Chromosome Conformation Capture Techniques Research Articles

ParticleChromo3D: a Particle Swarm Optimization algorithm for chromosome 3D structure prediction from Hi-C data

BackgroundThe three-dimensional (3D) structure of chromatin has a massive effect on its function. Because of this, it is desirable to have an understanding of the 3D structural organization of chromatin. To gain greater insight into the spatial organization of chromosomes and genomes and the functions they perform, chromosome conformation capture (3C) techniques, particularly Hi-C, have been developed. The Hi-C technology is widely used and well-known because of its ability to profile interactions for all read pairs in an entire genome. The advent of Hi-C has greatly expanded our understanding of the 3D genome, genome folding, gene regulation and has enabled the development of many 3D chromosome structure reconstruction methods.ResultsHere, we propose a novel approach for 3D chromosome and genome structure reconstruction from Hi-C data using Particle Swarm Optimization (PSO) approach called ParticleChromo3D. This algorithm begins with a grouping of candidate solution locations for each chromosome bin, according to the particle swarm algorithm, and then iterates its position towards a global best candidate solution. While moving towards the optimal global solution, each candidate solution or particle uses its own local best information and a randomizer to choose its path. Using several metrics to validate our results, we show that ParticleChromo3D produces a robust and rigorous representation of the 3D structure for input Hi-C data. We evaluated our algorithm on simulated and real Hi-C data in this work. Our results show that ParticleChromo3D is more accurate than most of the existing algorithms for 3D structure reconstruction.ConclusionsOur results also show that constructed ParticleChromo3D structures are very consistent, hence indicating that it will always arrive at the global solution at every iteration. The source code for ParticleChromo3D, the simulated and real Hi-C datasets, and the models generated for these datasets are available here: https://github.com/OluwadareLab/ParticleChromo3D

Models of topological barriers and molecular motors of bacterial DNA

ABSTRACT Bacterial genomes are partitioned into kilobases long domains that are topologically independent from each other, meaning that change of DNA superhelicity in one domain does not propagate to neighbours. This is made possible by proteins like the LacI repressor, which behave like topological barriers and block the diffusion of torsion along the DNA. Other proteins, like DNA gyrases and RNA polymerases, called molecular motors, use the energy released by the hydrolysis of ATP to apply forces and/or torques to the DNA and modify its superhelicity. Here, we report on simulation work aimed at enlightening the interplay between DNA supercoiling, topological barriers, and molecular motors. To this end, we developed a coarse-grained Hamiltonian model of topological barriers and a model of molecular motors and investigated their properties through Brownian dynamics simulations. We discuss their influence on the contact map of a model nucleoid and the steady state values of twist and writhe in the DNA. These coarse-grained models, which are able to predict the dynamics of plectonemes depending on the position of topological barriers and molecular motors, should prove helpful to back up experimental efforts, like the development of Chromosome Conformation Capture techniques, and decipher the organisational mechanisms of bacterial chromosomes.

Advances of enhancers in regulating craniomaxillofacial development in mammals

As a key regulatory element of gene differential expression, enhancer plays a crucial role in craniomaxillofacial development through regulating the spatiotemporal expression of target genes to promote tissue-specific differentiation. With the development of CRISPR and chromosome conformation capture technique, the function of enhancer and its regulatory mechanism has been explored in depth. This paper gave a systematic review on the mechanism of enhancer regulating target gene expression and the role of enhancer in oral craniofacial development and malformation.

Can abnormal chromatin folding cause high-penetrance cancer predisposition?

Sequencing cancer predisposing genes (CPGs) in evocative patients (i.e., patients with personal and family history of multiple/early-onset/unusual cancers) allows follow-up in their relatives to be adapted when a causative pathogenic variant is identified. Unfortunately, many evocative families remain unexplained. Part of this "missing heritability" could be due to CPG dysregulations caused by remote noncoding genomic alterations. Transcription levels are regulated through the ability of promoters to physically interact with their distant cis-regulatory elements. Three-dimensional chromatin contacts, mediated by a dynamic loop extrusion process, are uncovered by chromosome conformation capture (3C) and 3C-derived techniques, which have enabled the discovery of new pathological mechanisms in developmental diseases and cancers. High-penetrance cancer predisposition is caused by germline hereditary alterations otherwise found at the somatic level in sporadic cancers. Thus, data from both developmental diseases and cancers provide information about possible unknown cancer predisposition mechanisms. This mini-review aims to deduce from these data whether abnormal chromatin folding can cause high-penetrance cancer predisposition.

475 High resolution chromatin loops associate with gene targets for psoriasis susceptibility regions

There are over 80 psoriasis-associated loci identified, with a majority playing regulatory roles; however, their gene targets are yet to be identified. Chromatin loops play an important role in gene regulation, and can be detected by chromosome conformation capture techniques like Hi-C. In this study, we deep sequenced Hi-C libraries (∼1 billion reads/reaction) to generate nine PBMC-derived subsets from 2 individuals (∼50,000 cells each): 4 unstimulated CD3+CD45RO+ memory T cells (CD4+CLA-, CD4+CLA+, CD8+CLA-, CD8+CLA+); the same 4 subsets after 24 hours of CD3/CD28 stimulation of CD1c- cells (primarily T cells); and mDC. Using a 5k-10k contact map resolution, we identified 13,186 ± 2,857 Hi-C loops per library, ranging from 30 to 1,980 kb long, using Mustache. Notably, we found that ∼50% of the identified loops have at least one end overlapping a transcription start site, providing a unique resource for mapping long-range promoter interactions in immunocytes. We identified 27 ± 6 loops per library that linked to promoters and whose loop ends could also be mapped to known psoriasis signals, defined by the 95% credible intervals generated from a recent transethnic GWAS (HGG Adv 2022). Of 47 target genes involved in such loops, we found 8 previously suggested gene targets for psoriasis: ANXA6, ELMO1, ETS1, FASLG, MBD2, IFI44, PTGER4 and STARD6. Among them, ANXA6, ETS1, FASLG and IFI44 were present only in T cells, while the rest presented in both T cells and mDC. We further revealed other psoriasis gene candidates including KAT5, NHLRC3/PROSER1, and SUCO, which were linked to known psoriasis signals through loops in multiple Hi-C libraries for both individuals and in both T cells and mDC. This study provides complementary support for known psoriasis-related genes at the level of 3D genome structure, and nominates other genes of interest for future studies.

Dynamic Interplay between Structural Variations and 3D Genome Organization in Pancreatic Cancer (Adv. Sci. 18/2022)

Carcinogenesis of Structural Variations in Human Cancer In article number 2200818, Hebing Chen, Chengfeng Wang, and co-workers report carcinogenesis of structural variations (SVs) focusing on their dynamic interplay with three-dimensional (3D) genome organization with multi-omics strategies integrating long-read single-molecule real-time (SMRT) sequencing and high-throughput chromosome conformation capture (Hi-C) technique in deadly human pancreatic cancer. This work provides a genome-wide prospects of SVs and 3D genome, and expand current understanding of tumorigenesis, which might facilitate the identification of novel prognostic markers or therapeutic targets.

Three-dimensional genome structure shapes the recombination landscape of chromatin features during female germline stem cell development.

BackgroundDuring meiosis of mammalian cells, chromatin undergoes drastic reorganization. However, the dynamics of the three‐dimensional (3D) chromatin structure during the development of female germline stem cells (FGSCs) are poorly understood.MethodsThe high‐throughput chromosome conformation capture technique was used to probe the 3D structure of chromatin in mouse germ cells at each stage of FGSC development.ResultsThe global 3D genome was dramatically reorganized during FGSC development. In topologically associating domains, the chromatin structure was weakened in germinal vesicle stage oocytes and still present in meiosis I stage oocytes but had vanished in meiosis II oocytes. This switch between topologically associating domains was related to the biological process of FGSC development. Moreover, we constructed a landscape of chromosome X organization, which showed that the X chromosome occupied a smaller proportion of the active (A) compartment than the autosome during FGSC development. By comparing the high‐order chromatin structure between female and male germline development, we found that 3D genome organization was remodelled by two different potential mechanisms during gamete development, in which interchromosomal interactions, compartments, and topologically associating domain were decreased during FGSC development but reorganized and recovered during spermatogenesis. Finally, we identified conserved chromatin structures between FGSC development and early embryonic development.ConclusionsThese results provide a valuable resource to characterize chromatin organization and for further studies of FGSC development.

Sister chromatid-sensitive Hi-C to map the conformation of replicated genomes.

Chromosome conformation capture (Hi-C) techniques map the 3D organization of entire genomes. How sister chromatids fold in replicated chromosomes, however, cannot be determined with conventional Hi-C because of the identical DNA sequences of sister chromatids. Here, we present a protocol for sister chromatid-sensitive Hi-C (scsHi-C) that enables the distinction of DNA contacts within individual sister chromatids (cis sister contacts) from those between sister chromatids (trans sister contacts), thereby allowing investigation of the organization of replicated genomes. scsHi-C is based on live-cell labeling of nascent DNA by the synthetic nucleoside 4-thio-thymidine (4sT), which incorporates into a distinct DNA strand on each sister chromatid because of semi-conservative DNA replication. After purification of genomic DNA and in situ Hi-C library preparation, 4sT is chemically converted into 5-methyl-cytosine in the presence of OsO4/NH4Cl to introduce T-to-C signature point mutations on 4sT-labeled DNA. The Hi-C library is then sequenced, and ligated fragments are assigned to sister chromatids on the basis of strand orientation and the presence of signature mutations. The ensemble of scsHi-C contacts thereby represents genome-wide contact probabilities within and across sister chromatids. scsHi-C can be completed in 2 weeks, has been successfully applied in HeLa cells and can potentially be established for any cell type that allows proper cell cycle synchronization and incorporation of sufficient amounts of 4sT. The genome-wide maps of replicated chromosomes detected by scsHi-C enable investigation of the molecular mechanisms shaping sister chromatid topologies and the relevance of sister chromatid conformation in crucial processes like DNA repair, mitotic chromosome formation and potentially other biological processes.

A chromosome-scale genome assembly of the false clownfish, Amphiprion ocellaris.

The false clownfish Amphiprion ocellaris is a popular fish species and an emerging model organism for studying the ecology, evolution, adaptation, and developmental biology of reef fishes. Despite this, high-quality genomic resources for this species are scarce, hindering advanced genomic analyses. Leveraging the power of PacBio long-read sequencing and Hi-C chromosome conformation capture techniques, we constructed a high-quality chromosome-scale genome assembly for the clownfish A. ocellaris. The initial genome assembly comprised of 1,551 contigs of 861.42 Mb, with an N50 of 863.85 kb. Hi-C scaffolding of the genome resulted in 24 chromosomes containing 856.61 Mb. The genome was annotated with 26,797 protein-coding genes and had 96.62% completeness of conserved actinopterygian genes, making this genome the most complete and high quality among published anemonefish genomes. Transcriptomic analysis identified tissue-specific gene expression patterns, with the brain and optic lobe having the largest number of expressed genes. Further, comparative genomic analysis revealed 91 genome elements conserved only in A. ocellaris and its sister species Amphiprion percula, and not in other anemonefish species. These elements are close to genes that are involved in various nervous system functions and exhibited distinct expression patterns in brain tissue, potentially highlighting the genetic toolkits involved in lineage-specific divergence and behaviors of the clownfish branch. Overall, our study provides the highest quality A. ocellaris genome assembly and annotation to date, whilst also providing a valuable resource for understanding the ecology and evolution of reef fishes.

The Chromosome-Scale Reference Genome of Macadamia tetraphylla Provides Insights Into Fatty Acid Biosynthesis.

Macadamia is an evergreen tree belonging to the Proteaceae family. The two commercial macadamia species, Macadamia integrifolia and M. tetraphylla, are highly prized for their edible kernels. The M. integrifolia genome was recently sequenced, but the genome of M. tetraphylla has to date not been published, which limits the study of biological research and breeding in this species. This study reports a high-quality genome sequence of M. tetraphylla based on the Oxford Nanopore Technologies technology and high-throughput chromosome conformation capture techniques (Hi-C). An assembly of 750.87 Mb with 51.11 Mb N50 length was generated, close to the 740 and 758 Mb size estimates by flow cytometry and k-mer analysis, respectively. Genome annotation indicated that 61.42% of the genome is composed of repetitive sequences and 34.95% is composed of long terminal repeat retrotransposons. Up to 31,571 protein-coding genes were predicted, of which 92.59% were functionally annotated. The average gene length was 6,055 bp. Comparative genome analysis revealed that the gene families associated with defense response, lipid transport, steroid biosynthesis, triglyceride lipase activity, and fatty acid metabolism are expanded in the M. tetraphylla genome. The distribution of fourfold synonymous third-codon transversion showed a recent whole-genome duplication event in M. tetraphylla. Genomic and transcriptomic analysis identified 187 genes encoding 33 crucial oil biosynthesis enzymes, depicting a comprehensive map of macadamia lipid biosynthesis. Besides, the 55 identified WRKY genes exhibited preferential expression in root as compared to that in other tissues. The genome sequence of M. tetraphylla provides novel insights for breeding novel varieties and genetic improvement of agronomic traits.

Dynamic self-organization of the human genome during the cell cycle

The hierarchical organization of the chromatin fiber, the functional form of DNA in cells, has been extensively studied from a single nucleosome to the genome-wide scale. Chromosome conformation capture techniques revealed a detailed static picture of the genome's 3D folded state inside the cell nucleus, revealing presence of loops, topologically associated domains, A/B compartments and chromosome territories [1]. In addition, chromatin organization was found to resemble a fractal globule inside the nucleus [2].

Detection of Allele-Specific 3D Chromatin Interactions Using High-Resolution In-Nucleus 4C-seq.

Chromosome conformation capture techniques are a set of methods used to determine 3D genome organization through the capture and identification of physical contacts between pairs of genomic loci. Among them, 4C-seq (circular chromosome conformation capture coupled to high-throughput sequencing) allows for the identification and quantification of the sequences interacting with a preselected locus of interest. 4C-seq has been widely used in the literature, mainly to study chromatin loops between enhancers and promoters or between CTCF binding sites and to identify chromatin domain boundaries. As 3D-contacts may be established in an allele-specific manner, we describe an up-to-date allele-specific 4C-seq protocol, starting from the selection of allele-specific viewpoints to Illumina sequencing. This protocol has mainly been optimized for cultured mammalian cells, but can be adapted for other cell types with relatively minor changes in initial steps.

Chromosome Conformation Capture in Bacteria and Archaea.

The three-dimensional structure of the chromosome is encoded within its sequence and regulates activities such as replication and transcription. This necessitates the study of the spatial organization of the chromosome in relation to the underlying sequence. Chromosome conformation capture (3C) techniques are proximity ligation-based approaches that simplify the three-dimensional architecture of the chromosome into a one-dimensional library of hybrid ligation junctions. Deciphering the information contained in these libraries resolves chromosome architecture in a sequence-specific manner. This chapter describes the preparation of 3C libraries for bacteria and archaea. It details how the three-dimensional architecture of local chromatin can be extracted from the 3C library using qPCR (3C-qPCR), and it summarizes the processing of 3C libraries for next-generation sequencing (3C-Seq) for a study of global chromosome organization.

Single-cell Hi-C data analysis: safety in numbers.

Over the past decade, genome-wide assays for chromatin interactions in single cells have enabled the study of individual nuclei at unprecedented resolution and throughput. Current chromosome conformation capture techniques survey contacts for up to tens of thousands of individual cells, improving our understanding of genome function in 3D. However, these methods recover a small fraction of all contacts in single cells, requiring specialised processing of sparse interactome data. In this review, we highlight recent advances in methods for the interpretation of single-cell genomic contacts. After discussing the strengths and limitations of these methods, we outline frontiers for future development in this rapidly moving field.

High-throughput chromosome conformation capture-based analysis of higher-order chromatin structure in nasopharyngeal carcinoma.

BackgroundFirstly, we aimed to compare the differences of higher-order chromatin structure between nasopharyngeal carcinoma (NPC) and normal nasopharyngeal tissues. The second objective was to analyze the specific chromatin interaction site of NPC and the NPC-related genes regulated by this interaction site.MethodsWe included 6 NPC patients and 6 healthy controls to obtain the sequencing results of highest-throughput chromosome conformation capture (Hi-C) technique, followed by further analysis of the specific chromatin interaction sites in NPC.ResultsWe found an abnormal ultra-long distance interaction site on the chromosome 7p in the CNE210 sample, which was caused by a fusion gene SEPT7P2-PSPH. Additionally, a significant interaction site between chromosome 8q and 3p was revealed in the samples CNE25, CNE29, and CNE211, which was the interaction between 1.5 kb downstream of ASAP1 and 0.8 kb upstream of CTNNB1 gene. Further quantitative polymerase chain reaction (qPCR) revealed that ASAP1 and CTNNB1 genes were more highly expressed in CNE25, CNE29, and CNE211 than in the Np group, preliminarily indicating that this interaction site was likely related to the high expression of ASAP1 and CTNNB1 in NPC.ConclusionsThrough Hi-C analysis, we analyzed the specific chromatin interaction sites associated with NPC, and found the chromosomal translocation and chromatin interaction sites associated with NPC based on statistical analysis. This study has certain guiding significance for in-depth study of the mechanism of NPC occurrence and development.

Chromosome Modeling on Downsampled Hi-C Maps Enhances the Compartmentalization Signal.

The human genome is organized within a nucleus where chromosomes fold into an ensemble of different conformations. Chromosome conformation capture techniques such as Hi-C provide information about the genome architecture by creating a 2D heat map. Initially, Hi-C map experiments were performed in human interphase cell lines. Recently, efforts were expanded to several different organisms, cell lines, tissues, and cell cycle phases where obtaining high-quality maps is challenging. Poor sampled Hi-C maps present high sparse matrices where compartments located far from the main diagonal are difficult to observe. Aided by recently developed models for chromatin folding and dynamics investigation, we introduce a framework to enhance the compartments' information far from the diagonal observed in experimental sparse matrices. The simulations were performed using the Open-MiChroM platform aided by new trained parameters in the minimal chromatin model (MiChroM) energy function. The simulations optimized on a downsampled experimental map (10% of the original data) allow the prediction of a contact frequency similar to that of the complete (100%) experimental Hi-C. The modeling results open a discussion on how simulations and modeling can increase the statistics and help fill in some Hi-C regions not captured by poor sampling experiments. Open-MiChroM simulations allow us to explore the 3D genome organization of different organisms, cell lines, and cell phases that often do not produce high-quality Hi-C maps.

EnHiC: learning fine-resolution Hi-C contact maps using a generative adversarial framework

MotivationThe high-throughput chromosome conformation capture (Hi-C) technique has enabled genome-wide mapping of chromatin interactions. However, high-resolution Hi-C data requires costly, deep sequencing; therefore, it has only been achieved for a limited number of cell types. Machine learning models based on neural networks have been developed as a remedy to this problem.ResultsIn this work, we propose a novel method, EnHiC, for predicting high-resolution Hi-C matrices from low-resolution input data based on a generative adversarial network (GAN) framework. Inspired by non-negative matrix factorization, our model fully exploits the unique properties of Hi-C matrices and extracts rank-1 features from multi-scale low-resolution matrices to enhance the resolution. Using three human Hi-C datasets, we demonstrated that EnHiC accurately and reliably enhanced the resolution of Hi-C matrices and outperformed other GAN-based models. Moreover, EnHiC-predicted high-resolution matrices facilitated the accurate detection of topologically associated domains and fine-scale chromatin interactions.Availability and implementationEnHiC is publicly available at https://github.com/wmalab/EnHiC.Supplementary informationSupplementary data are available at Bioinformatics online.

Chromosome-scale genome assembly of the transformation-amenable common wheat cultivar 'Fielder'.

We have established a high-quality, chromosome-level genome assembly for the hexaploid common wheat cultivar ‘Fielder’, an American, soft, white, pastry-type wheat released in 1974 and known for its amenability to Agrobacterium tumefaciens-mediated transformation and genome editing. Accurate, long-read sequences were obtained using PacBio circular consensus sequencing with the HiFi approach. Sequence reads from 16 SMRT cells assembled using the hifiasm assembler produced assemblies with N50 greater than 20 Mb. We used the Omni-C chromosome conformation capture technique to order contigs into chromosome-level assemblies, resulting in 21 pseudomolecules with a cumulative size of 14.7 and 0.3 Gb of unanchored contigs. Mapping of published short reads from a transgenic wheat plant with an edited seed-dormancy gene, TaQsd1, identified four positions of transgene insertion into wheat chromosomes. Detection of guide RNA sequences in pseudomolecules provided candidates for off-target mutation induction. These results demonstrate the efficiency of chromosome-scale assembly using PacBio HiFi reads and their application in wheat genome-editing studies.

Chromosome-scale assembly of the Sparassis latifolia genome obtained using long-read and Hi-C sequencing.

Sparassis latifolia is a valuable edible mushroom cultivated in China. In 2018, our research group reported an incomplete and low-quality genome of S. latifolia obtained by Illumina HiSeq 2500 sequencing. These limitations in the available genome have constrained genetic and genomic studies in this mushroom resource. Herein, an updated draft genome sequence of S. latifolia was generated by Oxford Nanopore sequencing and the high-through chromosome conformation capture (Hi-C) technique. A total of 8.24 Gb of Oxford Nanopore long reads representing ∼198.08X coverage of the S. latifolia genome were generated. Subsequently, a high-quality genome of 41.41 Mb, with scaffold and contig N50 sizes of 3.31 and 1.51 Mb, respectively, was assembled. Hi-C scaffolding of the genome resulted in 12 pseudochromosomes containing 93.56% of the bases in the assembled genome. Genome annotation further revealed that 17.47% of the genome was composed of repetitive sequences. In addition, 13,103 protein-coding genes were predicted, among which 98.72% were functionally annotated. BUSCO assay results further revealed that there were 92.07% complete BUSCOs. The improved chromosome-scale assembly and genome features described here will aid further molecular elucidation of various traits, breeding of S. latifolia, and evolutionary studies with related taxa.

Diploid, triploid, tetraploid - chromatin organization in polyploid watermelon.

The first half of the 20th century was an important time for the scientific discipline of genetics in Japan (Iida, 2010). Hithoshi Kihara (1893–1986) was one of the pioneering researchers who helped to develop the field. He was the first to define the genome as the minimal set of chromosomes that is required for the fitness of a species and he also discovered sex chromosomes in plants. However, the achievement that made him internationally renowned was the development of seedless watermelon (Kihara, 1951). Kihara realized that triploid plants produce seedless fruits, which could be a desirable trait for certain crops. Therefore, he set out to create a triploid watermelon. To do this, he first obtained a tetraploid plant by treating a normal diploid with colchicine, a technique that was discovered shortly before Kiara’s watermelon experiment (Nebel, 1937). Next, he pollinated the tetraploid with pollen from a diploid. This yielded triploid progeny that did not produce seeds. Nowadays, seedless watermelons are still produced similarly. However, there is still a lot that we do not understand. The tetraploid progenitor of triploid watermelon exhibits many phenotypic differences as compared with their diploid counterparts. As yet, it remains enigmatic what causes these differences. Duplication of the genome might lead to rearrangements of the chromatin, because the nucleus needs to accommodate a doubled number of chromosomes. Although the exact effect of chromatin organization on gene regulation is not fully unraveled, it is clear that transcriptional activity is affected by the way the genome is folded (Doğan and Liu, 2018). Hence, altered chromatin organization might (partly) underlie the phenotypic differences in polyploids. In this issue, Marleny Garcia-Lozano and colleagues investigated the effect of genome doubling on chromatin conformation in watermelon. To understand the effect of doubling the number of chromosomes, the authors compared diploid watermelon with an isogenic tetraploid line. Compared to their diploid counterparts, the tetraploid plants had bigger seeds, bigger leaves, and a thicker stem with more and larger trichomes (Figure 1), phenotypes often seen in polyploids. To investigate how the chromatin is organized in diploid and tetraploid plants, the authors employed the Hi-C chromosome conformation capture technique. This technique crosslinks and ligates interacting chromatin fragments, followed by high-throughput sequencing, and is used to identify long-range physical interactions. They found that the interactions between genomic regions were different: in the tetraploid the chromosomes showed a higher number of interactions than in the diploid, especially between regions from different chromosomes. In addition to the chromatin interaction differences, the Hi-C results showed that the chromatin of tetraploid watermelon was organized differently than the chromatin in diploid plants. In eukaryotes, chromatin is divided into two spatially segregated compartments. The chromatin that occupies the interior nuclear space forms the transcriptionally active A compartment, whereas chromatin that resides at the nuclear periphery forms the less active B compartment. In tetraploid watermelon, several chromosomal regions had switched compartments. The authors found that chromatin organization also differed at lower levels of organization. Within the A and B compartments, chromatin is organized in megabase-sized clusters called topologically associated domains (TADs). TADs are demarcated by boundaries that function as insulators. As a result, DNA sequences within a TAD physically interact with each other more frequently than with sequences outside the TAD. The authors identified the boundaries of TADs in diploid and tetraploid watermelon and found that of 346 boundaries, 93 were differential between the two watermelon lines. Besides improved traits such as larger leaves, fruits, and flowers, polyploids can better deal with stressful environments. Therefore, polyploidy has played a key role in plant breeding for many years, and as climate change continues, polyploid crops might become increasingly important. Garcia-Lozano and colleagues found that the presence of a greater number of chromosomes alters the three-dimensional structure of the genome and affects interactions between genomic regions in watermelon. However, it is unclear how the altered chromatin organization affects the phenotype of watermelon after genome duplication. In addition to chromatin organization differences, the authors observed differences in the transcriptome and methylome between diploid and tetraploid watermelon. Seventy years after Kihara’s discovery, there is still a lot to learn about polyploid watermelon.