Functional Genomics Research Articles

Sensitivity of transcriptomics: Different samples and methodology alter conclusions in Gulf pipefish (Syngnathus scovelli).

Transcriptome analysis has become a central tool in evolutionary and functional genomics. However, variation among biological samples and analysis techniques can greatly influence results, potentially compromising insights into the phenomenon under study. Here, we evaluate differences in the brain transcriptome between female and male Gulf pipefish (Syngnathus scovelli). We perform comparisons between results from entire pipelines for brain transcriptome assembly, quantification, and analysis. We also offer a unique biological comparison between two sampling instances (Redfish Bay: n = 15, Port Lavaca: n = 7). Our results demonstrate crucial shortcomings with current experimental approaches. We found high variation within our results that was driven by both technical differences between pipelines and biological differences between pipefish samples. In our analysis of highly expressed genes, we found that the choice of methods influenced the degree of contamination or noise included in the identified genes. Notably, genes identified within the same pipeline were more similar than any other comparison. Our differential expression analysis revealed that both methodology and sampling location influenced the quantity and consistency of statistically significant transcripts. In the context of these results, we offer modifications to current practices that may increase the robustness of transcriptome-based conclusions. In particular, the use of a reference-guided assembly and an increase in sample sizes are likely to improve resistance to noise or error.

Interplay of human ABCC11 transporter gene variants with axillary skin microbiome functional genomics.

The human armpit microbiome is metabolically entangled with skin cell physiology. This "meta-organism" symbiotic mutualism results in sweat either with or without odor (osmidrosis), depending on host ABCC11 gene haplotypes. Apocrine metabolism produces odorless S-glutathione conjugate that is transferred by ABCC11 transporters into secretory vesicles, deglutamylated to S-Cys-Gly-3M3SH thiol, and exuded to skin surface. An anthropogenic clade of skin bacteria then takes up the thiol and bioconverts it to malodorous 3-methyl-3-sulfanylhexan-1-ol (3M3SH). We hypothesized a familial meta-organism association of human ABCC11 gene non-synonymous SNP rs17822931 interplaying with skin microbiome 3M3SH biosynthesis. Subjects were genotyped for ABCC11 SNPs, and their haplotypes were correlated with axilla microbiome DNA sequencing profiles and predicted metagenome functions. A multigeneration family pedigree revealed a Mendelian autosomal recessive pattern: the C allele of ABCC11 correlated with bacterial Cys-S-conjugate β-lyase (PatB) gene known for Staphylococcus hominis biosynthesis of 3M3SH from human precursor; PatB was rescinded in hosts with homozygous TT alleles encoding ABCC11 loss-of-function mutation. We posit that a C allele encoding functional ABCC11 is key to delivering host conjugate precursors that shape heritable skin niche conditions favorable to harboring Staphylococcus having genomics of odor thiol production. This provides existential insights into human evolution and global regional population ancestries.

Functional characteristics and computational model of abundant hyperactive loci in the human genome.

Enhancers and promoters are classically considered to be bound by a small set of transcription factors (TFs) in a sequence-specific manner. This assumption has come under increasing skepticism as the datasets of ChIP-seq assays of TFs have expanded. In particular, high-occupancy target (HOT) loci attract hundreds of TFs with often no detectable correlation between ChIP-seq peaks and DNA-binding motif presence. Here, we used a set of 1003 TF ChIP-seq datasets (HepG2, K562, H1) to analyze the patterns of ChIP-seq peak co-occurrence in combination with functional genomics datasets. We identified 43,891 HOT loci forming at the promoter (53%) and enhancer (47%) regions. HOT promoters regulate housekeeping genes, whereas HOT enhancers are involved in tissue-specific process regulation. HOT loci form the foundation of human super-enhancers and evolve under strong negative selection, with some of these loci being located in ultraconserved regions. Sequence-based classification analysis of HOT loci suggested that their formation is driven by the sequence features, and the density of mapped ChIP-seq peaks across TF-bound loci correlates with sequence features and the expression level of flanking genes. Based on the affinities to bind to promoters and enhancers we detected five distinct clusters of TFs that form the core of the HOT loci. We report an abundance of HOT loci in the human genome and a commitment of 51% of all TF ChIP-seq binding events to HOT locus formation thus challenging the classical model of enhancer activity and propose a model of HOT locus formation based on the existence of large transcriptional condensates.

Functional characteristics and computational model of abundant hyperactive loci in the human genome

Enhancers and promoters are classically considered to be bound by a small set of transcription factors (TFs) in a sequence-specific manner. This assumption has come under increasing skepticism as the datasets of ChIP-seq assays of TFs have expanded. In particular, high-occupancy target (HOT) loci attract hundreds of TFs with often no detectable correlation between ChIP-seq peaks and DNA-binding motif presence. Here, we used a set of 1003 TF ChIP-seq datasets (HepG2, K562, H1) to analyze the patterns of ChIP-seq peak co-occurrence in combination with functional genomics datasets. We identified 43,891 HOT loci forming at the promoter (53%) and enhancer (47%) regions. HOT promoters regulate housekeeping genes, whereas HOT enhancers are involved in tissue-specific process regulation. HOT loci form the foundation of human super-enhancers and evolve under strong negative selection, with some of these loci being located in ultraconserved regions. Sequence-based classification analysis of HOT loci suggested that their formation is driven by the sequence features, and the density of mapped ChIP-seq peaks across TF-bound loci correlates with sequence features and the expression level of flanking genes. Based on the affinities to bind to promoters and enhancers we detected five distinct clusters of TFs that form the core of the HOT loci. We report an abundance of HOT loci in the human genome and a commitment of 51% of all TF ChIP-seq binding events to HOT locus formation thus challenging the classical model of enhancer activity and propose a model of HOT locus formation based on the existence of large transcriptional condensates.

Multi-omics analysis reveals the positive impact of differential chloroplast activity during in vitro regeneration of barley.

Existence of potent in vitro regeneration system is a prerequisite for efficient genetic transformation and functional genomics of crop plants. In this study, two contrasting cultivars differencing in their in vitro regeneration efficiency were identified. Tissue culture friendly cultivar Golden Promise (GP) and tissue culture resistant DWRB91(D91) were selected as contrasting cultivars to investigate the molecular basis of regeneration efficiency through multiomics analysis. Transcriptomics analysis revealed 1487 differentially expressed genes (DEGs), in which 795 DEGs were upregulated and 692 DEGs were downregulated in the GP-D91 transcriptome. Genes encoding proteins localized in chloroplast and involved in ROS generation were upregulated in the embryogenic calli of GP. Moreover, proteome analysis by LC-MS/MS revealed 3062 protein groups and 16,989 peptide groups, out of these 1586 protein groups were differentially expressed proteins (DEPs). Eventually, GC-MS based metabolomics analysis revealed the higher activity of plastids and alterations in key metabolic processes such as sugar metabolism, fatty acid biosynthesis, and secondary metabolism. TEM analysis also revealed differential plastid development. Higher accumulation of sugars, amino acids and metabolites corresponding to lignin biosynthesis were observed in GP as compared to D91. A comprehensive examination of gene expression, protein profiling and metabolite patterns unveiled a significant increase in the genes encompassing various functions, such as ion homeostasis, chlorophyll metabolic process, ROS regulation, and the secondary metabolic pathway.

MolluscDB 2.0: a comprehensive functional and evolutionary genomics database for over 1400 molluscan species.

Mollusca represents the second-largest animal phylum but remains less explored genomically. The increase in high-quality genomes and diverse functional genomic data holds great promise for advancing our understanding of molluscan biology and evolution. To address the opportunities and challenges facing the molluscan research community in managing vast multi-omics resources, we developed MolluscDB 2.0 (http://mgbase.qnlm.ac), which integrates extensive functional genomic data and offers user-friendly tools for multilevel integrative and comparative analyses. MolluscDB 2.0 covers 1450 species across all eight molluscan classes and compiles ∼4200 datasets, making it the most comprehensive multi-omics resource for molluscs to date. MolluscDB 2.0 expands the layers of multi-omics data, including genomes, bulk transcriptomes, single-cell transcriptomes, proteomes, epigenomes and metagenomes. MolluscDB 2.0 also more than doubles the number of functional modules and analytical tools, updating 14 original modules and introducing 20 new, specialized modules. Overall, MolluscDB 2.0 provides highly valuable, open-access multi-omics platform for the molluscan research community, expediting scientific discoveries and deepening our understanding of molluscan biology and evolution.

Molecular Dynamics Simulations of Nucleosomes Containing Histone Variant H2A.J

Histone proteins form the building blocks of chromatin—nucleosomes. Incorporation of alternative histone variants instead of the major (canonical) histones into nucleosomes is a key mechanism enabling epigenetic regulation of genome functioning. In humans, H2A.J is a constitutively expressed histone variant whose accumulation is associated with cell senescence, inflammatory gene expression, and certain cancers. It is sequence-wise very similar to the canonical H2A histones, and its effects on the nucleosome structure and dynamics remain elusive. This study employed all-atom molecular dynamics simulations to reveal atomistic mechanisms of structural and dynamical effects conferred by the incorporation of H2A.J into nucleosomes. We showed that the H2A.J C-terminal tail and its phosphorylated form have unique dynamics and interaction patterns with the DNA, which should affect DNA unwrapping and the availability of nucleosomes for interactions with other chromatin effectors. The dynamics of the L1-loop and the hydrogen bonding patterns inside the histone octamer were shown to be sensitive to single amino acid substitutions, potentially explaining the higher thermal stability of H2A.J nucleosomes. Taken together, our study demonstrated unique dynamical features of H2A.J-containing nucleosomes, which contribute to further understanding of the molecular mechanisms employed by H2A.J in regulating genome functioning.

FunCoup 6: advancing functional association networks across species with directed links and improved user experience.

FunCoup 6 (https://funcoup.org) represents a significant advancement in global functional association networks, aiming to provide researchers with a comprehensive view of the functional coupling interactome. This update introduces novel methodologies and integrated tools for improved network inference and analysis. Major new developments in FunCoup 6 include vastly expanding the coverage of gene regulatory links, a new framework for bin-free Bayesian training and a new website. FunCoup 6 integrates a new tool for disease and drug target module identification using the TOPAS algorithm. To expand the utility of the resource for biomedical research, it incorporates pathway enrichment analysis using the ANUBIX and EASE algorithms. The unique comparative interactomics analysis in FunCoup provides insights of network conservation, now allowing users to align orthologs only or query each species network independently. Bin-free training was applied to 23 primary species, and in addition, networks were generated for all remaining 618 species in InParanoiDB 9. Accompanying these advancements, FunCoup 6 features a new redesigned website, together with updated API functionalities, and represents a pivotal step forward in functional genomics research, offering unique capabilities for exploring the complex landscape of protein interactions.

Active enhancers: recent research advances and insights into disease.

Precise regulation of gene expression is crucial to development. Enhancers, the core of gene regulation, determine the spatiotemporal pattern of gene transcription. Since many disease-associated mutations are characterized in enhancers, the research on enhancer will provide clues to precise medicine. Rapid advances in high-throughput sequencing technology facilitate the characterization of enhancers at genome wide, but understanding the functional mechanisms of enhancers remains challenging. Herein, we provide a panorama of enhancer characteristics, including epigenetic modifications, enhancer transcripts, and enhancer-promoter interaction patterns. Furthermore, we outline the applications of high-throughput sequencing technology and functional genomics methods in enhancer research. Finally, we discuss the role of enhancers in human disease and their potential as targets for disease prevention and treatment strategies.

Genome-Wide Profiling of Histone Modifications in Fission Yeast Using CUT&Tag.

Eukaryotic DNA is organized in the nucleus in the form of chromatin. Nucleosomes, the fundamental unit of chromatin, are subject to many posttranslational modifications (PTMs) as well as compositional variations through incorporation of histone variants. These alterations play important roles in regulation of genome structure and activity. Genome-wide profiling of these regulatory features is essential for understanding of genome function. Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-Seq) is a widely used method to assay genome-wide localization in fission yeast but suffers from the requirement for a large amount of input chromatin, antibodies, and a cumbersome experimental pipeline. New methods such as Cleavage Under Targets and Tagmentation (CUT&Tag), which combine the specificity of targeted cleavage and adapter insertion with the sensitivity of next-generation sequencing, enable identification and characterization of various epigenetic marks affording low input requirement as well as more streamlined protocols. However, these approaches have not been adapted for use in fission yeast, Schizosaccharomyces pombe. Here, we describe an adapted CUT&Tag protocol for epigenomic profiling in fission yeast using the heterochromatin-associated histone H3K9 methylation PTM for benchmarking.

Engineering structural variants to interrogate genome function.

Structural variation, such as deletions, duplications, inversions and complex rearrangements, can have profound effects on gene expression, genome stability, phenotypic diversity and disease susceptibility. Structural variants can encompass up to millions of bases and have the potential to rearrange substantial segments of the genome. They contribute considerably more to genetic diversity in human populations and have larger effects on phenotypic traits than point mutations. Until recently, our understanding of the effects of structural variants was driven mainly by studying naturally occurring variation. New genome-engineering tools capable of generating deletions, insertions, inversions and translocations, together with the discovery of new recombinases and advances in creating synthetic DNA constructs, now enable the design and generation of an extended range of structural variation. Here, we discuss these tools and examples of their application and highlight existing challenges that will need to be overcome to fully harness their potential.

Exploring the Role of Tools and Technologies in Enhancing Human Knowledge Acquisition: The Flora of Cambodia as a Pilot Project.

The globalization of the world profoundly impacts the academic and scientific domains. Technological advances have enabled an immeasurable progress in many scientific fields, for example structural or functional genomics, bioinformatics or even the development of artificial intelligence, which is becoming a catalyst for disciplines such as taxonomy. However, we are witnessing an unprecedented paradox. We have entered a period described as the 6th mass extinction (e.g., Ceballos et al. 2015, Ceballos et al. 2020, Cowie et al. 2022), where biodiversity has been in decline for many years because of anthropogenic activity (Myers et al. 2000, Sachs et al. 2009, Butchart et al. 2010) and yet, techniques have never been so advanced (e.g., online databases, metabarcording and eDNA identification methods). They produce a deluge of results and data that must be managed to facilitate their access and re-use and benefit to all users, i.e., scientists and stake-holders or policy makers on a global scale. Currently, the flora of Cambodia is part of the Flora of Cambodia, Laos and Viêt-nam*1 and is jointly edited by the Royal Botanic Garden of Edinburgh and the Muséum national d’Histoire naturelle in Paris since 2013. Given Cambodia's historical ties to French Indochina, France's long-term presence has significantly contributed to the documentation of Cambodia's biodiversity. The largest collection of Cambodian specimens is housed at the Paris Herbarium. The Flora of Cambodia project (Ung 2023) started in 2023 as a pilot project for testing the effectiveness of different open tools developped for taxonomists, e.g., the echinopscis platform*2 developped at the Royal Botanical Garden Kew; Bionomia*3; data made available online through platforms such as the Global Biodiversity Information Facility, Atlas of Living Australia, and World Flora Online. Other sources will also be used, like publications (Joyce et al. 2020) and "les herbonautes"*4, a citizen science platform. The short-term result is to produce a checklist of the vascular plants of Cambodia in multiple languages including Khmer. This project aligns with the FAIR (Findable, Accessible, Interoperable, Reusable) (Wilkinson et al. 2016) and CARE (Collective benefit, Authority to control, Responsability, Ethics) (Carroll et al. 2020) principles.

SoIR: a comprehensive Solanaceae information resource for comparative and functional genomic study.

The Solanaceae family, which includes economically important crops such as tomatoes, potatoes and peppers, has experienced a rapid expansion in genomic data due to advancements in sequencing technologies. However, existing databases are limited by incomplete species representation, a lack of comprehensive comparative genomic tools and the absence of systematic pan-genomic analyses. To address these gaps, we developed the Solanaceae Information Resource (SoIR, https://soir.bio2db.com), a comprehensive genomics database for the Solanaceae family. SoIR integrates genomic data from 81 species and transcriptomic data from 41 species, encompassing a total of 3908408 gene annotations derived from Gene Ontology, nonredundant protein, Pfam, Swiss-Prot and TrEMBL databases. The resource also includes 3437115 CRISPR guide sequences, 212395 transcription factorsand 19086 genes associated with methylation modification. In addition to species-specific analyses, SoIR provides extensive bioinformatics tools for investigating gene family evolution, phylogenetic relationships and karyotype reconstruction across 25 fully sequenced genomes. With advanced tools such as Blast, Synteny and Sequence Alignment, the platform provides users with interactive and intuitive visualizations for conducting cross-species comparative genomics. As the first comprehensive pan-genomic resource for the entire Solanaceae family, SoIR facilitates in-depth cross-species analysis, supporting global research initiatives in plant evolution, functional genomics and crop improvement.

DDDR-26. IDENTIFICATION OF MODULATORS OF IDH INHIBITOR SENSITIVITY INIDH-MUTATED DIFFUSE GLIOMAS

Abstract Driver mutations in IDH1 and IDH2 characterize a substantial proportion of lower-grade diffuse gliomas in adults. Mutated IDH molecules cause the accumulation of D-2-hydroxyglutarate (D-2-HG), which perturbs many cellular functions, including metabolism, epigenetic regulation, and the ability to differentiate. This is notably associated with DNA and histone hyper-methylation. Recently, the newly developed IDH inhibitor, vorasidenib, was shown to delay tumor progression and the need for additional treatment in the groundbreaking INDIGO clinical trial. Nevertheless, the responses to vorasidenib were variable between patients, and so far have been established only for less aggressive disease. In addition, it is unclear to what extent vorasidenib can cause tumor regression. This highlights the need to identify molecules and pathways that control IDH inhibitor sensitivity. Towards this goal, we generated a new mouse model that combines Idh1R132H and Trp53 loss-of-function, targeted to oligodendrocyte progenitors. All the mutant mice develop diffuse gliomas that recapitulate the cardinal features of the corresponding human disease. We established multiple cell lines from these tumors, which retained expression of mutant IDH. The cells robustly produced D-2-HG, which was concentration-dependently suppressed by vorasidenib. Accordingly, vorasidenib could lower DNA methylation, and induce the expression of mature glial cell markers. To identify determinants of vorasidenib sensitivity, we performed genome-wide loss-of-function CRISPR/Cas9 functional genomics screens. In those experiments, targeting genes associated with astrocyte differentiation or Notch signaling enhanced cell fitness in the presence of vorasidenib, consistent with the anticipated effects of the drug on inducing cell differentiation. Conversely, vorasidenib-treated cells were more sensitive to the loss of regulators of cell growth and metabolism, and of some epigenetic regulators. These studies point to strategies that may enhance the efficacy of IDH inhibitors for the treatment of IDH mutated gliomas.

Assessing Protein Surface-Based Scoring for Interpreting Genomic Variants

Clinical genomics sequencing is rapidly expanding the number of variants that need to be functionally elucidated. Interpreting genetic variants (i.e., mutations) usually begins by identifying how they affect protein-coding sequences. Still, the three-dimensional (3D) protein molecule is rarely considered for large-scale variant analysis, nor in analyses of how proteins interact with each other and their environment. We propose a standardized approach to scoring protein surface property changes as a new dimension for functionally and mechanistically interpreting genomic variants. Further, it directs hypothesis generation for functional genomics research to learn more about the encoded protein’s function. We developed a novel method leveraging 3D structures and time-dependent simulations to score and statistically evaluate protein surface property changes. We evaluated positive controls composed of eight thermophilic versus mesophilic orthologs and variants that experimentally change the protein’s solubility, which all showed large and statistically significant differences in charge distribution (p < 0.01). We scored static 3D structures and dynamic ensembles for 43 independent variants (23 pathogenic and 20 uninterpreted) across four proteins. Focusing on the potassium ion channel, KCNK9, the average local surface potential shifts were 0.41 kBT/ec with an average p-value of 1 × 10−2. In contrast, dynamic ensemble shifts averaged 1.15 kBT/ec with an average p-value of 1 × 10−5, enabling the identification of changes far from mutated sites. This study demonstrates that an objective assessment of how mutations affect electrostatic distributions of protein surfaces can aid in interpreting genomic variants discovered through clinical genomic sequencing.

The genome landscape of the Xinglong buffalo

BackgroundXinglong buffalo, as an indigenous breed in Hainan province of China, possesses characteristics such as high humidity tolerance, disease resistance and high reproductive capacity. Combined with whole genome sequencing technology, comprehensive investigation can be undertaken to elucidate the genomic characteristics, functions and genetic variation of Xinglong buffalo population.ResultsXinglong buffalo has the highest genetic diversity, lowest runs of homozygosity average length, and fasted decay of linkage disequilibrium in our study population. Phylogenetic tree results revealed that Xinglong buffalo was gathered together with Fuzhong buffalo firstly. The population genetic structure analysis indicates that at K = 3, the Xinglong buffalo for the first time showed a distinct ancestral origin from other water buffalo. Furthermore, compared to different populations, candidate genes displaying significantly distinct patterns of single nucleotide polymorphisms (SNPs) (e.g., RYR2, COX15, PCDH9, DTWD2, FCRL5) distribution have been identified in the Xinglong buffalo.ConclusionsBased on the whole genome sequencing data, this study identified a substantial number of SNPs and assessed the genetic diversity and selection signatures within the Xinglong buffalo population. These results contribute to understanding the genomic characteristics of Xinglong buffalo and their genetic evolutionary status. However, the practical significance of these signatures for genetic enhancement still requires confirmation through additional samples and further experimental validation.

Functional genomics of human skeletal development and the patterning of height heritability.

Underlying variation in height are regulatory changes to chondrocytes, cartilage cells comprising long-bone growth plates. Currently, we lack knowledge on epigenetic regulation and gene expression of chondrocytes sampled across the human skeleton, and therefore we cannot understand basic regulatory mechanisms controlling height biology. We first rectify this issue by generating extensive epigenetic and transcriptomic maps from chondrocytes sampled from different growth plates across developing human skeletons, discovering novel regulatory networks shaping human bone/joint development. Next, using these maps in tandem with height genome-wide association study (GWAS) signals, we disentangle the regulatory impacts that skeletal element-specific versus global-acting variants have on skeletal growth, revealing the prime importance of regulatory pleiotropy in controlling height variation. Finally, as height is highly heritable, and thus often the test case for complex-trait genetics, we leverage these datasets within a testable omnigenic model framework to discover novel chondrocyte developmental modules and peripheral-acting factors shaping height biology and skeletal growth.

The design and engineering of synthetic genomes.

Synthetic genomics seeks to design and construct entire genomes to mechanistically dissect fundamental questions of genome function and to engineer organisms for diverse applications, including bioproduction of high-value chemicals and biologics, advanced cell therapies, and stress-tolerant crops. Recent progress has been fuelled by advancements in DNA synthesis, assembly, delivery and editing. Computational innovations, such as the use of artificial intelligence to provide prediction of function, also provide increasing capabilities to guide synthetic genome design and construction. However, translating synthetic genome-scale projects from idea to implementation remains highly complex. Here, we aim to streamline this implementation process by comprehensively reviewing the strategies for design, construction, delivery, debugging and tailoring of synthetic genomes as well as their potential applications.

Quantifying defective and wild-type viruses from high-throughput RNA sequencing.

Defective viral genomes (DVGs) are variants of the wild-type (wt) virus that lack the ability to complete autonomously an infectious cycle. However, in the presence of their parental (helper) wt virus, DVGs can interfere with the replication, encapsidation and spread of functional genomes, acting as a significant selective force in viral evolution. DVGs also affect the host's immune responses and are linked to chronic infections and milder symptoms. Thus, identifying and characterizing DVGs is crucial for understanding infection prognosis. Quantifying DVGs is challenging due to their inability to sustain themselves, which makes it difficult to distinguish them from the helper virus, especially using high-throughput RNA sequencing (RNA-seq). An accurate quantification is essential for understanding their very dynamical interactions with the helper virus. We present a method to simultaneously estimate the abundances of DVGs and wt genomes within a sample by identifying genomic regions with significant deviations from the expected sequencing depth. Our approach involves reconstructing the depth profile through a linear system of equations, which provides an estimate of the number of wt and DVG genomes of each type. Until now, in silico methods have only estimated the DVG-to-wt ratio for localized genomic regions. This is the first method that simultaneously estimates the proportions of wt and DVGs genome wide from short-reads RNA sequencing. The MATLAB code and the synthetic datasets are freely available at https://github.com/jmusan/wtDVGquantific.

Targeting Noncoding cis-Regulatory Elements for Cancer Therapy in the Context of the 3D Genome.

Significant efforts have been made to identify and validate oncoproteins and ncRNAs as therapeutic targets for cancer therapy; however, emerging observations suggest that noncoding cis-regulatory elements, which orchestrate the 3D organization of the genome and thus the transcriptional landscape, are potential therapeutic targets as well. In this commentary, we envisage that further efforts to decipher the noncoding cis-regulatory code and performing systematic surveys of functional noncoding cis-regulatory elements and recurrent 3D genome alterations in both cancerous and nonmalignant cells within tumor tissues will pave the way to the development of novel therapeutic strategies.