Inter-chromosomal transcription hubs shape the 3D genome architecture of African trypanosomes

Three-dimensional (3D) genome organization becomes altered during development, aging, and disease1–23, but the factors regulating chromatin topology are incompletely understood and currently no technology can efficiently screen for new regulators of multiscale chromatin organization. Here, we developed an image-based high-content screening platform (Perturb-tracing) that combines pooled CRISPR screen, a new cellular barcode readout method (BARC-FISH), and chromatin tracing. We performed a loss-of-function screen in human cells, and visualized alterations to their genome organization from 13,000 imaging target-perturbation combinations, alongside perturbation-paired barcode readout in the same single cells. Using 1.4 million 3D positions along chromosome traces, we discovered tens of new regulators of chromatin folding at different length scales, ranging from chromatin domains and compartments to chromosome territory. A subset of the regulators exhibited 3D genome effects associated with loop-extrusion and A-B compartmentalization mechanisms, while others were largely unrelated to these known 3D genome mechanisms. We found that the ATP-dependent helicase CHD7, the loss of which causes the congenital neural crest syndrome CHARGE24 and a chromatin remodeler previously shown to promote local chromatin openness25–27, counter-intuitively compacts chromatin over long range in different genomic contexts and cell backgrounds including neural crest cells, and globally represses gene expression. The DNA compaction effect of CHD7 is independent of its chromatin remodeling activity and does not require other protein partners. Finally, we identified new regulators of nuclear architectures and found a functional link between chromatin compaction and nuclear shape. Altogether, our method enables scalable, high-content identification of chromatin and nuclear topology regulators that will stimulate new insights into the 3D genome functions, such as global gene and nuclear regulation, in health and disease.

In Trypanosoma brucei, genes are arranged in Polycistronic Transcription Units (PTUs), which are demarcated by transcription start and stop sites. Transcription start sites are also binding sites of Origin Recognition Complex 1 (ORC1). This spatial coincidence implies that transcription and replication in trypanosomes must occur in a highly ordered and cooperative manner. Interestingly, a previously published genetic screen identified the T. brucei MCM-BP, which interacts with subunits of MCM helicase, as a protein whose downregulation results in the loss of transcriptional silencing at subtelomeric loci. Here, I show that TbMCM-BP is required for DNA replication and transcription. TbMCM-BP depletion causes a significant reduction of replicating cells in S phase and genome-wide impairments of replication origin activation. Moreover, levels of sense and antisense transcripts increase at boundaries of PTUs in the absence of TbMCM-BP. TbMCM-BP is also important for transcriptional repression of the specialized subtelomeric PTUs, the Bloodstream-form Expression-Sites (BESs), which house the major antigenic determinant (the Variant Surface Glycoprotein, VSG gene) as well as TbORC1 binding sites. Overall, this study reveals that TbMCM-BP, a replication initiation protein, also guides the initiation, termination and directionality of transcription.

Acetylcholinesterase in man is encoded by a single gene, ACHE, located on chromosome 7q22. In this study, the transcription start sites and major DNA promoter elements controlling the expression of this gene have been characterized by structural and functional studies. Immediately upstream of the first untranslated exon of the gene are GC-rich sequences containing consensus binding sites for several transcription factors, including Sp1, EGR-1 and AP2. In vitro transcription studies and RNase protection analyses of mRNA isolated from human NT2/D1 teratocarcinoma cells reveal that two closely spaced transcription cap sites are located at a consensus initiator (Inr) element similar to that found in the terminal transferase gene. Transient transfection of mutant genes shows that removal of three bases of this initiator sequence reduces promoter activity by 98% in NT2/D1 cells. In vitro transcription studies and transient transfection of a series of 5' deletion mutants of the ACHE promoter linked to a luciferase reporter show an Sp1 site at -71 to be essential for promoter activity. Purified Sp1 protein protects this site from DNase cleavage during in vitro footprinting experiments. A conserved AP2 consensus binding site, located between the GC box elements and the Inr, is protected by recombinant AP2 protein in DNase footprinting experiments, induces a mobility shift with AP2 protein and AP2-containing cell extracts, and fosters inhibition of transcription by AP2 as measured by transient transfection in mouse and human cell lines and in in vitro transcription reactions. These results indicate that AP2 functions as a repressor of human ACHE and mouse Ache transcription.

Ankylosing Spondylitis (AS) is a chronic inflammatory arthritis of the spine exhibiting a strong genetic background. The mechanistic and functional understanding of the AS-associated genomic loci, identified with Genome Wide Association Studies (GWAS), remains challenging. Chromosome conformation capture (3C) and derivatives are recent techniques which are of great help in elucidating the spatial genome organization and of enormous support in uncover a mechanistic explanation for disease-associated genetic variants. The perturbation of three-dimensional (3D) genome hierarchy may lead to a plethora of human diseases, including rheumatological disorders. Here we illustrate the latest approaches and related findings on the field of genome organization, highlighting how the instability of 3D genome conformation may be among the causes of rheumatological disease phenotypes. We suggest a new perspective on the inclusive potential of a 3C approach to inform GWAS results in rheumatic diseases. 3D genome organization may ultimately lead to a more precise and comprehensive functional interpretation of AS association, which is the starting point for emerging and more specific therapies.

DNA methylation is required to maintain both DNA replication timing precision and 3D genome organization integrity.

ABSTRACTThe folding and dynamics of three-dimensional (3D) genome organization are fundamental for eukaryotes executing genome functions but have been largely unexplored in nonmodel fungi. Using high-throughput sequencing coupled with chromosome conformation capture (Hi-C) data, we generated two chromosome-level assemblies for Pucciniastriiformis f. sp. tritici, a fungus causing stripe rust disease on wheat, for studying 3D genome architectures of plant pathogenic fungi. The chromatin organization of the fungus followed a combination of the fractal globule model and the equilibrium globule model. Surprisingly, chromosome compartmentalization was not detected. Dynamics of 3D genome organization during two developmental stages of P. striiformis f. sp. tritici indicated that regulation of gene activities might be independent of the changes of genome organization. In addition, chromatin conformation conservation was found to be independent of genome sequence synteny conservation among different fungi. These results highlighted the distinct folding principles of fungal 3D genomes. Our findings should be an important step toward a holistic understanding of the principles and functions of genome architecture across different eukaryotic kingdoms.IMPORTANCE Previously, our understanding of 3D genome architecture has mainly come from model mammals, insects, and plants. However, the organization and regulatory functions of 3D genomes in fungi are largely unknown. In this study, we comprehensively investigated P. striiformis f. sp. tritici, a plant fungal pathogen, and revealed distinct features of the 3D genome, comparing it with the universal folding feature of 3D genomes in higher eukaryotic organisms. We further suggested that there might be distinct regulatory mechanisms of gene expression that are independent of chromatin organization changes during the developmental stages of this rust fungus. Moreover, we showed that the evolutionary pattern of 3D genomes in this fungus is also different from the cases in mammalian genomes. In addition, the genome assembly pipeline and the generated two chromosome-level genomes will be valuable resources. These results highlighted the unexplored distinct features of 3D genome organization in fungi. Therefore, our study provided complementary knowledge to holistically understand the organization and functions of 3D genomes across different eukaryotes.

Three-dimensional (3D) genome organization is tightly coupled with gene regulation in various biological processes and diseases. In cancer, various types of large-scale genomic rearrangements can disrupt the 3D genome, leading to oncogenic gene expression. However, unraveling the pathogenicity of the 3D cancer genome remains a challenge since closer examinations have been greatly limited due to the lack of appropriate tools specialized for disorganized higher-order chromatin structure. Here, we updated a 3D-genome Interaction Viewer and database named 3DIV by uniformly processing ∼230 billion raw Hi-C reads to expand our contents to the 3D cancer genome. The updates of 3DIV are listed as follows: (i) the collection of 401 samples including 220 cancer cell line/tumor Hi-C data, 153 normal cell line/tissue Hi-C data, and 28 promoter capture Hi-C data, (ii) the live interactive manipulation of the 3D cancer genome to simulate the impact of structural variations and (iii) the reconstruction of Hi-C contact maps by user-defined chromosome order to investigate the 3D genome of the complex genomic rearrangement. In summary, the updated 3DIV will be the most comprehensive resource to explore the gene regulatory effects of both the normal and cancer 3D genome. ‘3DIV’ is freely available at http://3div.kr.

Three-dimensional (3D) genome organization becomes altered during development, aging and disease, but the factors regulating chromatin topology are incompletely understood and currently no technology can efficiently screen for new regulators of multi-scale chromatin organization. Here, we developed an image-based high-content screening platform (Perturb-tracing) that combines pooled CRISPR screens, a cellular barcode readout method (BARC-FISH) and chromatin tracing. We performed a loss-of-function screen in human cells, and visualized alterations to their 3D chromatin folding conformations, alongside perturbation-paired barcode readout in the same single cells. We discovered tens of new regulators of chromatin folding at different length scales, ranging from chromatin domains and compartments to chromosome territory. A subset of the regulators exhibited 3D genome effects associated with loop extrusion and A–B compartmentalization mechanisms, while others were largely unrelated to these known 3D genome mechanisms. Finally, we identified new regulators of nuclear architectures and found a functional link between chromatin compaction and nuclear shape. Altogether, our method enables scalable, high-content identification of chromatin and nuclear topology regulators that will stimulate new insights into the 3D genome.

MotivationThree-dimensional (3D) genome organization plays important functional roles in cells. User-friendly tools for reconstructing 3D genome models from chromosomal conformation capturing data and analyzing them are needed for the study of 3D genome organization.ResultsWe built a comprehensive graphical tool (GenomeFlow) to facilitate the entire process of modeling and analysis of 3D genome organization. This process includes the mapping of Hi-C data to one-dimensional (1D) reference genomes, the generation, normalization and visualization of two-dimensional (2D) chromosomal contact maps, the reconstruction and the visualization of the 3D models of chromosome and genome, the analysis of 3D models and the integration of these models with functional genomics data. This graphical tool is the first of its kind in reconstructing, storing, analyzing and annotating 3D genome models. It can reconstruct 3D genome models from Hi-C data and visualize them in real-time. This tool also allows users to overlay gene annotation, gene expression data and genome methylation data on top of 3D genome models.Availability and implementationThe source code and user manual: https://github.com/jianlin-cheng/GenomeFlow.Supplementary informationSupplementary data are available at Bioinformatics online.

To investigate three-dimensional (3D) genome organization in prokaryotic and eukaryotic cells, three main strategies are employed, namely nuclear proximity ligation-based methods, imaging tools (such as fluorescence in situ hybridization (FISH) and its derivatives), and computational/visualization methods. Proximity ligation-based methods are based on digestion and re-ligation of physically proximal cross-linked chromatin fragments accompanied by massively parallel DNA sequencing to measure the relative spatial proximity between genomic loci. Imaging tools enable direct visualization and quantification of spatial distances between genomic loci, and advanced implementation of (super-resolution) microscopy helps to significantly improve the resolution of images. Computational methods are used to map global 3D genome structures at various scales driven by experimental data, and visualization methods are used to visualize genome 3D structures in virtual 3D space-based on algorithms. In this review, we focus on the introduction of novel imaging and visualization methods to study 3D genomes. First, we introduce the progress made recently in 3D genome imaging in both fixed cell and live cells based on long-probe labeling, short-probe labeling, RNA FISH, and the CRISPR system. As the fluorescence-capturing capability of a particular microscope is very important for the sensitivity of bioimaging experiments, we also introduce two novel super-resolution microscopy methods, SDOM and low-power super-resolution STED, which have potential for time-lapse super-resolution live-cell imaging of chromatin. Finally, we review some software tools developed recently to visualize proximity ligation-based data. The imaging and visualization methods are complementary to each other, and all three strategies are not mutually exclusive. These methods provide powerful tools to explore the mechanisms of gene regulation and transcription in cell nuclei.

The book is intended for students studying medical and biological specialties. CHAPTER I. EPIGENETICS INTRODUCTION The science of epigenetics looks at the mechanisms of molecular modifications of histones and DNA that can regulate gene activity without affecting the nucleotide sequences in the DNA molecule. Recognized epigenetic regulators are DNA methylation, post-translational modifications of histones, and non-coding RNAs (nkRNAs). One of the most important differences between eukaryotic cells and prokaryotes is the presence of a complex nucleo-protein chromatin complex in eukaryotes. It is in this form that the DNA molecule is stored in our cells. On the one hand, the complex structural organization of chromatin provides a compact arrangement of DNA in the cell nucleus. On the other hand, chromatin is directly involved in the process of regulating gene expression. At the same time, the nucleosome depicted in Fig. 1 (a structural and functional unit of chromatin) is considered as a key component in the processes of regulating gene expression. The nucleus of the nucleosome is 8 histone proteins (octamers). The nucleus of the nucleosome consists of two copies of each of the histone proteins H2A, H2B, H3 and H4. The DNA chain, which includes 147 nucleotides, folds 1.65 times around the octamer of histones. The nucleosomes are arranged as a linear array along the DNA molecule in the form of "beads on a string". The linker section of DNA connecting adjacent nucleosomes (transcriptionally inactive) is sealed with H1-histone protein. The length of the linker section is 30 nm. Moreover, the site of the beginning of transcription is usually located inside the nucleosome. Consequently, the nucleosome serves as a gene repressor, preventing the initiation of transcription. That is, chromatin provides a total repression of genes. In contrast, transcription becomes possible as a result of chromatin remodeling factors that enable the "dismantling" of nucleosomes or otherwise alter their structure and organization. Thus, the repression (inactivation) of genes begins with wrapping the DNA molecule around the histones in the nucleosome, and the liberation of genes from repression (activation) involves freeing DNA from binding to histone proteins and unfolding DNA by chromatin remodeling factors (Lorch Y., Kornberg R. D., 2017). Thanks to this mechanism, selective expression of only those genes that are needed at a given time by the cell or tissue is possible. It should be emphasized that nucleosome repression extends not only to transcription, but also to most other biological processes associated with the DNA molecule, such as replication, mitotic division, repair of double-strand breaks, and maintenance of telomeres. Thus, epigenetic mechanisms control various physiological and pathological processes by regulating the expression of the corresponding genes by changing the availability of epigenetic control systems to chromatin. The scope of application of epigenetic research methods is rapidly expanding. Currently, we are witnessing the active introduction of epigenetic approaches in the field of practical medicine aimed at diagnosing and treating dangerous human diseases. CHAPTER II. TRANSCRIPTION FACTORS INTRODUCTION For the first time, the existence of transcription factors was revealed on the basis of a discovery that made it possible to establish in vitro purified RNA polymerase-II can initiate transcription on the DNA template in the presence of a cell extract (Weil P. A. et al., 1979). Further research aimed at the fractionation and identification of the general transcription factors (GTF) required to initiate transcription in vitro has identified similar factors in rats, Drosophila, and yeast and substantiated the assumption that GTFs are indeed "common" factors necessary for the expression of genes transcribed by RNA polymerase II. is highly conserved in a number of eukaryotic organisms (Matsui T. et al., 1980). We only mention RNA polymerase II because only this type of enzyme has the ability to synthesize mRNA. Whereas RNA polymerase I is responsible for the synthesis of pro-rRNA, and RNA polymerase III is responsible for the synthesis of tRNA and other non-coding cell RNAs. Meanwhile, the regulation of transcription in eukaryotes is quite complex, since it depends on chromatin remodeling complexes (Burns L. G., Peterson C. L., 1997) and covalent modification of histone proteins (Natsume-Kitatani Y., Mamitsuka H., 2016). In transcription initiation, the immediate target of GTF is a well-defined promo zone of a structural gene. In the structure of the promotra of eukaryotes, the main elements and regulatory elements can be distinguished. The main elements of the promotra (bark promoter, see Fig. 2.1) can be attributed to the site for assembling the transcription initiation complex (PIC), including the TATA sequence located above from the transcription start site (TSS ), and an initiating sequence (Inr) covering the start site. Promoters may include a TATA unit, an initiator sequence (Inr), or both (Hampsey M., 1998). A third major element, the downstream promoter element (DPE), was originally described in Drosophila and is located about 30 p.p. below TSS. The DPE promoter element appears to function in conjunction with the Inr element as a binding site for the transcription factor TFIID on non-TATA promoters. According to current research, the cellular (main) promoters of multicellular organisms that control the initiation of transcription by RNA polymerase II may contain short sequences of nucleotides called cow promoter elements (motifs) (e.g., the TATA block, the initiator (Inr), and the lower element of the cow promoter (DPE)) that recruit RNA polymerase II through a common transcription initiation mechanism (Dreos R. et al., 2021). The authors report that the classes of Promoters of Inr+DPE are not only present in the genome of Drosophila and humans and are structurally similar to each other, but may also be common to different species of multicellular organisms. The most studied element of the cow promoter is the TATA box, but the TATA box is found only in about 10-20% of multicellular cortical promoters. Therefore, along with the TATA sequence, it is necessary to name other possible key DNA sequences known as cortical promoter elements, which include: BRE, MTE, TST and DPE sequences. The two BRE (TFIIB recognition element) motifs are located either above (BREu) or below (BREd) elements of the TATA box. It should be emphasized that TBP, TATA box, and BRE demonstrate high levels of conservatism in the range from archaebacteria to humans (Kadonaga J. T., 2012). In doing so, BREu as well as BREd have both positive and negative effects on transcription activity. The downstream core promoter element (DPE) was detected in the analysis of non-TATA gene promoters in Drosophila. The MTE element (motif ten element), which is located directly in front of the DPE, was identified as an overrepresented sequence of a cow promoter called "motif 10" and then discovered, that it is a functional element of a cow promoter. The MTE and DPE motifs exhibit high conservatism in the range from Drosophila to humans, and both motifs appear to be directly recognized by the subunits of the main transcription factor TFIID, TAF proteins that resemble histone proteins in structure. In turn, the TCT sequence regulates the transcription of ribosomal protein genes in Drosophila and humans. Although there are no universal cortical promoter elements that are present in all promoters, the concept of a cow promoter of nuclear RNA polymerase II can be defined as a minimum stretch of DNA that is sufficient to accurately initiate transcription by RNA polymerase II (Kadonaga J. T., 2012; Haberle V., Stark A., 2018). It should be noted that the results of modern research will constantly supplement the list of all new components of the cow promoter, for example, DNA-replicatedrelated element (DRE), Ohler 1,6 and 7 motifs (Danino Y. M. et al., 2015; Haberle V., Stark A., 2018). According to the authors, the bark promoter may be transformed in the course of evolution. Due to this, gene expression levels can be modulated by the composition of cow promoter elements. Such modulation is directly achieved through the emergence of combinations of new elements of the cow promoter, as a result of which an additional level of transcription regulation is realized. To summarize the above facts, transcription is usually initiated at a specific position, the Transcription Initiation Site (TSS), at the 5' end of the gene. The TSS site is embedded in a bark promoter, which is a short sequence spanning 50 base pairs above and 50 below TSS. The cortical promoter serves as a binding platform for the components required to initiate transcription, including RNA polymerase II and related common transcription factors (GTFs). Regulatory elements. The cortical promoter is sufficient to initiate transcription, but such transcription has low basal activity, which can be further activated, generally by more distally arranged regulatory elements called enhancers (discussed below). Enhancers bind regulatory proteins known as transcription factors, recruit transcription cofactors, and can further enhance transcription. CHAPTER III. CELL SIGNALING PATHWAYS INTRODUCTION In a multicellular organism, the work of each cell is regulated by a large number of signals. These signals can be formed both in the organism itself, reflecting the specific needs of a living organism (metabolic state, stages of development, differentiation, reproduction), and in the form of a reaction to the effects of the external environment. The implementation of each of these signals encompasses all the biological and biochemical processes that lead from the cell's perception of the signal to the cell's response. A signal to a cell is something that is recogni

Toward understanding the dynamic state of 3D genome

Chromatin architecture is a fundamental mediator of genome function. Fasting is a major environmental cue across the animal kingdom, yet how it impacts three-dimensional (3D) genome organization is unknown. Here we show that fasting induces an intestine-specific, reversible and large-scale spatial reorganization of chromatin in Caenorhabditis elegans. This fasting-induced 3D genome reorganization requires inhibition of the nutrient-sensing mTOR pathway, acting through the regulation of RNA Pol I, but not Pol II nor Pol III, and is accompanied by remodelling of the nucleolus. By uncoupling the 3D genome configuration from the animal’s nutritional status, we find that the expression of metabolic and stress-related genes increases when the spatial reorganization of chromatin occurs, showing that the 3D genome might support the transcriptional response in fasted animals. Our work documents a large-scale chromatin reorganization triggered by fasting and reveals that mTOR and RNA Pol I shape genome architecture in response to nutrients.

The detailed principles of the hierarchical folding of eukaryotic chromosomes have been revealed during the last two decades. Along with structures composing three-dimensional (3D) genome organization (chromatin compartments, topologically associating domains, chromatin loops, etc.), the molecular mechanisms that are involved in their establishment and maintenance have been characterized. Generally, protein–protein and protein–DNA interactions underlie the spatial genome organization in eukaryotes. However, it is becoming increasingly evident that weak interactions, which exist in biological systems, also contribute to the 3D genome. Here, we provide a snapshot of our current understanding of the role of the weak interactions in the establishment and maintenance of the 3D genome organization. We discuss how weak biological forces, such as entropic forces operating in crowded solutions, electrostatic interactions of the biomolecules, liquid-liquid phase separation, DNA supercoiling, and RNA environment participate in chromosome segregation into structural and functional units and drive intranuclear functional compartmentalization.

Large scale structural variation in the genome is known to be linked to cancer pathways. Breast cancer progression from normal epithelium to localized, and metastatic cancer involves genomic perturbations such as mutations, deletions, and duplications. Emerging research has shown that the structure of the 3D genome, specifically how chromosomes and genes are folded and organized in the nucleus, directly impacts gene expression and thus, may contribute to disease progression. In fact, local folding around critical genes, especially those involved in important biological pathways, may have direct impact on cancer initiation and progression. Limits in technology has restricted a more complete understanding linking 3D genome to disease. Biochemical methods require many cells and are unable to preserve single cell and sub-population level information. Previous imaging methods are limited by the number of analyzable targets, reducing the generalizability of results across other genomic loci and limited a comprehensive genome-wide understanding. Here, we use jebFISHTM and the PaintScapeTM platform to resolve 3D structural features of genomic loci at high fidelity. With our Biological Pathways Panel, we focus on genes that are known to play critical roles in DNA repair, cell cycle, apoptosis, chromatin modifications, and transcriptional regulation. Using breast cancer progression as the model, we apply jebFISHTM to cell lines from normal epithelium, localized cancer, and metastatic cancer. We observe dramatic changes in 3D genome at both single cell and population levels. At each stage of breast cancer progression, we identify specific alterations to 3D genome features including changes in structure and switching of A/B chromatin compartments. Structural alterations are also present at whole chromosome level. We see disruption and repositioning of chromosome territories as well as interchromosomal interactions. jebFISHTM single-cell, single-homolog resolution enables identification of cellular sub-populations at each stage of breast cancer progression. We observe changes in the frequency of sub-populations with disease progression. Our results suggest that genome organization, at the local gene as well as chromosome level is modulating the expression of genes important in biological pathways. The presence of sub-populations suggests that the heterogeneous nature of 3D genome organization within the population is an important feature, impacting our understanding and treatment of diseases. While this study focuses on breast cancer, we envisage that these results are generalizable and further the understanding of genome organization and gene expression. Citation Format: Huy Nguyen, Shyamtanu Chattoraj, Jude Dunne. 3D genome structural alterations in biological pathway genes drive the progression of cells from normal epithelium to metastatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2746.