Systematic functional characterization of antisense eRNA of protocadherin α composite enhancer.

Enhancers generate bidirectional noncoding enhancer RNAs (eRNAs) that may regulate gene expression. At present, the eRNA function remains enigmatic. Here, we report a 5' capped antisense eRNA PEARL (Pcdh eRNA associated with R-loop formation) that is transcribed from the protocadherin (Pcdh) α HS5-1 enhancer region. Through loss- and gain-of-function experiments with CRISPR/Cas9 DNA fragment editing, CRISPRi, and CRISPRa, as well as locked nucleic acid strategies, in conjunction with ChIRP, MeDIP, DRIP, QHR-4C, and HiChIP experiments, we found that PEARL regulates Pcdhα gene expression by forming local RNA-DNA duplexes (R-loops) in situ within the HS5-1 enhancer region to promote long-distance chromatin interactions between distal enhancers and target promoters. In particular, increased levels of eRNA PEARL via perturbing transcription elongation factor SPT6 lead to strengthened local three-dimensional chromatin organization within the Pcdh superTAD. These findings have important implications regarding molecular mechanisms by which the HS5-1 enhancer regulates stochastic Pcdhα promoter choice in single cells in the brain.

Reprogramming of Meiotic Chromatin Architecture during Spermatogenesis.

Chromatin organization undergoes drastic reconfiguration during gametogenesis. However, the molecular reprogramming of three-dimensional chromatin structure in this process remains poorly understood for mammals, including primates. Here, we examined three-dimensional chromatin architecture during spermatogenesis in rhesus monkey using low-input Hi-C. Interestingly, we found that topologically associating domains (TADs) undergo dissolution and reestablishment in spermatogenesis. Strikingly, pachytene spermatocytes, where synapsis occurs, are strongly depleted for TADs despite their active transcription state but uniquely show highly refined local compartments that alternate between transcribing and non-transcribing regions (refined-A/B). Importantly, such chromatin organization is conserved in mouse, where it remains largely intact upon transcription inhibition. Instead, it is attenuated in mutant spermatocytes, where the synaptonemal complex failed to be established. Intriguingly, this is accompanied by the restoration of TADs, suggesting that the synaptonemal complex may restrict TADs and promote local compartments. Thus, these data revealed extensive reprogramming of higher-order meiotic chromatin architecture during mammalian gametogenesis.

Hi-C as a molecular rangefinder to examine genomic rearrangements.

The mammalian genome is highly packed into the nucleus. Over the past decade, the development of Hi-C has contributed significantly to our understanding of the three-dimensional (3D) chromatin structure, uncovering the principles and functions of higher-order chromatin organizations. Recent studies have repositioned its property in spatial proximity measurement to address challenging problems in genome analyses including genome assembly, haplotype phasing, and the detection of genomic rearrangements. In particular, the power of Hi-C in detecting large-scale structural variations (SVs) in the cancer genome has been demonstrated, which is challenging to be addressed solely with short-read-based whole-genome sequencing analyses. In this review, we first provide a comprehensive view of Hi-C as an intuitive and effective SV detection tool. Then, we introduce recently developed bioinformatics tools utilizing Hi-C to investigate genomic rearrangements. Finally, we discuss the potential application of single-cell Hi-C to address the heterogeneity of genomic rearrangements and sub-population identification in the cancer genome.

Three-Dimensional Gene Regulation Network in Glioblastoma Ferroptosis.

Ferroptosis is an iron-dependent form of cell death, which is reported to be associated with glioma progression and drug sensitivity. Targeting ferroptosis is a potential therapeutic approach for glioma. However, the molecular mechanism of glioma cell ferroptosis is not clear. In this study, we profile the change of 3D chromatin structure in glioblastoma ferroptosis by using HiChIP and study the 3D gene regulation network in glioblastoma ferroptosis. A combination of an analysis of HiChIP and RNA-seq data suggests that change of chromatin loops mediated by 3D chromatin structure regulates gene expressions in glioblastoma ferroptosis. Genes that are regulated by 3D chromatin structures include genes that were reported to function in ferroptosis, like HDM2 and TXNRD1. We propose a new regulatory mechanism governing glioblastoma cell ferroptosis by 3D chromatin structure.

The three-dimensional genome: regulating gene expression during pluripotency and development.

The precise expression of genes in time and space during embryogenesis is largely influenced by communication between enhancers and promoters, which is propagated and governed by the physical proximity of these elements in the nucleus. Here, we review how chromatin domains organize the genome by guiding enhancers to their target genes thereby preventing non-specific interactions with other neighboring regions. We also discuss the dynamics of chromatin interactions between enhancers and promoters, as well as the consequent changes in gene expression, that occur in pluripotent cells and during development. Finally, we evaluate how genomic changes such as deletions, inversions and duplications affect 3D chromatin configuration overall and lead to ectopic enhancer-promoter contacts, and thus gene misexpression, which can contribute to abnormal development and disease.

Epigenetic and Transcriptional Variability Shape Phenotypic Plasticity.

Epigenetic and transcriptional variability contribute to the vast diversity of cellular and organismal phenotypes and are key in human health and disease. In this review, we describe different types, sources, and determinants of epigenetic and transcriptional variability, enabling cells and organisms to adapt and evolve to a changing environment. We highlight the latest research and hypotheses on how chromatin structure and the epigenome influence gene expression variability. Further, we provide an overview of challenges in the analysis of biological variability. An improved understanding of the molecular mechanisms underlying epigenetic and transcriptional variability, at both the intra- and inter-individual level, provides great opportunity for disease prevention, better therapeutic approaches, and personalized medicine.

A new class of constitutively active super-enhancers is associated with fast recovery of 3D chromatin loops.

BACKGROUND: Super-enhancers or stretch enhancers are clusters of active enhancers that often coordinate cell-type specific gene regulation during development and differentiation. In addition, the enrichment of disease-associated single nucleotide polymorphism in super-enhancers indicates their critical function in disease-specific gene regulation. However, little is known about the function of super-enhancers beyond gene regulation. RESULTS: In this study, through a comprehensive analysis of super-enhancers in 30 human cell/tissue types, we identified a new class of super-enhancers which are constitutively active across most cell/tissue types. These 'common' super-enhancers are associated with universally highly expressed genes in contrast to the canonical definition of super-enhancers that assert cell-type specific gene regulation. In addition, the genome sequence of these super-enhancers is highly conserved by evolution and among humans, advocating their universal function in genome regulation. Integrative analysis of 3D chromatin loops demonstrates that, in comparison to the cell-type specific super-enhancers, the cell-type common super-enhancers present a striking association with rapidly recovering loops. CONCLUSIONS: In this study, we propose that a new class of super-enhancers may play an important role in the early establishment of 3D chromatin structure.

Chromatin Conformation Links Distal Target Genes to CKD Loci.

Genome-wide association studies (GWASs) have identified many genetic risk factors for CKD. However, linking common variants to genes that are causal for CKD etiology remains challenging. By adapting self-transcribing active regulatory region sequencing, we evaluated the effect of genetic variation on DNA regulatory elements (DREs). Variants in linkage with the CKD-associated single-nucleotide polymorphism rs11959928 were shown to affect DRE function, illustrating that genes regulated by DREs colocalizing with CKD-associated variation can be dysregulated and therefore, considered as CKD candidate genes. To identify target genes of these DREs, we used circular chromosome conformation capture (4C) sequencing on glomerular endothelial cells and renal tubular epithelial cells. Our 4C analyses revealed interactions of CKD-associated susceptibility regions with the transcriptional start sites of 304 target genes. Overlap with multiple databases confirmed that many of these target genes are involved in kidney homeostasis. Expression quantitative trait loci analysis revealed that mRNA levels of many target genes are genotype dependent. Pathway analyses showed that target genes were enriched in processes crucial for renal function, identifying dysregulated geranylgeranyl diphosphate biosynthesis as a potential disease mechanism. Overall, our data annotated multiple genes to previously reported CKD-associated single-nucleotide polymorphisms and provided evidence for interaction between these loci and target genes. This pipeline provides a novel technique for hypothesis generation and complements classic GWAS interpretation. Future studies are required to specify the implications of our dataset and further reveal the complex roles that common variants have in complex diseases, such as CKD.

[Attraction of Likenesses: Mechanisms of Self-Association and Compartmentalization of Eukaryotic Chromatin].

Chromatin packing in eukaryotic chromosomes has been traditionally viewed as a hierarchical process, in which nucleosome chains fold into helical chromatin fibers. These fibers would then fold into more complex regular structures. However, recent chromatin imaging studies and analyses of chromosomal DNA contacts within the 3D space of the cell nucleus have necessitated a radical revision of the hierarchical chromatin packing model. According to the new studies, the nucleosome chain has a free spatial configuration without regular helical fibers in most cell types. The overall 3D organization of DNA in the cell nucleus includes chromatin loops and contact domains of up to several million base pairs in size. During cell differentiation, individual structure-functional chromatin domains marked by similar types of histone modifications and functional states can merge together and form chromosomal subcompartments suited for local gene activation or repression. This "attraction of likenesses" may be mediated by direct self-association of nucleosome chains as well as by architectural chromatin proteins making oligomeric protein "bridges" between nucleosomes as well as larger dynamic condensates leading to liquid-liquid phase separation inside the cell nucleus. Future studies of mechanisms of chromatin self-association and compartmentalization will require a combination of molecular, imaging, and computational approaches capable of revealing the 3D organization of the eukaryotic genome with nucleosomal resolution.

pyHiM: a new open-source, multi-platform software package for spatial genomics based on multiplexed DNA-FISH imaging.

Genome-wide ensemble sequencing methods improved our understanding of chromatin organization in eukaryotes but lack the ability to capture single-cell heterogeneity and spatial organization. To overcome these limitations, new imaging-based methods have emerged, giving rise to the field of spatial genomics. Here, we present pyHiM, a user-friendly python toolbox specifically designed for the analysis of multiplexed DNA-FISH data and the reconstruction of chromatin traces in individual cells. pyHiM employs a modular architecture, allowing independent execution of analysis steps and customization according to sample specificity and computing resources. pyHiM aims to facilitate the democratization and standardization of spatial genomics analysis.

Hi-C, a chromatin 3D structure technique advancing the functional genomics of immune cells.

The functional performance of immune cells relies on a complex transcriptional regulatory network. The three-dimensional structure of chromatin can affect chromatin status and gene expression patterns, and plays an important regulatory role in gene transcription. Currently available techniques for studying chromatin spatial structure include chromatin conformation capture techniques and their derivatives, chromatin accessibility sequencing techniques, and others. Additionally, the recently emerged deep learning technology can be utilized as a tool to enhance the analysis of data. In this review, we elucidate the definition and significance of the three-dimensional chromatin structure, summarize the technologies available for studying it, and describe the research progress on the chromatin spatial structure of dendritic cells, macrophages, T cells, B cells, and neutrophils.

A graph neural network-based interpretable framework reveals a novel DNA fragility-associated chromatin structural unit.

BACKGROUND: DNA double-strand breaks (DSBs) are among the most deleterious DNA lesions, and they can cause cancer if improperly repaired. Recent chromosome conformation capture techniques, such as Hi-C, have enabled the identification of relationships between the 3D chromatin structure and DSBs, but little is known about how to explain these relationships, especially from global contact maps, or their contributions to DSB formation. RESULTS: Here, we propose a framework that integrates graph neural network (GNN) to unravel the relationship between 3D chromatin structure and DSBs using an advanced interpretable technique GNNExplainer. We identify a new chromatin structural unit named the DNA fragility-associated chromatin interaction network (FaCIN). FaCIN is a bottleneck-like structure, and it helps to reveal a universal form of how the fragility of a piece of DNA might be affected by the whole genome through chromatin interactions. Moreover, we demonstrate that neck interactions in FaCIN can serve as chromatin structural determinants of DSB formation. CONCLUSIONS: Our study provides a more systematic and refined view enabling a better understanding of the mechanisms of DSB formation under the context of the 3D genome.

Epigenetic Regulations in Mammalian Cells: Roles and Profiling Techniques.

The genome is almost identical in all the cells of the body. However, the functions and morphologies of each cell are different, and the factors that determine them are the genes and proteins expressed in the cells. Over the past decades, studies on epigenetic information, such as DNA methylation, histone modifications, chromatin accessibility, and chromatin conformation have shown that these properties play a fundamental role in gene regulation. Furthermore, various diseases such as cancer have been found to be associated with epigenetic mechanisms. In this study, we summarized the biological properties of epigenetics and single-cell epigenomic profiling techniques, and discussed future challenges in the field of epigenetics.

Stage-specific dynamic reorganization of genome topology shapes transcriptional neighborhoods in developing human retinal organoids.

We have generated a high-resolution Hi-C map of developing human retinal organoids to elucidate spatiotemporal dynamics of genomic architecture and its relationship with gene expression patterns. We demonstrate progressive stage-specific alterations in DNA topology and correlate these changes with transcription of cell-type-restricted gene markers during retinal differentiation. Temporal Hi-C reveals a shift toward A compartment for protein-coding genes and B compartment for non-coding RNAs, displaying high and low expression, respectively. Notably, retina-enriched genes are clustered near lost boundaries of topologically associated domains (TADs), and higher-order assemblages (i.e., TAD cliques) localize in active chromatin regions with binding sites for eye-field transcription factors. These genes gain chromatin contacts at their transcription start site as organoid differentiation proceeds. Our study provides a global view of chromatin architecture dynamics associated with diversification of cell types during retinal development and serves as a foundational resource for in-depth functional investigations of retinal developmental traits.

Methods for the Differential Analysis of Hi-C Data.

The 3D organization of chromatin within the nucleus enables dynamic regulation and cell type-specific transcription of the genome. This is true at multiple levels of resolution: on a large scale, with chromosomes occupying distinct volumes (chromosome territories); at the level of individual chromatin fibers, which are organized into compartmentalized domains (e.g., Topologically Associating Domains-TADs), and at the level of short-range chromatin interactions between functional elements of the genome (e.g., enhancer-promoter loops).The widespread availability of Chromosome Conformation Capture (3C)-based high-throughput techniques has been instrumental in advancing our knowledge of chromatin nuclear organization. In particular, Hi-C has the potential to achieve the most comprehensive characterization of chromatin 3D interactions, as it is theoretically able to detect any pair of restriction fragments connected as a result of ligation by proximity.This chapter will illustrate how to compare the chromatin interactome in different experimental conditions, starting from pre-computed Hi-C contact matrices, how to visualize the results, and how to correlate the observed variations in chromatin interaction strength with changes in gene expression.

3D chromatin structure in chondrocytes identifies putative osteoarthritis risk genes.

Genome-wide association studies have identified over 100 loci associated with osteoarthritis risk, but the majority of osteoarthritis risk variants are noncoding, making it difficult to identify the impacted genes for further study and therapeutic development. To address this need, we used a multiomic approach and genome editing to identify and functionally characterize potential osteoarthritis risk genes. Computational analysis of genome-wide association studies and ChIP-seq data revealed that chondrocyte regulatory loci are enriched for osteoarthritis risk variants. We constructed a chondrocyte-specific regulatory network by mapping 3D chromatin structure and active enhancers in human chondrocytes. We then intersected these data with our previously collected RNA-seq dataset of chondrocytes responding to fibronectin fragment, a known osteoarthritis trigger. Integration of the 3 genomic datasets with recently reported osteoarthritis genome-wide association study variants revealed a refined set of putative causal osteoarthritis variants and their potential target genes. One of the putative target genes identified was SOCS2, which was connected to a putative causal variant by a 170-kb loop and is differentially regulated in response to fibronectin fragment. CRISPR-Cas9-mediated deletion of SOCS2 in primary human chondrocytes from 3 independent donors led to heightened expression of inflammatory markers after fibronectin fragment treatment. These data suggest that SOCS2 plays a role in resolving inflammation in response to cartilage matrix damage and provides a possible mechanistic explanation for its influence on osteoarthritis risk. In total, we identified 56 unique putative osteoarthritis risk genes for further research and potential therapeutic development.

A Tremendous Reorganization Journey for the 3D Chromatin Structure from Gametes to Embryos.

The 3D chromatin structure within the nucleus is important for gene expression regulation and correct developmental programs. Recently, the rapid development of low-input chromatin conformation capture technologies has made it possible to study 3D chromatin structures in gametes, zygotes and early embryos in a variety of species, including flies, vertebrates and mammals. There are distinct 3D chromatin structures within the male and female gametes. Following the fertilization of male and female gametes, fertilized eggs undergo drastic epigenetic reprogramming at multi levels, including the 3D chromatin structure, to convert the terminally differentiated gamete state into the totipotent state, which can give rise to an individual. However, to what extent the 3D chromatin structure reorganization is evolutionarily conserved and what the underlying mechanisms are for the tremendous reorganization in early embryos remain elusive. Here, we review the latest findings on the 3D chromatin structure reorganization during embryogenesis, and discuss the convergent and divergent reprogramming patterns and key molecular mechanisms for the 3D chromatin structure reorganization from gametes to embryos in different species. These findings shed light on how the 3D chromatin structure reorganization contribute to embryo development in different species. The findings also indicate the role of the 3D chromatin structure on the acquisition of totipotent developmental potential.

InsuLock: A Weakly Supervised Learning Approach for Accurate Insulator Prediction, and Variant Impact Quantification.

Mapping chromatin insulator loops is crucial to investigating genome evolution, elucidating critical biological functions, and ultimately quantifying variant impact in diseases. However, chromatin conformation profiling assays are usually expensive, time-consuming, and may report fuzzy insulator annotations with low resolution. Therefore, we propose a weakly supervised deep learning method, InsuLock, to address these challenges. Specifically, InsuLock first utilizes a Siamese neural network to predict the existence of insulators within a given region (up to 2000 bp). Then, it uses an object detection module for precise insulator boundary localization via gradient-weighted class activation mapping (~40 bp resolution). Finally, it quantifies variant impacts by comparing the insulator score differences between the wild-type and mutant alleles. We applied InsuLock on various bulk and single-cell datasets for performance testing and benchmarking. We showed that it outperformed existing methods with an AUROC of ~0.96 and condensed insulator annotations to ~2.5% of their original size while still demonstrating higher conservation scores and better motif enrichments. Finally, we utilized InsuLock to make cell-type-specific variant impacts from brain scATAC-seq data and identified a schizophrenia GWAS variant disrupting an insulator loop proximal to a known risk gene, indicating a possible new mechanism of action for the disease.

Epigenomic and enhancer dysregulation in uterine leiomyomas.

BACKGROUND: Uterine leiomyomas, also known as uterine fibroids or myomas, are the most common benign gynecological tumors and are found in women of reproductive and postmenopausal age. There is an exceptionally high prevalence of this tumor in women by the age of 50 years. Black women are particularly affected, with an increased incidence, earlier age of onset, larger and faster growing fibroids and greater severity of symptoms as compared to White women. Although advances in identifying genetic and environmental factors to delineate these fibroids have already been made, only recently has the role of epigenomics in the pathogenesis of this disease been considered. OBJECTIVE AND RATIONALE: Over recent years, studies have identified multiple epigenomic aberrations that may contribute to leiomyoma development and growth. This review will focus on the most recent discoveries in three categories of epigenomic changes found in uterine fibroids, namely aberrant DNA methylation, histone tail modifications and histone variant exchange, and their translation into altered target gene architecture and transcriptional outcome. The findings demonstrating how the altered 3D shape of the enhancer can regulate gene expression from millions of base pairs away will be discussed. Additionally, translational implications of these discoveries and potential roadblocks in leiomyoma treatment will be addressed. SEARCH METHODS: A comprehensive PubMed search was performed to identify published articles containing keywords relevant to the focus of the review, such as: uterine leiomyoma, uterine fibroids, epigenetic alterations, epigenomics, stem cells, chromatin modifications, extracellular matrix [ECM] organization, DNA methylation, enhancer, histone post-translational modifications and dysregulated gene expression. Articles until September 2021 were explored and evaluated to identify relevant updates in the field. Most of the articles focused on in the discussion were published between 2015 and 2021, although some key discoveries made before 2015 were included for background information and foundational purposes. We apologize to the authors whose work was not included because of space restrictions or inadvertent omission. OUTCOMES: Chemical alterations to the DNA structure and of nucleosomal histones, without changing the underlying DNA sequence, have now been implicated in the phenotypic manifestation of uterine leiomyomas. Genome-wide DNA methylation analysis has revealed subsets of either suppressed or overexpressed genes accompanied by aberrant promoter methylation. Furthermore, differential promoter access resulting from altered 3D chromatin structure and histone modifications plays a role in regulating transcription of key genes thought to be involved in leiomyoma etiology. The dysregulated genes function in tumor suppression, apoptosis, angiogenesis, ECM formation, a variety of cancer-related signaling pathways and stem cell differentiation. Aberrant DNA methylation or histone modification is also observed in altering enhancer architecture, which leads to changes in enhancer-promoter contact strength, producing novel explanations for the overexpression of high mobility group AT-hook 2 and gene dysregulation found in mediator complex subunit 12 mutant fibroids. While many molecular mechanisms and epigenomic features have been investigated, the basis for the racial disparity observed among those in the Black population remains unclear. WIDER IMPLICATIONS: A comprehensive understanding of the exact pathogenesis of uterine leiomyoma is lacking and requires attention as it can provide clues for prevention and viable non-surgical treatment. These findings will widen our knowledge of the role epigenomics plays in the mechanisms related to uterine leiomyoma development and highlight novel approaches for the prevention and identification of epigenome targets for long-term non-invasive treatment options of this significantly common disease.

Computational Analysis of Hi-C Data.

The chromatin organization in the 3D nuclear space is essential for genome functionality. This spatial organization encompasses different topologies at diverse scale lengths with chromosomes occupying distinct volumes and individual chromosomes folding into compartments, inside which the chromatin fiber is packed in large domains (as the topologically associating domains, TADs) and forms short-range interactions (as enhancer-promoter loops). The widespread adoption of high-throughput techniques derived from chromosome conformation capture (3C) has been instrumental in investigating the nuclear organization of chromatin. In particular, Hi-C has the potential to achieve the most comprehensive characterization of chromatin 3D structures, as in principle it can detect any pair of restriction fragments connected as a result of ligation by proximity. However, the analysis of the enormous amount of genomic data produced by Hi-C techniques requires the application of complex, multistep computational procedures that may constitute a difficult task also for expert computational biologists. In this chapter, we describe the computational analysis of Hi-C data obtained from the lymphoblastoid cell line GM12878, detailing the processing of raw data, the generation and normalization of the Hi-C contact map, the detection of TADs and chromatin interactions, and their visualization and annotation.