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Type I diabetes is an autoimmune disease mediated by T-cell destruction of b cells in pancreatic islets. Currently, there is no known cure, and treatment consists of daily insulin injections. Genome-wide association studies and twin studies have indicated a strong genetic heritability for type I diabetes and implicated several genes. As most strongly associated variants are noncoding, there is still a lack of identification of functional and, therefore, likely causal variants. Given that many of these genetic variants reside in enhancer elements, we have tested 121 CD4+ Tcell enhancer variants associated with T1D. We found four to be functional through massively parallel reporter assays. Three of the enhancer variants weaken activity, while the fourth strengthens activity. We link these to their cognate genes using 3D genome architecture or eQTL data and validate them using CRISPR editing. Validated target genes include CLEC16A and SOCS1. While these genes have been previously implicated in type 1 diabetes and other autoimmune diseases, we show that enhancers controlling their expression harbor functional variants. These variants, therefore, may act as causal type 1 diabetic variants. [ABSTRACT FROM AUTHOR]

Secondary trastuzumab resistance represents an evolutionary adaptation of HER2‐positive breast cancer during anti‐HER2 treatment. Most current studies have tended to prioritize HER2 and its associated signaling pathways, often overlooking broader but seemingly less relevant cellular processes, along with their associated genetic and epigenetic mechanisms. Here, transcriptome data is not only characterized but also examined epigenomic and 3D genome architecture information in both trastuzumab‐sensitive and secondary‐resistant breast cancer cells. The findings reveal that the global metabolic reprogramming associated with trastuzumab resistance may stem from genome‐wide alterations in both histone modifications and chromatin structure. Specifically, the transcriptional activities of key genes involved in lipid metabolism appear to be regulated by variant promoter H3K27me3 and H3K4me3 modifications, as well as promoter‐enhancer interactions. These discoveries offer valuable insights into how cancer cells adapt to anti‐tumor drugs and have the potential to impact future diagnostic and treatment strategies. [ABSTRACT FROM AUTHOR]

Molecular and cellular characterization of tumors is essential due to the complex and heterogeneous nature of cancer. In recent decades, many bioinformatic tools and experimental techniques have been developed to achieve personalized characterization of tumors. However, sample handling continues to be a major challenge as limitations such as prior treatments before sample acquisition, the amount of tissue obtained, transportation, or the inability to process fresh samples pose a hurdle for experimental strategies that require viable cell suspensions. Here, we present an optimized protocol that allows the recovery of highly viable cell suspensions from breast cancer primary tumor biopsies. Using these cell suspensions we have successfully characterized genome architecture through Hi-C. Also, we have evaluated single-cell gene expression and the tumor cellular microenvironment through single-cell RNAseq. Both technologies are key in the detailed and personalized molecular characterization of tumor samples. The protocol described here is a cost-effective alternative to obtain viable cell suspensions from biopsies simply and efficiently.

Type I diabetes is an autoimmune disease mediated by T-cell destruction of β cells in pancreatic islets. Currently, there is no known cure, and treatment consists of daily insulin injections. Genome-wide association studies and twin studies have indicated a strong genetic heritability for type I diabetes and implicated several genes. As most strongly associated variants are noncoding, there is still a lack of identification of functional and, therefore, likely causal variants. Given that many of these genetic variants reside in enhancer elements, we have tested 121 CD4+ T-cell enhancer variants associated with T1D. We found four to be functional through massively parallel reporter assays. Three of the enhancer variants weaken activity, while the fourth strengthens activity. We link these to their cognate genes using 3D genome architecture or eQTL data and validate them using CRISPR editing. Validated target genes include CLEC16A and SOCS1. While these genes have been previously implicated in type 1 diabetes and other autoimmune diseases, we show that enhancers controlling their expression harbor functional variants. These variants, therefore, may act as causal type 1 diabetic variants.

Research on the three-dimensional (3D) structure of the genome and its distribution within the nuclear space has made a big leap in the last two decades. Work in the animal field has led to significant advances in our general understanding on eukaryotic genome organization. This did not only bring along insights into how the 3D genome interacts with the epigenetic landscape and the transcriptional machinery but also how 3D genome architecture is relevant for fundamental developmental processes, such as cell differentiation. In parallel, the 3D organization of plant genomes have been extensively studied, which resulted in both congruent and novel findings, contributing to a more complete view on how eukaryotic genomes are organized in multiple dimensions. Plant genomes are remarkably diverse in size, composition, and ploidy. Furthermore, as intrinsically sessile organisms without the possibility to relocate to more favorable environments, plants have evolved an elaborate epigenetic repertoire to rapidly respond to environmental challenges. The diversity in genome organization and the complex epigenetic programs make plants ideal study subjects to acquire a better understanding on universal features and inherent constraints of genome organization. Furthermore, considering a wide range of species allows us to study the evolutionary crosstalk between the various levels of genome architecture. In this article, we aim at summarizing important findings on 3D genome architecture obtained in various plant species. These findings cover many aspects of 3D genome organization on a wide range of levels, from gene loops to topologically associated domains and to global 3D chromosome configurations. We present an overview on plant 3D genome organizational features that resemble those in animals and highlight facets that have only been observed in plants to date.

In eukaryotes, the genome does not emerge in a specific shape but rather as a hierarchial bundle within the nucleus. This multifaceted genome organization consists of multiresolution cellular structures, such as chromosome territories, compartments, and topologically associating domains, which are frequently defined by architecture, design proteins including CTCF and cohesin, and chromatin loops. This review briefly discusses the advances in understanding the basic rules of control, chromatin folding, and functional areas in early embryogenesis. With the use of chromosome capture techniques, the latest advancements in technologies for visualizing chromatin interactions come close to revealing 3D genome formation frameworks with incredible detail throughout all genomic levels, including at single-cell resolution. The possibility of detecting variations in chromatin architecture might open up new opportunities for disease diagnosis and prevention, infertility treatments, therapeutic approaches, desired exploration, and many other application scenarios. [ABSTRACT FROM AUTHOR]

Three-dimensional (3D) genome architecture is characterized by multi-scale patterns and plays an essential role in gene regulation. Chromatin conformation capturing experiments have revealed many properties underlying 3D genome architecture, such as the compartmentalization of chromatin based on transcriptional states. However, they are complex, costly and time consuming, and therefore only a limited number of cell types have been examined using these techniques. Increasing effort is being directed towards deriving computational methods that can predict chromatin conformation and associated structures. Here we present DNA-delay differential analysis (DDA), a purely sequence-based method based on chaos theory to predict genome-wide A and B compartments. We show that DNA-DDA models derived from a 20 Mb sequence are sufficient to predict genome wide compartmentalization at the scale of 100 kb in four different cell types. Although this is a proof-of-concept study, our method shows promise in elucidating the mechanisms responsible for genome folding as well as modeling the impact of genetic variation on 3D genome architecture and the processes regulated thereby. [ABSTRACT FROM AUTHOR]

Over the past 15 years, a variety of next-generation sequencing assays have been developed for measuring the 3D conformation of DNA in the nucleus. Each of these assays gives, for a particular cell or tissue type, a distinct picture of 3D chromatin architecture. Accordingly, making sense of the relationship between genome structure and function requires teasing apart two closely related questions: how does chromatin 3D structure change from one cell type to the next, and how do different measurements of that structure differ from one another, even when the two assays are carried out in the same cell type? In this work, we assemble a collection of chromatin 3D datasets-each represented as a 2D contact map- spanning multiple assay types and cell types. We then build a machine learning model that predicts missing contact maps in this collection. We use the model to systematically explore how genome 3D architecture changes, at the level of compartments, domains, and loops, between cell type and between assay types.

The simultaneous observation of three-dimensional (3D) chromatin structure and transcription in single cells is critical to understand how DNA is organized inside cells and how this organization influences or is affected by other processes, such as transcription. We have recently introduced an innovative technology known as Hi-M, which enables the sequential tagging, 3D visualization, and precise localization of multiple genomic DNA regions alongside RNA expression within individual cells. In this chapter, we present a comprehensive guide outlining the creation of probes, as well as sample preparation and labeling. Finally, we provide a step-by-step guide to conduct a complete Hi-M acquisition using our open-source software package, Qudi-HiM, which controls the robotic microscope handling the entire acquisition procedure., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Enhancer clusters overlapping disease-associated mutations in Pierre Robin sequence (PRS) patients regulate SOX9 expression at genomic distances over 1.25 Mb. We applied optical reconstruction of chromatin architecture (ORCA) imaging to trace 3D locus topology during PRS-enhancer activation. We observed pronounced changes in locus topology between cell types. Subsequent analysis of single-chromatin fiber traces revealed that these ensemble-average differences arise through changes in the frequency of commonly sampled topologies. We further identified two CTCF-bound elements, internal to the SOX9 topologically associating domain, which promote stripe formation, are positioned near the domain's 3D geometric center, and bridge enhancer-promoter contacts in a series of chromatin loops. Ablation of these elements results in diminished SOX9 expression and altered domain-wide contacts. Polymer models with uniform loading across the domain and frequent cohesin collisions recapitulate this multi-loop, centrally clustered geometry. Together, we provide mechanistic insights into architectural stripe formation and gene regulation over ultra-long genomic ranges. [Display omitted] • SOX9 domain topology dynamically changes during a developmental transition • Structural elements promote TAD-wide interactions, stripe formation, and transcription • Structural elements are CTCF dependent and situated centrally in the 3D TAD structure • Polymer simulations of the multi-loop model best recapitulate the topological features Enhancer clusters overlapping disease-associated mutations in Pierre Robin sequence (PRS) patients regulate SOX9 expression at genomic distances over 1.25 Mb. Chen and Long et al. trace the 3D topology of the SOX9 locus during PRS-enhancer activation and identify structural elements that promote domain-wide interactions and facilitate ultra-long-range gene regulation. [ABSTRACT FROM AUTHOR]

Special AT-rich sequence binding protein 1 (SATB1) has long been proposed to act as a global chromatin loop organizer in T cells. However, the exact functions of SATB1 in spatial genome organization remain elusive. Here we show that the depletion of SATB1 in human and murine T cells leads to transcriptional dysregulation for genes involved in T cell activation, as well as alterations of 3D genome architecture at multiple levels, including compartments, topologically associating domains, and loops. Importantly, SATB1 extensively colocalizes with CTCF throughout the genome. Depletion of SATB1 leads to increased chromatin contacts among and across the SATB1/CTCF co-occupied sites, thereby affecting the transcription of critical regulators of T cell activation. The loss of SATB1 does not affect CTCF occupancy but significantly reduces the retention of CTCF in the nuclear matrix. Collectively, our data show that SATB1 contributes to 3D genome organization by constraining chromatin topology surrounding CTCF-binding sites. [Display omitted] • SATB1 regulates the transcriptional program associated with T cell activation • SATB1 extensively colocalizes with CTCF throughout the genome • SATB1 constrains chromatin interactions surrounding the CTCF-bound sites • SATB1 promotes the association of CTCF with the nuclear matrix Wang et al. report that SATB1 regulates genome organization in T cells via functional interplay with chromatin architectural protein CTCF. SATB1 extensively co-localizes with CTCF and influences chromatin folding surrounding their binding sites. Depletion of SATB1 rearranges chromatin loops at genes critical for T cell activation, resulting in their dysregulation. [ABSTRACT FROM AUTHOR]

Animal genomes are compartmentalized into insulated regulatory units named topology-associated domains (TADs). TADs insulate gene promoters from enhancers that occupy neighboring TADs. Chromosomal rearrangements that disrupt TAD structure can generate new regulatory interactions between enhancers and promoters that were once separated into different TADs, which might lead to new gene expression patterns. On the one hand, TAD rearrangements are known to cause deleterious phenotypes, but, on the other hand, rearrangements can also create novel expression patterns that may be selected during evolution because they generate advantageous phenotypes. Here, we review recent studies that explore the effects of chromosomal rearrangements and genetic perturbations on TAD structure and gene regulation in the context of development and evolution. We discuss the possible contribution of evolutionary breakpoints (EBRs) that affect TAD structure to the evolution of gene regulation and the phenotype., (© 2022 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)