3D Genome Research Articles

Disorganized chromatin hierarchy and stem cell aging in a male patient of atypical laminopathy-based progeria mandibuloacral dysplasia type A

Studies of laminopathy-based progeria offer insights into aging-associated diseases and highlight the role of LMNA in chromatin organization. Mandibuloacral dysplasia type A (MAD) is a largely unexplored form of atypical progeria that lacks lamin A post-translational processing defects. Using iPSCs derived from a male MAD patient carrying homozygous LMNA p.R527C, premature aging phenotypes are recapitulated in multiple mesenchymal lineages, including mesenchymal stem cells (MSCs). Comparison with 26 human aging MSC expression datasets reveals that MAD-MSCs exhibit the highest similarity to senescent primary human MSCs. Lamina-chromatin interaction analysis reveals reorganization of lamina-associating domains (LADs) and repositioning of non-LAD binding peaks may contribute to the observed accelerated senescence. Additionally, 3D genome organization further supports hierarchical chromatin disorganization in MAD stem cells, alongside dysregulation of genes involved in epigenetic modification, stem cell fate maintenance, senescence, and geroprotection. Together, these findings suggest LMNA missense mutation is linked to chromatin alterations in an atypical progeroid syndrome.

Comparative study of the three-dimensional genomes of granulosa cells in germinal vesicle and metaphase II follicles.

Follicle development is a critical process in the female reproductive system, with significant implications for fertility and reproductive health. Germinal vesicle (GV) oocytes are primary oocytes that are arrested in the dictyate stage, also known as the diplotene stage of meiotic prophase I. Metaphase II (MII) is the stage at which the oocyte is typically retrieved for assisted reproductive technologies such as in vitro fertilization (IVF). The granulosa cells play a pivotal role in follicle development processes. 3D chromatin organization is a fundamental aspect of cellular biology that has significant implications for gene regulation and cellular function. In this study, we investigated 3D chromatin organization in granulosacells from GV and MII follicles, which is essential for understanding the regulatory mechanisms governing oocyte development. The results revealed distinct compartmentalization patterns,including stable genomic regions and transitions during oocyte maturation. Notably, there was a significant shift in functional gene activation, particularly in processes related to hormone metabolic pathways. Furthermore, alterations in topologically associating domains (TADs) were observed, with differential expression observed in genes that are involved in crucial biological processes. The analysis also identified a subset of genes with altered promoter-enhancer interactions (PEIs), reflecting a regulatory shift in gene expression related to reproductive processes. These findings provide valuable insights into 3D genome organization in granulosa cells with implications for reproductive health and the development of assisted reproductive technologies. Understanding spatial genome organization at different stages of follicular development may help realize novel strategies for enhancing success rates in assisted reproductive technologies.

Enhancers and genome conformation provide complex transcriptional control of a herpesviral gene.

Complex transcriptional control is a conserved feature of both eukaryotes and the viruses that infect them. Despite viral genomes being smaller and more gene dense than their hosts, we generally lack a sense of scope for the features governing the transcriptional output of individual viral genes. Even having a seemingly simple expression pattern does not imply that a gene's underlying regulation is straightforward. Here, we illustrate this by combining high-density functional genomics, expression profiling, and viral-specific chromosome conformation capture to define with unprecedented detail the transcriptional regulation of a single gene from Kaposi's sarcoma-associated herpesvirus (KSHV). We used as our model KSHV ORF68 - which has simple, early expression kinetics and is essential for viral genome packaging. We first identified seven cis-regulatory regions involved in ORF68 expression by densely tiling the ~154 kb KSHV genome with dCas9 fused to a transcriptional repressor domain (CRISPRi). A parallel Cas9 nuclease screen indicated that three of these regions act as promoters of genes that regulate ORF68. RNA expression profiling demonstrated that three more of these regions act by either repressing or enhancing other distal viral genes involved in ORF68 transcriptional regulation. Finally, we tracked how the 3D structure of the viral genome changes during its lifecycle, revealing that these enhancing regulatory elements are physically closer to their targets when active, and that disrupting some elements caused large-scale changes to the 3D genome. These data enable us to construct a complete model revealing that the mechanistic diversity of this essential regulatory circuit matches that of human genes.

Correction: ProbC: joint modeling of epigenome and transcriptome effects in 3D genome

Correction: ProbC: joint modeling of epigenome and transcriptome effects in 3D genome

Multiscale 3D genome rewiring during PTF1A-mediated somatic cell reprogramming into neural stem cells

The genome is intricately folded into chromatin compartments, topologically associating domains (TADs) and loops unique to each cell type. How this higher-order genome organization regulates cell fate transition remains elusive. Here we show how a single non-neural progenitor transcription factor, PTF1A, reorchestrates the 3D genome during fibroblast transdifferentiation into neural stem cells (NSCs). Multiomics analyses integrating Hi-C data, PTF1A and CTCF DNA-binding profiles, H3K27ac modification, and gene expression, demonstrate that PTF1A binds to subTAD boundaries subsequently associated with elevated CTCF binding and enhanced boundary insulation, and reorganizes chromatin loops, leading to gene expression changes that drive transdifferentiation into NSCs. Moreover, PTF1A activates enhancers and super-enhancers near low-insulation boundaries and modulates H3K27ac deposition, promoting cell fate transitions. Together, our data implicate an involvement of 3D genome in transcriptional and cell fate alterations, and highlight an essential role for PTF1A in gene expression control and multiscale 3D genome remodeling during cell reprogramming.

"Transcriptional Regulation of human NMNAT2: Insights from 3D Genome Sequencing and Bioinformatics".

Nicotinamide mononucleotide adenylyl transferases 2 (NMNAT2) is a crucial nicotinamide adenine dinucleotide (NAD)-synthesizing enzyme essential for neuronal health. In the Religious Orders Study/Memory and Aging Project (ROSMAP), human brain levels of NMNAT2 mRNA positively correlated with cognitive capabilities in older adults. NMNAT2 mRNA abundance is significantly reduced following various insults or proteinopathies. To elucidate the transcriptional regulation of NMNAT2, we employed circular chromosome conformation capture followed by high-throughput sequencing (4C-seq) to identify potential NMNAT2 enhancer and silencer regions by determining genomic regions interacting with the NMNAT2 promoter in human SH-SY5Y cells. We discovered distinct NMNAT2 promoter interactomes in undifferentiated versus neuron-like SH-SY5Y cells. Utilizing bioinformatics analyses, we identified putative transcriptional factors and NMNAT2-associated genes. Notably, the mRNA levels of many of these genes showed a significant correlation with NMNAT2 mRNA levels in ∼400 single-nuclei RNA-seq datasets from ROSMAP. Additionally, using CRISPR-Cas9 strategies, we confirmed the requirement of two specific genomic regions within the interactomes and four transcription factors in regulating NMNAT2 transcription. In summary, our study identifies genomic loci containing NMNAT2 regulatory elements and predicts associated genes and transcription factors through computational analyses.

A mini-review of single-cell Hi-C embedding methods

Single-cell Hi-C (scHi-C) techniques have significantly advanced our understanding of the 3D genome organization, providing crucial insights into the spatial genome architecture within individual nuclei. Numerous computational and statistical methods have been developed to analyze scHi-C data, with embedding methods playing a key role. Embedding reduces the dimensionality of complex scHi-C contact maps, making it easier to extract biologically meaningful patterns. These methods not only enhance cell clustering based on chromatin structures but also facilitate visualization and other downstream analyses. Most scHi-C embedding methods incorporate strategies such as normalization and imputation to address the inherent sparsity of scHi-C data, thereby further improving data quality and interpretability. In this review, we systematically examine the existing methods designed for scHi-C embedding, outlining their methodologies and discussing their capabilities in handling normalization and imputation. Additionally, we present a comprehensive benchmarking analysis to compare both embedding techniques and their clustering performances. This review serves as a practical guide for researchers seeking to select suitable scHi-C embedding tools, ultimately contributing to the understanding of the 3D organization of the genome.

3D chromatin maps of a brown alga reveal U/V sex chromosome spatial organization.

Nuclear three dimensional (3D) folding of chromatin structure has been linked to gene expression regulation and correct developmental programs, but little is known about the 3D architecture of sex chromosomes within the nucleus, and how that impacts their role in sex determination. Here, we determine the sex-specific 3D organization of the model brown alga Ectocarpus chromosomes at 2 kb resolution, by mapping long-range chromosomal interactions using Hi-C coupled with Oxford Nanopore long reads. We report that Ectocarpus interphase chromatin exhibits a non-Rabl conformation, with strong contacts among telomeres and among centromeres, which feature centromere-specific LTR retrotransposons. The Ectocarpus chromosomes do not contain large local interactive domains that resemble TADs described in animals, but their 3D genome organization is largely shaped by post-translational modifications of histone proteins. We show that the sex determining region (SDR)within the U and V chromosomes are insulated and span the centromeres andwe link sex-specific chromatin dynamics and gene expression levels to the 3D chromatin structure of the U and V chromosomes. Finally, we uncover the unique conformation of a large genomic region on chromosome 6 harboring an endogenous viral element, providing insights regarding the impact of a latent giant dsDNA virus on the host genome's 3D chromosomal folding.

EXPRESSO: a multi-omics database to explore multi-layered 3D genomic organization.

The three-dimensional (3D) organization of the human genome plays a crucial role in gene regulation. EXPloration of Regulatory Epigenome with Spatial and Sequence Observations (EXPRESSO) is a novel multi-omics database for exploration and visualization of multi-layered 3D genomic features across 46 different human tissues. Integrating 1360 3D genomic datasets (Hi-C, HiChIP, ChIA-PET) and 842 1D genomic and transcriptomic datasets (ChIP-seq, ATAC-seq, RNA-seq) from the same biosample, EXPRESSO provides a comprehensive resource for studying the interplay between 3D genome architecture and transcription regulation. This database offers diverse 3D genomic feature types (compartments, contact matrix, contact domains, stripes as diagonal lines extending from a genomic locus in contact matrix, chromatin loops, etc.) and user-friendly interface for both data exploration and download. Other key features include REpresentational State Transfer application programming interfaces for programmatic access, advanced visualization tools for 3D genomic features and web-based applications that correlate 3D genomic features with gene expression and epigenomic modifications. By providing extensive datasets and tools, EXPRESSO aims to deepen our understanding of 3D genomic architecture and its implications for human health and disease, serving as a vital resource for the research community. EXPRESSO is freely available at https://expresso.sustech.edu.cn.

Disparate pathways for extrachromosomal DNA biogenesis and genomic DNA repair.

Oncogene amplification on extrachromosomal DNA (ecDNA) is a pervasive driver event in cancer, yet our understanding of how ecDNA forms is limited. Here, we couple a CRISPR-based method for ecDNA induction with extensive characterization of newly formed ecDNA to examine their biogenesis. We find that DNA circularization is efficient, irrespective of 3D genome context, with formation of 800kb, 1 Mb, and 1.8 Mb ecDNAs reaching or exceeding 15%. We show non-homologous end joining and microhomology-mediated end joining both contribute to ecDNA formation, while inhibition of DNA-PKcs and ATM have opposing impacts on ecDNA formation. EcDNA and the corresponding chromosomal excision scar can form at significantly different rates and respond differently to DNA-PKcs and ATM inhibition. Taken together, our results support a model of ecDNA formation in which double strand break ends dissociate from their legitimate ligation partners prior to joining of illegitimate ends to form the ecDNA and excision scar.

Single-cell DNA methylome and 3D genome atlas of the human subcutaneous adipose tissue.

Human subcutaneous adipose tissue (SAT) contains a diverse array of cell-types; however, the epigenomic landscape among the SAT cell-types has remained elusive. Our integrative analysis of single-cell resolution DNA methylation and chromatin conformation profiles (snm3C-seq), coupled with matching RNA expression (snRNA-seq), systematically cataloged the epigenomic, 3D topology, and transcriptomic dynamics across the SAT cell-types. We discovered that the SAT CG methylation (mCG) landscape is characterized by pronounced hyper-methylation in myeloid cells and hypo-methylation in adipocytes and adipose stem and progenitor cells (ASPCs), driving nearly half of the 705,063 detected differentially methylated regions (DMRs). In addition to the enriched cell-type-specific transcription factor binding motifs, we identified TET1 and DNMT3A as plausible candidates for regulating cell-type level mCG profiles. Furthermore, we observed that global mCG profiles closely correspond to SAT lineage, which is also reflected in cell-type-specific chromosome compartmentalization. Adipocytes, in particular, display significantly more short-range chromosomal interactions, facilitating the formation of complex local 3D genomic structures that regulate downstream transcriptomic activity, including those associated with adipogenesis. Finally, we discovered that variants in cell-type level DMRs and A compartments significantly predict and are enriched for variance explained in abdominal obesity. Together, our multimodal study characterizes human SAT epigenomic landscape at the cell-type resolution and links partitioned polygenic risk of abdominal obesity to SAT epigenome.

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.

ChromaFold predicts the 3D contact map from single-cell chromatin accessibility

Identifying cell-type-specific 3D chromatin interactions between regulatory elements can help decipher gene regulation and interpret disease-associated non-coding variants. However, achieving this resolution with current 3D genomics technologies is often infeasible given limited input cell numbers. We therefore present ChromaFold, a deep learning model that predicts 3D contact maps, including regulatory interactions, from single-cell ATAC sequencing (scATAC-seq) data alone. ChromaFold uses pseudobulk chromatin accessibility, co-accessibility across metacells, and a CTCF motif track as inputs and employs a lightweight architecture to train on standard GPUs. Trained on paired scATAC-seq and Hi-C data in human samples, ChromaFold accurately predicts the 3D contact map and peak-level interactions across diverse human and mouse test cell types. Compared to leading contact map prediction models that use ATAC-seq and CTCF ChIP-seq, ChromaFold achieves state-of-the-art performance using only scATAC-seq. Finally, fine-tuning ChromaFold on paired scATAC-seq and Hi-C in a complex tissue enables deconvolution of chromatin interactions across cell subpopulations.

Development and evolution of Drosophila chromatin landscape in a 3D genome context

Little is known about how the epigenomic states change during development and evolution in a 3D genome context. Here we use Drosophila pseudoobscura with complex turnover of sex chromosomes as a model to address this, by collecting massive epigenomic and Hi-C data from five developmental stages and three adult tissues. We reveal that over 60% of the genes and transposable elements (TE) exhibit at least one developmental transition of chromatin state. Transitions on specific but not housekeeping enhancers are associated with specific chromatin loops and topologically associated domain borders (TABs). While evolutionarily young TEs are generally silenced, old TEs more often have been domesticated as interacting TABs or specific enhancers. But on the recently evolved X chromosome, young TEs are instead often active and recruited as TABs, due to acquisition of dosage compensation. Overall we characterize how Drosophila epigenomic landscapes change during development and in response to chromosome evolution, and highlight the important roles of TEs in genome organization and regulation.

Global loss of promoter–enhancer connectivity and rebalancing of gene expression during early colorectal cancer carcinogenesis

Although three-dimensional (3D) genome architecture is crucial for gene regulation, its role in disease remains elusive. We traced the evolution and malignant transformation of colorectal cancer (CRC) by generating high-resolution chromatin conformation maps of 33 colon samples spanning different stages of early neoplastic growth in persons with familial adenomatous polyposis (FAP). Our analysis revealed a substantial progressive loss of genome-wide cis-regulatory connectivity at early malignancy stages, correlating with nonlinear gene regulation effects. Genes with high promoter–enhancer (P–E) connectivity in unaffected mucosa were not linked to elevated baseline expression but tended to be upregulated in advanced stages. Inhibiting highly connected promoters preferentially represses gene expression in CRC cells compared to normal colonic epithelial cells. Our results suggest a two-phase model whereby neoplastic transformation reduces P–E connectivity from a redundant state to a rate-limiting one for transcriptional levels, highlighting the intricate interplay between 3D genome architecture and gene regulation during early CRC progression.

A 3D genome view of colon cancer initiation.

A 3D genome view of colon cancer initiation.

Integrative 3D genomics with multi-omics analysis and functional validation of genetic regulatory mechanisms of abdominal fat deposition in chickens

Chickens are the most abundant agricultural animals globally, with controlling abdominal fat deposition being a key objective in poultry breeding. While GWAS can identify genetic variants associated with abdominal fat deposition, the precise roles and mechanisms of these variants remain largely unclear. Here, we use male chickens from two lines divergently selected for abdominal fat deposition as experimental models. Through the integration of genomic, epigenomic, 3D genomic, and transcriptomic data, we build a comprehensive chromatin 3D regulatory network map to identify the genetic regulatory mechanisms that influence abdominal fat deposition in chickens. Notably, we find that the rs734209466 variant functions as an allele-specific enhancer, remotely enhancing the transcription of IGFBP2 and IGFBP5 by the binding transcription factor IRF4. This interaction influences the differentiation and proliferation of preadipocytes, which ultimately affects phenotype. This work presents a detailed genetic regulatory map for chicken abdominal fat deposition, offering molecular targets for selective breeding.

An integrated view of the structure and function of the human 4D nucleome.

The dynamic three-dimensional (3D) organization of the human genome (the "4D Nucleome") is closely linked to genome function. Here, we integrate a wide variety of genomic data generated by the 4D Nucleome Project to provide a detailed view of human 3D genome organization in widely used embryonic stem cells (H1-hESCs) and immortalized fibroblasts (HFFc6). We provide extensive benchmarking of 3D genome mapping assays and integrate these diverse datasets to annotate spatial genomic features across scales. The data reveal a rich complexity of chromatin domains and their sub-nuclear positions, and over one hundred thousand structural loops and promoter-enhancer interactions. We developed 3D models of population-based and individual cell-to-cell variation in genome structure, establishing connections between chromosome folding, nuclear organization, chromatin looping, gene transcription, and DNA replication. We demonstrate the use of computational methods to predict genome folding from DNA sequence, uncovering potential effects of genetic variants on genome structure and function. Together, this comprehensive analysis contributes insights into human genome organization and enhances our understanding of connections between the regulation of genome function and 3D genome organization in general.

Cohesin and CTCF emerge as building blocks of 3D genome structure.

Cohesin and CTCF emerge as building blocks of 3D genome structure.

Image-based 3D genomics through chromatin tracing

Image-based 3D genomics through chromatin tracing