Three-dimensional Genome Organization Research Articles

Organismal evolution of the 3D genome

Genomic Evolution The conformation of chromosomes within the nucleus can reflect a cell's type or state. However, studies of the conservation and evolutionary history of the mechanisms regulating genome structure across species are lacking. Hoencamp et al. mapped three-dimensional (3D) genome organization in 24 eukaryote species, including animals, fungi, and plants. At interphase, species' telomeres and centromeres either clustered across chromosomes or oriented in a polarized state maintaining individual chromosomal territories within the cell, a difference attributed to condensin II. An experimental loss of condensin II in human cells promotes the formation of centromere clusters but has no effect on loop or compartment formation. Whether the structure of the 3D genome varies across species may thus depend on whether they carry a functional condensin II gene. Science , abe2218, this issue p. [984][1] [1]: /lookup/doi/10.1126/science.abe2218

The Myc‐Associated Zinc Finger Protein (MAZ) Controls Cohesin Positioning and Genome Organization Independently OR In Concert With CTCF

The Myc-associated zinc finger protein (MAZ) is a transcription factor that has been shown to regulate transcriptional initiation and termination. However, the underlying mechanisms of MAZ function in the context of chromatin is not fully understood. By applying biochemistry and genetics approaches, we show that MAZ binds to many genomic sites adjacent to the CCCTC-binding factor (CTCF), a protein that is best known for its insulation function in three-dimensional organization of eukaryotic genomes. We demonstrate that, similar to CTCF, MAZ physically interacts with the cohesin subunit Rad21. In addition, we find that MAZ shares with CTCF the ability to independently pause the elongating form of RNA polymerase II, and subsequently affects RNA alternative splicing. Our ChIP-seq analysis of the genome wide distribution of cohesins and CTCF reveals that depletion of CTCF results in preferential loss of CTCF from sites not occupied by MAZ, showing that the binding of both CTCF and cohesin is more stable at the CTCF/MAZ double sites than at the sites occupied by CTCF alone. In an insulation assay like that used for CTCF, we find that binding of MAZ to sites between an enhancer and promoter results in downregulation of reporter gene expression, supporting a role for MAZ as a novel insulator protein. Hi-C analysis indicates that depletion of MAZ in cells not only disrupts local interactions within Topologically associating domains (TADs) but also reduces contacts that establish the boundaries of individual TADs. Taken together, our results suggest that MAZ shares properties with CTCF that allow it to act independently or synergistically in organizing higher order genome architectures, thereby controlling gene expression.

Chromatin coordinates stemness

Stem Cells Blood cells are produced from stem cells in the bone marrow and thymus and transition between quiescence and activation depending on the body's needs. Although generally quiescent, long-term hematopoietic stem cells (LT-HSCs) self-renew and can transform into short-term HSCs (ST-HSCs) to produce multilineage blood cells. Takayama et al. used single-cell analysis and a sequencing approach called ATAC-seq on various human hematopoietic cell populations. The method identified chromatin accessibility signatures that distinguish LT-HSCs and activated hematopoietic stem and progenitor cells (HSPCs). The activated HSPC signature displays binding to sites on the transcription factor CTCF, which is a mediator of three-dimensional (3D) genome organization. As HSCs differentiate, CTCF interacts with chromatin to silence loops that enclose genes. Thus, CTCF acts as a gatekeeper for coordinating the 3D chromatin interactions that regulate hematopoietic cell dynamics. Cell Stem Cell 28 , P488 (2021).

At the Crossroad of Gene Regulation and Genome Organization: Potential Roles for ATP-Dependent Chromatin Remodelers in the Regulation of CTCF-Mediated 3D Architecture.

Simple SummaryThe way DNA is packaged in the nucleus of a cell is important for when and how genes are expressed. There are many levels of packaging, and new techniques have revealed that long-range interactions are important for both promoting and restricting the transcription of genes. Some long-range interactions are mediated by physical loops in the genome where, like a rubber band, the ring-shaped cohesin complex loops sections of DNA bound by CCCTC-binding factor (CTCF). Both cohesin and CTCF act on DNA, and increasing evidence indicates that their function is inhibited by nucleosomes bound to the DNA. In this review, we summarize the current knowledge of how individual chromatin remodelers, which utilize ATP to move nucleosomes on DNA, facilitate or inhibit cohesin/CTCF-dependent looping interactions.In higher order organisms, the genome is assembled into a protein-dense structure called chromatin. Chromatin is spatially organized in the nucleus through hierarchical folding, which is tightly regulated both in cycling cells and quiescent cells. Assembly and folding are not one-time events in a cell’s lifetime; rather, they are subject to dynamic shifts to allow changes in transcription, DNA replication, or DNA damage repair. Chromatin is regulated at many levels, and recent tools have permitted the elucidation of specific factors involved in the maintenance and regulation of the three-dimensional (3D) genome organization. In this review/perspective, we aim to cover the potential, but relatively unelucidated, crosstalk between 3D genome architecture and the ATP-dependent chromatin remodelers with a specific focus on how the architectural proteins CTCF and cohesin are regulated by chromatin remodeling.

ReHiC: Enhancing Hi-C data resolution via residual convolutional network.

High-throughput chromosome conformation capture (Hi-C) is one of the most popular methods for studying the three-dimensional organization of genomes. However, Hi-C protocols can be expensive since they require large amounts of sample material and may be time-consuming. Most commonly used Hi-C data are low-resolution. Such data can only be used to identify large-scale genomic interactions and are not sufficient to identify the small-scale patterns. We propose a novel deep learning-based computational approach (named ReHiC) that enhances the resolution of Hi-C data and allows us to achieve high-resolution Hi-C data at a relatively low cost. Our model only requires 1/16 down-sampling ratio of the original sequence reading to predict higher resolution Hi-C data. This is very close to high-resolution data in terms of numerical distribution and interaction distribution. More importantly, our framework stacks deeper and converges faster due to residual blocks in the core of the network. Extensive experiments show that ReHiC performs better than HiCPlus and HiCNN, two recently developed and frequently used methods to look at the spatial organization of chromatin structure in the cell. Moreover, the portability of our framework verified by extensive experiments shows that the trained model can also enhance the Hi-C matrix of other cell types efficiently. In conclusion, ReHiC offers more accurate high-resolution image reconstruction in a broad field.

RNA Biogenesis Instructs Functional Inter-Chromosomal Genome Architecture.

Three-dimensional (3D) genome organization has emerged as an important layer of gene regulation in development and disease. The functional properties of chromatin folding within individual chromosomes (i.e., intra-chromosomal or in cis) have been studied extensively. On the other hand, interactions across different chromosomes (i.e., inter-chromosomal or in trans) have received less attention, being often regarded as background noise or technical artifacts. This viewpoint has been challenged by emerging evidence of functional relationships between specific trans chromatin interactions and epigenetic control, transcription, and splicing. Therefore, it is an intriguing possibility that the key processes involved in the biogenesis of RNAs may both shape and be in turn influenced by inter-chromosomal genome architecture. Here I present the rationale behind this hypothesis, and discuss a potential experimental framework aimed at its formal testing. I present a specific example in the cardiac myocyte, a well-studied post-mitotic cell whose development and response to stress are associated with marked rearrangements of chromatin topology both in cis and in trans. I argue that RNA polymerase II clusters (i.e., transcription factories) and foci of the cardiac-specific splicing regulator RBM20 (i.e., splicing factories) exemplify the existence of trans-interacting chromatin domains (TIDs) with important roles in cellular homeostasis. Overall, I propose that inter-molecular 3D proximity between co-regulated nucleic acids may be a pervasive functional mechanism in biology.

A Newly Assigned Role of CTCF in Cellular Response to Broken DNAs.

Best known as a transcriptional factor, CCCTC-binding factor (CTCF) is a highly conserved multifunctional DNA-binding protein with 11 zinc fingers. It functions in diverse genomic processes, including transcriptional activation/repression, insulation, genome imprinting and three-dimensional genome organization. A big surprise has recently emerged with the identification of CTCF engaging in the repair of DNA double-strand breaks (DSBs) and in the maintenance of genome fidelity. This discovery now adds a new dimension to the multifaceted attributes of this protein. CTCF facilitates the most accurate DSB repair via homologous recombination (HR) that occurs through an elaborate pathway, which entails a chain of timely assembly/disassembly of various HR-repair complexes and chromatin modifications and coordinates multistep HR processes to faithfully restore the original DNA sequences of broken DNA sites. Understanding the functional crosstalks between CTCF and other HR factors will illuminate the molecular basis of various human diseases that range from developmental disorders to cancer and arise from impaired repair. Such knowledge will also help understand the molecular mechanisms underlying the diverse functions of CTCF in genome biology. In this review, we discuss the recent advances regarding this newly assigned versatile role of CTCF and the mechanism whereby CTCF functions in DSB repair.

Insights into the Links between MYC and 3D Chromatin Structure and Epigenetics Regulation: Implications for Cancer Therapy.

MYC is embedded in the transcriptional oasis of the 8q24 gene desert. A plethora of genomic elements has roles in MYC aberrant expression in cancer development by interacting with transcription factors and epigenetics regulators as well as altering the structure of chromatin at the MYC locus and tissue-specific long-range enhancer-promoter contacts. Furthermore, MYC is a master regulator of several human cancers by modulating the transcription of numerous cancer-related genes through epigenetic mechanisms. This review provides a comprehensive overview of the three-dimensional genomic organization around MYC and the role of epigenetic machinery in transcription and function of MYC as well as discusses various epigenetic-targeted therapeutic strategies in MYC-driven cancers.

Deep Learning of Sequence Patterns for CCCTC-Binding Factor-Mediated Chromatin Loop Formation

The three-dimensional (3D) organization of the human genome is of crucial importance for gene regulation, and the CCCTC-binding factor (CTCF) plays an important role in chromatin interactions. However, it is still unclear what sequence patterns in addition to CTCF motif pairs determine chromatin loop formation. To discover the underlying sequence patterns, we have developed a deep learning model, called DeepCTCFLoop, to predict whether a chromatin loop can be formed between a pair of convergent or tandem CTCF motifs using only the DNA sequences of the motifs and their flanking regions. Our results suggest that DeepCTCFLoop can accurately distinguish the CTCF motif pairs forming chromatin loops from the ones not forming loops. It significantly outperforms CTCF-MP, a machine learning model based on word2vec and boosted trees, when using DNA sequences only. Furthermore, we show that DNA motifs binding to several transcription factors, including ZNF384, ZNF263, ASCL1, SP1, and ZEB1, may constitute the complex sequence patterns for CTCF-mediated chromatin loop formation. DeepCTCFLoop has also been applied to disease-associated sequence variants to identify candidates that may disrupt chromatin loop formation. Therefore, our results provide useful information for understanding the mechanism of 3D genome organization and may also help annotate and prioritize the noncoding sequence variants associated with human diseases.

A dynamic actin-dependent nucleoskeleton and cell identity.

Actin is an essential regulator of cellular functions. In the eukaryotic cell nucleus, actin regulates chromatin as a bona fide component of chromatin remodelling complexes, it associates with nuclear RNA polymerases to regulate transcription and is involved in co-transcriptional assembly of nascent RNAs into ribonucleoprotein complexes. Actin dynamics are, therefore, emerging as a major regulatory factor affecting diverse cellular processes. Importantly, the involvement of actin dynamics in nuclear functions is redefining the concept of nucleoskeleton from a rigid scaffold to a dynamic entity that is likely linked to the three-dimensional organization of the nuclear genome. In this review, we discuss how nuclear actin, by regulating chromatin structure through phase separation may contribute to the architecture of the nuclear genome during cell differentiation and facilitate the expression of specific gene programs. We focus specifically on mitochondrial genes and how their dysregulation in the absence of actin raises important questions about the role of cytoskeletal proteins in regulating chromatin structure. The discovery of a novel pool of mitochondrial actin that serves as 'mitoskeleton' to facilitate organization of mtDNA supports a general role for actin in genome architecture and a possible function of distinct actin pools in the communication between nucleus and mitochondria.

The Nucleome Data Bank: web-based resources to simulate and analyze the three-dimensional genome.

We introduce the Nucleome Data Bank (NDB), a web-based platform to simulate and analyze the three-dimensional (3D) organization of genomes. The NDB enables physics-based simulation of chromosomal structural dynamics through the MEGABASE + MiChroM computational pipeline. The input of the pipeline consists of epigenetic information sourced from the Encode database; the output consists of the trajectories of chromosomal motions that accurately predict Hi-C and fluorescence insitu hybridization data, as well as multiple observations of chromosomal dynamics in vivo. As an intermediate step, users can also generate chromosomal sub-compartment annotations directly from the same epigenetic input, without the use of any DNA–DNA proximity ligation data. Additionally, the NDB freely hosts both experimental and computational structural genomics data. Besides being able to perform their own genome simulations and download the hosted data, users can also analyze and visualize the same data through custom-designed web-based tools. In particular, the one-dimensional genetic and epigenetic data can be overlaid onto accurate 3D structures of chromosomes, to study the spatial distribution of genetic and epigenetic features. The NDB aims to be a shared resource to biologists, biophysicists and all genome scientists. The NDB is available at https://ndb.rice.edu.

Somatic mutation distributions in cancer genomes vary with three-dimensional chromatin structure.

Somatic mutations in driver genes may ultimately lead to the development of cancer. Understanding how somatic mutations accumulate in cancer genomes and the underlying factors that generate somatic mutations is therefore crucial for developing novel therapeutic strategies. To understand the interplay between spatial genome organization and specific mutational processes, we studied 3,000 tumor-normal-pair whole-genome datasets from 42 different human cancer types. Our analyses reveal that the change in somatic mutational load in cancer genomes is co-localized with topologically-associating-domain boundaries. Domain boundaries constitute a better proxy to track mutational load change than replication timing measurements. We show that different mutational processes lead to distinct somatic mutation distributions where certain processes generate mutations in active domains, and others generate mutations in inactive domains. Overall, the interplay between three-dimensional genome organization and active mutational processes has a substantial influence on the large-scale mutation-rate variations observed in human cancers.

Cancer-specific CTCF binding facilitates oncogenic transcriptional dysregulation

BackgroundThe three-dimensional genome organization is critical for gene regulation and can malfunction in diseases like cancer. As a key regulator of genome organization, CCCTC-binding factor (CTCF) has been characterized as a DNA-binding protein with important functions in maintaining the topological structure of chromatin and inducing DNA looping. Among the prolific binding sites in the genome, several events with altered CTCF occupancy have been reported as associated with effects in physiology or disease. However, hitherto there is no comprehensive survey of genome-wide CTCF binding patterns across different human cancers.ResultsTo dissect functions of CTCF binding, we systematically analyze over 700 CTCF ChIP-seq profiles across human tissues and cancers and identify cancer-specific CTCF binding patterns in six cancer types. We show that cancer-specific lost and gained CTCF binding events are associated with altered chromatin interactions, partially with DNA methylation changes, and rarely with sequence mutations. While lost bindings primarily occur near gene promoters, most gained CTCF binding events exhibit enhancer activities and are induced by oncogenic transcription factors. We validate these findings in T cell acute lymphoblastic leukemia cell lines and patient samples and show that oncogenic NOTCH1 induces specific CTCF binding and they cooperatively activate expression of target genes, indicating transcriptional condensation phenomena.ConclusionsSpecific CTCF binding events occur in human cancers. Cancer-specific CTCF binding can be induced by other transcription factors to regulate oncogenic gene expression. Our results substantiate CTCF binding alteration as a functional epigenomic signature of cancer.

A Comparative Study of Supervised Machine Learning Algorithms for the Prediction of Long-Range Chromatin Interactions

The role of three-dimensional genome organization as a critical regulator of gene expression has become increasingly clear over the last decade. Most of our understanding of this association comes from the study of long range chromatin interaction maps provided by Chromatin Conformation Capture-based techniques, which have greatly improved in recent years. Since these procedures are experimentally laborious and expensive, in silico prediction has emerged as an alternative strategy to generate virtual maps in cell types and conditions for which experimental data of chromatin interactions is not available. Several methods have been based on predictive models trained on one-dimensional (1D) sequencing features, yielding promising results. However, different approaches vary both in the way they model chromatin interactions and in the machine learning-based strategy they rely on, making it challenging to carry out performance comparison of existing methods. In this study, we use publicly available 1D sequencing signals to model cohesin-mediated chromatin interactions in two human cell lines and evaluate the prediction performance of six popular machine learning algorithms: decision trees, random forests, gradient boosting, support vector machines, multi-layer perceptron and deep learning. Our approach accurately predicts long-range interactions and reveals that gradient boosting significantly outperforms the other five methods, yielding accuracies of about 95%. We show that chromatin features in close genomic proximity to the anchors cover most of the predictive information, as has been previously reported. Moreover, we demonstrate that gradient boosting models trained with different subsets of chromatin features, unlike the other methods tested, are able to produce accurate predictions. In this regard, and besides architectural proteins, transcription factors are shown to be highly informative. Our study provides a framework for the systematic prediction of long-range chromatin interactions, identifies gradient boosting as the best suited algorithm for this task and highlights cell-type specific binding of transcription factors at the anchors as important determinants of chromatin wiring mediated by cohesin.

Targeted DNase Hi-C.

Technology advance during the past decade has greatly expanded our understanding of the higher-order structure of the genome. The various chromosome conformation capture (3C)-based techniques such as Hi-C have provided the most widely used tools for interrogating three-dimensional (3D) genome organization. We recently developed a Hi-C variant, DNase Hi-C, for characterizing 3D genome organization. DNase Hi-C employs DNase I for chromatin fragmentation, aiming to overcome restriction enzyme digestion-related limitations associated with traditional Hi-C methods. By combining DNase Hi-C with DNA capture technology, we further implemented a high-throughput approach, called targeted DNase Hi-C, which enables to map fine-scale chromatin architecture at exceptionally high resolution and thereby is an ideal tool for mapping the physical landscapes of cis-regulatory networks and for characterizing phenotype-associated chromatin 3D signatures. Here, I describe a detailed protocol of targeted DNase Hi-C library preparation, which covers experimental steps starting from cell cross-linking to library amplification.

RFX5 regulates gene expression of the Pcdhα cluster.

Gene expression and three-dimensional (3D) genome organization are thought to be closely related. The protocadherin (Pcdh) clusters are central for neuronal self-avoidance in brain development and have been used as model genes to explore the role of 3D genome structures in gene regulation. Transcription factor RFX5 (regulatory factor x 5) is a member of the winged-helix subfamily of the helix-turn-helix superfamily proteins. The RFX5 protein contains four domains: oligomerization, DNA-binding, helical, and transactivation domains. RFX5 plays an important role in regulating expression of the major histocompatibility complex class II (MHC II) gene complex. Here we report a role of RFX5 in the regulation of clustered Pcdh genes. We first knocked out the RFX5 gene by using CRISPR/Cas9 DNA-fragment editing in HEC-1-B cell line. By RNA-seq experiments, we found that RFX5 deletion results in a significant increase of expression levels of the Pcdhα6, Pcdhα12 and Pcdhαc2 genes. By ChIP-nexus experiments, we found that RFX5 deletion leads to the increased binding of CTCF and RAD21 in the Pcdhα cluster. Finally, through quantitative high-resolution chromosome conformation capture copy (QHR-4C) experiments, we found that RFX5 deletion results in a significant increase of long-distance chromatin interactions between the HS5-1 enhancer and its target promoters in the Pcdhα cluster. In conclusion, RFX5 regulates gene expression of the Pcdhα cluster through higher-order chromatin structure.

G9a/GLP-sensitivity of H3K9me2 Demarcates Two Types of Genomic Compartments

In the nucleus, chromatin is folded into hierarchical architecture that is tightly linked to various nuclear functions. However, the underlying molecular mechanisms that confer these architectures remain incompletely understood. Here, we investigated the functional roles of H3 lysine 9 dimethylation (H3K9me2), one of the abundant histone modifications, in three-dimensional (3D) genome organization. Unlike in mouse embryonic stem cells, inhibition of methyltransferases G9a and GLP in differentiated cells eliminated H3K9me2 predominantly at A-type (active) genomic compartments, and the level of residual H3K9me2 modifications was strongly associated with B-type (inactive) genomic compartments. Furthermore, chemical inhibition of G9a/GLP in mouse hepatocytes led to decreased chromatin-nuclear lamina interactions mainly at G9a/GLP-sensitive regions, increased degree of genomic compartmentalization, and up-regulation of hundreds of genes that were associated with alterations of the 3D chromatin. Collectively, our data demonstrated essential roles of H3K9me2 in 3D genome organization.

Cryo-EM structures of holo condensin reveal a subunit flip-flop mechanism.

Complexes containing a pair of structural maintenance of chromosomes (SMC) family proteins are fundamental for the three-dimensional (3D) organization of genomes in all domains of life. The eukaryotic SMC complexes cohesin and condensin are thought to fold interphase and mitotic chromosomes, respectively, into large loop domains, although the underlying molecular mechanisms have remained unknown. We used cryo-EM to investigate the nucleotide-driven reaction cycle of condensin from the budding yeast Saccharomyces cerevisiae. Our structures of the five-subunit condensin holo complex at different functional stages suggest that ATP binding induces the transition of the SMC coiled coils from a folded-rod conformation into a more open architecture. ATP binding simultaneously triggers the exchange of the two HEAT-repeat subunits bound to the SMC ATPase head domains. We propose that these steps result in the interconversion of DNA-binding sites in the catalytic core of condensin, forming the basis of the DNA translocation and loop-extrusion activities.

FreeHi-C spike-in simulations for benchmarking differential chromatin interaction detection.

High-throughput genome-wide chromatin conformation capture assay (Hi-C) is routinely used to profile long-range genomic interactions and three-dimensional organization of genomes. A key application of Hi-C is the comparative analysis of genomic interactions across different time points, cellular conditions, or multiple stimuli. While operating characteristics of methods for Hi-C data processing such as normalization, pairwise interaction and higher-order organization detection have been relatively well studied, properties of methods for differential chromatin interaction detection are less investigated. We have recently developed FreeHi-C to enable data-driven non-parametric simulations from Hi-C experiments. Here, we extend FreeHi-C with a user/data-driven spike-in module to facilitate comparisons of differential chromatin interaction detection methods where the ground truth differential chromatin interactions are known under a wide variety of settings. We use FreeHi-C to benchmark four differential chromatin interaction detection methods, namely HiCcompare, multiHiCcompare, diffHic, and Selfish, using three comparative analysis settings with different sequencing depths and spike-in proportions. This comparison reveals distinguished performances in terms of the standard metrics such as the false discovery rate control, detection power, significance order, precision-recall curve, and receiver operating characteristic curve as well as overall genomic properties of the types of differential chromatin interactions detectable by each method. Furthermore, it highlights the lack of power for all methods in small replication settings.

CTCF-mediated genome organization and leukemogenesis

Recent technological advancements and genome-wide studies provide compelling evidence that dynamic chromatin interaction and three-dimensional genome organization in nuclei play an important role in regulating gene expression. Mammalian genomes consist of many small functional domains termed topologically associated domains (TADs), many of them organized by CCCTC-binding factor (CTCF) and the cohesion complex. Changes in genome TADs might result in inappropriate promoter/enhancer communications leading to activation of oncogenes or suppression of tumor suppressors. During normal hematopoiesis and leukemogenesis, genome structure alters considerably to facilitate normal and malignant hematopoiesis, respectively. Delineating theses normal and abnormal processes will evolve our understanding of disease pathogenesis and development of potential treatment strategies. This review highlights the role of CTCF and its associated protein complexes in three-dimensional genome organization in development and leukemogenesis, as well as the roles of CTCF boundary defined TAD in transcription regulation. We further explore the function of chromatin modulators, such as CTCF, cohesin, and long noncoding RNAs (lncRNAs) in chromosomal interactions and hematopoietic genome organization. Finally, we focus on the implication of 3D genome alteration in the pathogenesis of leukemia and provide a scientific basis for targeted intervention.