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TAD boundaries are genomic elements that separate biological processes in neighboring domains by blocking DNA loops that are formed through Cohesin‐mediated loop extrusion. Most TAD boundaries consist of arrays of binding sites for the CTCF protein, whose interaction with the Cohesin complex blocks loop extrusion. TAD boundaries are not fully impermeable though and allow a limited amount of inter‐TAD loop formation. Based on the reanalysis of Nano‐C data, a multicontact Chromosome Conformation Capture assay, we propose a model whereby clustered CTCF binding sites promote the successive stalling of Cohesin and subsequent dissociation from the chromatin. A fraction of Cohesin nonetheless achieves boundary read‐through. Due to a constant rate of Cohesin dissociation elsewhere in the genome, the maximum length of inter‐TAD loops is restricted though. We speculate that the DNA‐encoded organization of stalling sites regulates TAD boundary permeability and discuss implications for enhancer–promoter loop formation and other genomic processes. [ABSTRACT FROM AUTHOR]

Within the three‐dimensional (3D) nuclear space, the genome organizes into a series of orderly structures that impose important influences on gene regulation. T lymphocytes, crucial players in adaptive immune responses, undergo intricate transcriptional remodeling upon activation, leading to differentiation into specific effector and memory T cell subsets. Recent evidence suggests that T cell activation is accompanied by dynamic changes in genome architecture at multiple levels, providing a unique biological context to explore the functional relevance and molecular mechanisms of 3D genome organization. Here, we summarize recent advances that link the reorganization of genome architecture to the remodeling of transcriptional programs and conversion of cell fates during T cell activation and differentiation. We further discuss how various chromatin architecture regulators, including CCCTC‐binding factor and several transcription factors, collectively modulate the genome architecture during this process. [ABSTRACT FROM AUTHOR]

Cellular senescence is acknowledged as a key contributor to organismal ageing and late‐life disease. Though popular, the study of senescence in vitro can be complicated by the prolonged and asynchronous timing of cells committing to it and by its paracrine effects. To address these issues, we repurposed a small molecule inhibitor, inflachromene (ICM), to induce senescence to human primary cells. Within 6 days of treatment with ICM, senescence hallmarks, including the nuclear eviction of HMGB1 and ‐B2, are uniformly induced across IMR90 cell populations. By generating and comparing various high throughput datasets from ICM‐induced and replicative senescence, we uncovered a high similarity of the two states. Notably though, ICM suppresses the pro‐inflammatory secretome associated with senescence, thus alleviating most paracrine effects. In summary, ICM rapidly and synchronously induces a senescent‐like phenotype thereby allowing the study of its core regulatory program without confounding heterogeneity. [ABSTRACT FROM AUTHOR]

Human life expectancy is constantly increasing and aging has become a major risk factor for many diseases, although the underlying gene regulatory mechanisms are still unclear. Using transcriptomic and chromosomal conformation capture (Hi‐C) data from human skin fibroblasts from individuals across different age groups, we identified a tight coupling between the changes in co‐regulation and co‐localization of genes. We obtained transcription factors, cofactors, and chromatin regulators that could drive the cellular aging process by developing a time‐course prize‐collecting Steiner tree algorithm. In particular, by combining RNA‐Seq data from different age groups and protein–protein interaction data we determined the key transcription regulators and gene regulatory changes at different life stage transitions. We then mapped these transcription regulators to the 3D reorganization of chromatin in young and old skin fibroblasts. Collectively, we identified key transcription regulators whose target genes are spatially rearranged and correlate with changes in their expression, thereby providing potential targets for reverting cellular aging. [ABSTRACT FROM AUTHOR]

Lamina‐associated domains are large regions of heterochromatin positioned at the nuclear periphery. These domains have been implicated in gene repression, especially in the context of development. In mammals, LAD organization is dependent on nuclear lamins, inner nuclear membrane proteins, and chromatin state. In addition, chromatin readers and modifier proteins have been implicated in this organization, potentially serving as molecular tethers that interact with both nuclear envelope proteins and chromatin. More recent studies have focused on teasing apart the rules that govern dynamic LAD organization and how LAD organization, in turn, relates to gene regulation and overall 3D genome organization. This review highlights recent studies in mammalian cells uncovering factors that instruct the choreography of LAD organization, re‐organization, and dynamics at the nuclear lamina, including LAD dynamics in interphase and through mitotic exit, when LAD organization is re‐established, as well as intra‐LAD subdomain variations. [ABSTRACT FROM AUTHOR]

The human genome is organized in an extremely complexed yet ordered way within the nucleus. Genome organization plays a critical role in the regulation of gene expression. Viruses manipulate the host machinery to influence host genome organization to favor their survival and promote disease development. Epstein‐Barr virus (EBV) is a common human virus, whose infection is associated with various diseases, including infectious mononucleosis, cancer, and autoimmune disorders. This review summarizes our current knowledge of how EBV uses different strategies to control the cellular 3D genome organization to affect cell gene expression to transform normal cells into lymphoblasts. [ABSTRACT FROM AUTHOR]

Deletions, duplications, insertions, inversions, and translocations, collectively called structural variations (SVs), affect more base pairs of the genome than any other sequence variant. The recent technological advancements in genome sequencing have enabled the discovery of tens of thousands of SVs per human genome. These SVs primarily affect non‐coding DNA sequences, but the difficulties in interpreting their impact limit our understanding of human disease etiology. The functional annotation of non‐coding DNA sequences and methodologies to characterize their three‐dimensional (3D) organization in the nucleus have greatly expanded our understanding of the basic mechanisms underlying gene regulation, thereby improving the interpretation of SVs for their pathogenic impact. Here, we discuss the various mechanisms by which SVs can result in altered gene regulation and how these mechanisms can result in rare genetic disorders. Beyond changing gene expression, SVs can produce novel gene‐intergenic fusion transcripts at the SV breakpoints. [ABSTRACT FROM AUTHOR]

Germ cells are unique in engendering totipotency, yet the mechanisms underlying this capacity remain elusive. Here, we perform comprehensive and in‐depth nucleome analysis of mouse germ‐cell development in vitro, encompassing pluripotent precursors, primordial germ cells (PGCs) before and after epigenetic reprogramming, and spermatogonia/spermatogonial stem cells (SSCs). Although epigenetic reprogramming, including genome‐wide DNA de‐methylation, creates broadly open chromatin with abundant enhancer‐like signatures, the augmented chromatin insulation safeguards transcriptional fidelity. These insulatory constraints are then erased en masse for spermatogonial development. Notably, despite distinguishing epigenetic programming, including global DNA re‐methylation, the PGCs‐to‐spermatogonia/SSCs development entails further euchromatization. This accompanies substantial erasure of lamina‐associated domains, generating spermatogonia/SSCs with a minimal peripheral attachment of chromatin except for pericentromeres—an architecture conserved in primates. Accordingly, faulty nucleome maturation, including persistent insulation and improper euchromatization, leads to impaired spermatogenic potential. Given that PGCs after epigenetic reprogramming serve as oogenic progenitors as well, our findings elucidate a principle for the nucleome programming that creates gametogenic progenitors in both sexes, defining a basis for nuclear totipotency. Synopsis: Multi‐omics profiling of an in vitro system for mouse germ‐cell development delineates key principles of nucleome programming, including progressive euchromatization and dynamic insulation, as a basis for nuclear totipotency. 3D genome matures unidirectionally over epigenetic reprogramming and programming.Epigenetic reprogramming creates broadly open chromatin with enhanced insulation.Spermatogonia show minimal, peripherally‐positioned, and pericentromeric LADs.Nucleome mis‐programming leads to impaired spermatogenic potential. [ABSTRACT FROM AUTHOR]

An extraordinary degree of condensation is required to fit the eukaryotic genome inside the nucleus. This compaction is attained by first coiling the DNA around structures called nucleosomes. Mammalian genomes are further folded into sophisticated three‐dimensional (3D) configurations, enabling the genetic code to dictate a diverse range of cell fates. Recent advances in molecular and computational technologies have enabled the query of higher‐order chromatin architecture at an unprecedented resolution and scale. In T lymphocytes, similar to other developmental programs, the hierarchical genome organization is shaped by a highly coordinated division of labor among different classes of sequence‐specific transcription factors. In this review, we will summarize the general principles of 1D and 3D genome organization, introduce the common experimental and computational techniques to measure the multilayer chromatin organization, and discuss the pervasive role of transcription factors on chromatin organization in T lymphocytes. [ABSTRACT FROM AUTHOR]

Chinese hamster ovary (CHO) cell lines are the pillars of a multibillion‐dollar biopharmaceutical industry producing recombinant therapeutic proteins. The effects of local chromatin organization and epigenetic repression within these cell lines result in unpredictable and unstable transgene expression following random integration. Limited knowledge of the CHO genome and its higher order chromatin organization has thus far impeded functional genomics approaches required to tackle these issues. Here, we present an integrative three‐dimensional (3D) map of genome organization within the CHOK1SV® 10E9 cell line in conjunction with an improved, less fragmented CHOK1SV 10E9 genome assembly. Using our high‐resolution chromatin conformation datasets, we have assigned ≈90% of sequence to a chromosome‐scale genome assembly. Our genome‐wide 3D map identifies higher order chromatin structures such as topologically associated domains, incorporates our chromatin accessibility data to enhance the identification of active cis‐regulatory elements, and importantly links these cis‐regulatory elements to target promoters in a 3D promoter interactome. We demonstrate the power of our improved functional annotation by evaluating the 3D landscape of a transgene integration site and two phenotypically different cell lines. Our work opens up further novel genome engineering targets, has the potential to inform vital improvements for industrial biotherapeutic production, and represents a significant advancement for CHO cell line development. [ABSTRACT FROM AUTHOR]

More than a decade of massive DNA sequencing efforts have generated a large body of genomic, transcriptomic and epigenomic information that has provided a more and more detailed view of the functional elements and transactions within the human genome. Considerable efforts have also focused on linking these elements with one another by mapping their interactions and by establishing 3‐dimensional (3D) genomic landscapes in various cell and tissue types. In parallel, multiple studies have associated genomic deletions, duplications and other rearrangements with human pathologies. In this review, we explore recent progresses that have allowed connecting disease‐causing alterations with perturbations of the 3D genome organization. [ABSTRACT FROM AUTHOR]

Abstract: The discovery of the CRISPR‐Cas9 endonuclease has enabled facile genome editing in living cells and organisms. Catalytically inactive Cas9 (dCas9) retains the ability to bind DNA in an RNA‐guided fashion, and has additionally been explored as a tool for transcriptional modulation, epigenetic editing, and genome imaging. This Review highlights recent progress and challenges in the development of dCas9 for imaging genomic loci. The emergence and maturation of this technology offers the potential to answer mechanistic questions about chromosome dynamics and three‐dimensional genome organization in vivo. [ABSTRACT FROM AUTHOR]

Cellular differentiation requires precisely regulated tissue-specific and developmental stage-specific gene expression patterns. Numerous studies have highlighted the predictive power of enhancers on lineage-specific gene expression programs and have started to unravel their mechanisms of action. We review here the dynamics of the enhancer landscape during hematopoietic differentiation and how enhancers function in the context of the 3D organization of the genome. We further discuss the involvement of aberrant enhancer activity in human diseases and emerging strategies aiming at controlling enhancer activity and chromosome topology for therapeutic purposes. [ABSTRACT FROM AUTHOR]