associating domains (TADs), chromatin loops, and different compartments existing in the chromosomes. However, less progress has been made in modelling chromosomes based on experimentally-driven approaches. Previously, polymer simulations were employed, incorporating one-dimension information and generating connectivity profiles for the chromosomes. Our method (4DHiC) extracts 2D information from their corresponding Hi-C maps and implements it as a harmonic constraint in the polymer model. Here, different Hi-C maps for MRC cell lines infected with and without seasonal coronavirus infection at varying stages of infection were generated to test the model's applicability. We performed Langevin dynamics to simulate the polymer model with HiC-map constraints using the 4DHiC method. On comparing the simulated and experimental HiC map, a strong correlation was observed (point-to-point RPearson∼0.75). Along with the model development, this study also revealed the critical structural difference that arises from viral infection in the chromosome organization. One important chromosome organization feature, A/B compartmentalization, was also studied. A clear separation was observed based on the principal component analysis, predicting the change in compartment contacts. Another advantage of the method is the scalability, i.e. can vary the number of beads based on the resolution of the HiC map. Lastly, we emphasize that the protocol is general and can be used to model the whole genome or individual chromosomes for any system.

Chromosome conformation capture approaches to investigate 3D genome architecture in Ankylosing Spondylitis.

Ankylosing Spondylitis (AS) is a chronic inflammatory arthritis of the spine exhibiting a strong genetic background. The mechanistic and functional understanding of the AS-associated genomic loci, identified with Genome Wide Association Studies (GWAS), remains challenging. Chromosome conformation capture (3C) and derivatives are recent techniques which are of great help in elucidating the spatial genome organization and of enormous support in uncover a mechanistic explanation for disease-associated genetic variants. The perturbation of three-dimensional (3D) genome hierarchy may lead to a plethora of human diseases, including rheumatological disorders. Here we illustrate the latest approaches and related findings on the field of genome organization, highlighting how the instability of 3D genome conformation may be among the causes of rheumatological disease phenotypes. We suggest a new perspective on the inclusive potential of a 3C approach to inform GWAS results in rheumatic diseases. 3D genome organization may ultimately lead to a more precise and comprehensive functional interpretation of AS association, which is the starting point for emerging and more specific therapies.

β-actin mediated H3K27ac changes demonstrate the link between compartment switching and enhancer-dependent transcriptional regulation

BackgroundRecent work has demonstrated that three-dimensional genome organization is directly affected by changes in the levels of nuclear cytoskeletal proteins such as β-actin. The mechanisms which translate changes in 3D genome structure into changes in transcription, however, are not fully understood. Here, we use a comprehensive genomic analysis of cells lacking nuclear β-actin to investigate the mechanistic links between compartment organization, enhancer activity, and gene expression.ResultsUsing HiC-Seq, ATAC-Seq, and RNA-Seq, we first demonstrate that transcriptional and chromatin accessibility changes observed upon β-actin loss are highly enriched in compartment-switching regions. Accessibility changes within compartment switching genes, however, are mainly observed in non-promoter regions which potentially represent distal regulatory elements. Our results also show that β-actin loss induces widespread accumulation of the enhancer-specific epigenetic mark H3K27ac. Using the ABC model of enhancer annotation, we then establish that these epigenetic changes have a direct impact on enhancer activity and underlie transcriptional changes observed upon compartment switching. A complementary analysis of fibroblasts undergoing reprogramming into pluripotent stem cells further confirms that this relationship between compartment switching and enhancer-dependent transcriptional change is not specific to β-actin knockout cells but represents a general mechanism linking compartment-level genome organization to gene expression.ConclusionsWe demonstrate that enhancer-dependent transcriptional regulation plays a crucial role in driving gene expression changes observed upon compartment-switching. Our results also reveal a novel function of nuclear β-actin in regulating enhancer function by influencing H3K27 acetylation levels.

Pan-3D genome analysis reveals structural and functional differentiation of soybean genomes

BackgroundHigh-order chromatin structure plays important roles in gene regulation. However, the diversity of the three-dimensional (3D) genome across plant accessions are seldom reported.ResultsHere, we perform the pan-3D genome analysis using Hi-C sequencing data from 27 soybean accessions and comprehensively investigate the relationships between 3D genomic variations and structural variations (SVs) as well as gene expression. We find that intersection regions between A/B compartments largely contribute to compartment divergence. Topologically associating domain (TAD) boundaries in A compartments exhibit significantly higher density compared to those in B compartments. Pan-3D genome analysis shows that core TAD boundaries have the highest transcription start site (TSS) density and lowest GC content and repeat percentage. Further investigation shows that non-long terminal repeat (non-LTR) retrotransposons play important roles in maintaining TAD boundaries, while Gypsy elements and satellite repeats are associated with private TAD boundaries. Moreover, presence and absence variation (PAV) is found to be the major contributor to 3D genome variations. Nevertheless, approximately 55% of 3D genome variations are not associated with obvious genetic variations, and half of them affect the flanking gene expression. In addition, we find that the 3D genome may also undergo selection during soybean domestication.ConclusionOur study sheds light on the role of 3D genomes in plant genetic diversity and provides a valuable resource for studying gene regulation and genome evolution.

Radiation-Induced 3D Genome Structure Reorganization Regulates Gene Transcription

Radiation can induce cellular three-dimensional (3D) genome reorganization. However, the exact functional relevance of spatial rearrangements to gene transcription remains unknown. First, we screened and profiled the global picture of the 3D genome structure reorganization in human hepatocellular carcinoma cells MHCC-LM3 that were irradiated with 4Gy X-ray at a concentration of 100 cGy/min for 4 min using the high-throughput chromosome conformation capture (Hi-C) technique. They were maped and analyzed at three levels: compartment, topologically associating domain (TAD) and TAD boundary, and loop. The regulatory relationships within gene transcription exerted by the related dynamic spatial structure were confirmed by ribonucleic acid sequencing (RNA-seq) and further experimentally confirmed by quantitative real-time polymerase chain reaction in the same treated cell samples. A significant increase in interchromosomal interactions was observed in the irradiated cells. The extent of the effect of dynamic compartment switches and disrupted and gained TAD boundaries on gene transcription was significantly greater than that of small differential chromatin loops. Genes in human leukocyte antigen (HLA) and C‑C motif chemokine ligand (CCL) gene clusters associated with TAD boundary alterations were considerably upregulated, and this effect was maintained 24 h post-radiation. We characterized the radiation-induced landscape map of the 3D genome in cells and elucidated the mechanism by which 3D genome reorganization spatially regulates gene transcription. This study provides a basis for further research directed toward understanding the role of structural alterations in gene expression.

Three-dimensional genome landscape comprehensively reveals patterns of spatial gene regulation in papillary and anaplastic thyroid cancers: a study using representative cell lines for each cancer type

BackgroundSpatial chromatin structure is intricately linked with somatic aberrations, and somatic mutations of various cancer-related genes, termed co-mutations (CoMuts), occur in certain patterns during cancer initiation and progression. The functional mechanisms underlying these genetic events remain largely unclear in thyroid cancer (TC). With discrepant differentiation, papillary thyroid cancer (PTC) and anaplastic thyroid cancer (ATC) differ greatly in characteristics and prognosis. We aimed to reveal the spatial gene alterations and regulations between the two TC subtypes.MethodsWe systematically investigated and compared the spatial co-mutations between ATC (8305C), PTC (BCPAP and TPC-1), and normal thyroid cells (Nthy-ori-3–1). We constructed a framework integrating whole-genome sequencing (WGS), high-throughput chromosome conformation capture (Hi-C), and transcriptome sequencing, to systematically detect the associations between the somatic co-mutations of cancer-related genes, structural variations (SVs), copy number variations (CNVs), and high-order chromatin conformation.ResultsSpatial co-mutation hotspots were enriched around topologically associating domains (TADs) in TC. A common set of 227 boundaries were identified in both ATC and PTC, with significant overlaps between them. The spatial proximities of the co-mutated gene pairs in the two TC types were significantly greater than in the gene-level and overall backgrounds, and ATC cells had higher TAD contact frequency with CoMuts > 10 compared with PTC cells. Compared with normal thyroid cells, in ATC the number of the created novel three-dimensional chromatin structural domains increased by 10%, and the number of shifted TADs decreased by 7%. We found five TAD blocks with CoMut genes/events specific to ATC with certain mutations in genes including MAST-NSUN4, AM129B/TRUB2, COL5A1/PPP1R26, PPP1R26/GPSM1/CCDC183, and PRAC2/DLX4. For the majority of ATC and PTC cells, the HOXA10 and HIF2α signals close to the transcription start sites of CoMut genes within TADs were significantly stronger than those at the background. CNV breakpoints significantly overlapped with TAD boundaries in both TC subtypes. ATCs had more CNV losses overlapping with TAD boundaries, and noncoding SVs involved in intrachromosomal SVs, amplified inversions, and tandem duplication differed between ATC and PTC. TADs with short range were more abundant in ATC than PTC. More switches of A/B compartment types existed in ATC cells compared with PTC. Gene expression was significantly synchronized, and orchestrated by complex epigenetics and regulatory elements.ConclusionChromatin interactions and gene alterations and regulations are largely heterogeneous in TC. CNVs and complex SVs may function in the TC genome by interplaying with TADs, and are largely different between ATC and PTC. Complexity of TC genomes, which are highly organized by 3D genome-wide interactions mediating mutational and structural variations and gene activation, may have been largely underappreciated. Our comprehensive analysis may provide key evidence and targets for more customized diagnosis and treatment of TC.

A spatial genome aligner for resolving chromatin architectures from multiplexed DNA FISH

Multiplexed fluorescence in situ hybridization (FISH) is a widely used approach for analyzing three-dimensional genome organization, but it is challenging to derive chromosomal conformations from noisy fluorescence signals, and tracing chromatin is not straightforward. Here we report a spatial genome aligner that parses true chromatin signal from noise by aligning signals to a DNA polymer model. Using genomic distances separating imaged loci, our aligner estimates spatial distances expected to separate loci on a polymer in three-dimensional space. Our aligner then evaluates the physical probability observed signals belonging to these loci are connected, thereby tracing chromatin structures. We demonstrate that this spatial genome aligner can efficiently model chromosome architectures from DNA FISH data across multiple scales and be used to predict chromosome ploidies de novo in interphase cells. Reprocessing of previous whole-genome chromosome tracing data with this method indicates the spatial aggregation of sister chromatids in S/G2 phase cells in asynchronous mouse embryonic stem cells and provides evidence for extranumerary chromosomes that remain tightly paired in postmitotic neurons of the adult mouse cortex.