Modeling epigenetic lesions that cause gliomas.

Epigenetic lesions that disrupt regulatory elements represent potential cancer drivers. However, we lack experimental models for validating their tumorigenic impact. Here, we model aberrations arising in isocitrate dehydrogenase-mutant gliomas, which exhibit DNA hypermethylation. We focus on a CTCF insulator near the PDGFRA oncogene that is recurrently disrupted by methylation in these tumors. We demonstrate that disruption of the syntenic insulator in mouse oligodendrocyte progenitor cells (OPCs) allows an OPC-specific enhancer to contact and induce Pdgfra, thereby increasing proliferation. We show that a second lesion, methylation-dependent silencing of the Cdkn2a tumor suppressor, cooperates with insulator loss in OPCs. Coordinate inactivation of the Pdgfra insulator and Cdkn2a drives gliomagenesis in vivo. Despite locus synteny, the insulator is CpG-rich only in humans, a feature that may confer human glioma risk but complicates mouse modeling. Our study demonstrates the capacity of recurrent epigenetic lesions to drive OPC proliferation in vitro and gliomagenesis in vivo.

Non-cell-autonomous disruption of nuclear architecture as a potential cause of COVID-19-induced anosmia.

SARS-CoV-2 infects less than 1% of cells in the human body, yet it can cause severe damage in a variety of organs. Thus, deciphering the non-cell-autonomous effects of SARS-CoV-2 infection is imperative for understanding the cellular and molecular disruption it elicits. Neurological and cognitive defects are among the least understood symptoms of COVID-19 patients, with olfactory dysfunction being their most common sensory deficit. Here, we show that both in humans and hamsters, SARS-CoV-2 infection causes widespread downregulation of olfactory receptors (ORs) and of their signaling components. This non-cell-autonomous effect is preceded by a dramatic reorganization of the neuronal nuclear architecture, which results in dissipation of genomic compartments harboring OR genes. Our data provide a potential mechanism by which SARS-CoV-2 infection alters the cellular morphology and the transcriptome of cells it cannot infect, offering insight to its systemic effects in olfaction and beyond.

The Self-Organizing Genome: Principles of Genome Architecture and Function.

Genomes have complex three-dimensional architectures. The recent convergence of genetic, biochemical, biophysical, and cell biological methods has uncovered several fundamental principles of genome organization. They highlight that genome function is a major driver of genome architecture and that structural features of chromatin act as modulators, rather than binary determinants, of genome activity. The interplay of these principles in the context of self-organization can account for the emergence of structural chromatin features, the diversity and single-cell heterogeneity of nuclear architecture in cell types and tissues, and explains evolutionarily conserved functional features of genomes, including plasticity and robustness.

Large-Scale Topological Changes Restrain Malignant Progression in Colorectal Cancer.

Widespread changes to DNA methylation and chromatin are well documented in cancer, but the fate of higher-order chromosomal structure remains obscure. Here we integrated topological maps for colon tumors and normal colons with epigenetic, transcriptional, and imaging data to characterize alterations to chromatin loops, topologically associated domains, and large-scale compartments. We found that spatial partitioning of the open and closed genome compartments is profoundly compromised in tumors. This reorganization is accompanied by compartment-specific hypomethylation and chromatin changes. Additionally, we identify a compartment at the interface between the canonical A and B compartments that is reorganized in tumors. Remarkably, similar shifts were evident in non-malignant cells that have accumulated excess divisions. Our analyses suggest that these topological changes repress stemness and invasion programs while inducing anti-tumor immunity genes and may therefore restrain malignant progression. Our findings call into question the conventional view that tumor-associated epigenomic alterations are primarily oncogenic.

Heterochromatin-Driven Nuclear Softening Protects the Genome against Mechanical Stress-Induced Damage.

Tissue homeostasis requires maintenance of functional integrity under stress. A central source of stress is mechanical force that acts on cells, their nuclei, and chromatin, but how the genome is protected against mechanical stress is unclear. We show that mechanical stretch deforms the nucleus, which cells initially counteract via a calcium-dependent nuclear softening driven by loss of H3K9me3-marked heterochromatin. The resulting changes in chromatin rheology and architecture are required to insulate genetic material from mechanical force. Failure to mount this nuclear mechanoresponse results in DNA damage. Persistent, high-amplitude stretch induces supracellular alignment of tissue to redistribute mechanical energy before it reaches the nucleus. This tissue-scale mechanoadaptation functions through a separate pathway mediated by cell-cell contacts and allows cells/tissues to switch off nuclear mechanotransduction to restore initial chromatin state. Our work identifies an unconventional role of chromatin in altering its own mechanical state to maintain genome integrity in response to deformation.

The Energetics and Physiological Impact of Cohesin Extrusion.

Cohesin extrusion is thought to play a central role in establishing the architecture of mammalian genomes. However, extrusion has not been visualized in vivo, and thus, its functional impact and energetics are unknown. Using ultra-deep Hi-C, we show that loop domains form by a process that requires cohesin ATPases. Once formed, however, loops and compartments are maintained for hours without energy input. Strikingly, without ATP, we observe the emergence of hundreds of CTCF-independent loops that link regulatory DNA. We also identify architectural "stripes," where a loop anchor interacts with entire domains at high frequency. Stripes often tether super-enhancers to cognate promoters, and in B cells, they facilitate Igh transcription and recombination. Stripe anchors represent major hotspots for topoisomerase-mediated lesions, which promote chromosomal translocations and cancer. In plasmacytomas, stripes can deregulate Igh-translocated oncogenes. We propose that higher organisms have coopted cohesin extrusion to enhance transcription and recombination, with implications for tumor development.

Lamina-Associated Domains: Links with Chromosome Architecture, Heterochromatin, and Gene Repression.

In metazoan cell nuclei, hundreds of large chromatin domains are in close contact with the nuclear lamina. Such lamina-associated domains (LADs) are thought to help organize chromosomes inside the nucleus and have been associated with gene repression. Here, we discuss the properties of LADs, the molecular mechanisms that determine their association with the nuclear lamina, their dynamic links with other nuclear compartments, and their proposed roles in gene regulation.

Genome Regulation by Polycomb and Trithorax: 70 Years and Counting.

Polycomb (PcG) and Trithorax (TrxG) group proteins are evolutionarily conserved chromatin-modifying factors originally identified as part of an epigenetic cellular memory system that maintains repressed or active gene expression states. Recently, they have been shown to globally control a plethora of cellular processes. This functional diversity is achieved by their ability to regulate chromatin at multiple levels, ranging from modifying local chromatin structure to orchestrating the three-dimensional organization of the genome. Understanding this system is a fascinating challenge of critical relevance for biology and medicine, since misexpression or mutation of multiple PcG components, as well as of TrxG members of the COMPASS family and of the SWI/SNF complex, is implicated in cancer and other diseases.

SAF-A Regulates Interphase Chromosome Structure through Oligomerization with Chromatin-Associated RNAs.

Higher eukaryotic chromosomes are organized into topologically constrained functional domains; however, the molecular mechanisms required to sustain these complex interphase chromatin structures are unknown. A stable matrix underpinning nuclear organization was hypothesized, but the idea was abandoned as more dynamic models of chromatin behavior became prevalent. Here, we report that scaffold attachment factor A (SAF-A), originally identified as a structural nuclear protein, interacts with chromatin-associated RNAs (caRNAs) via its RGG domain to regulate human interphase chromatin structures in a transcription-dependent manner. Mechanistically, this is dependent on SAF-A's AAA+ ATPase domain, which mediates cycles of protein oligomerization with caRNAs, in response to ATP binding and hydrolysis. SAF-A oligomerization decompacts large-scale chromatin structure while SAF-A loss or monomerization promotes aberrant chromosome folding and accumulation of genome damage. Our results show that SAF-A and caRNAs form a dynamic, transcriptionally responsive chromatin mesh that organizes large-scale chromosome structures and protects the genome from instability.

A nuclear architecture screen in Drosophila identifies Stonewall as a link between chromatin position at the nuclear periphery and germline stem cell fate.

The association of genomic loci to the nuclear periphery is proposed to facilitate cell type-specific gene repression and influence cell fate decisions. However, the interplay between gene position and expression remains incompletely understood, in part because the proteins that position genomic loci at the nuclear periphery remain unidentified. Here, we used an Oligopaint-based HiDRO screen targeting ∼1000 genes to discover novel regulators of nuclear architecture in Drosophila cells. We identified the heterochromatin-associated protein Stonewall (Stwl) as a factor promoting perinuclear chromatin positioning. In female germline stem cells (GSCs), Stwl binds and positions chromatin loci, including GSC differentiation genes, at the nuclear periphery. Strikingly, Stwl-dependent perinuclear positioning is associated with transcriptional repression, highlighting a likely mechanism for Stwl's known role in GSC maintenance and ovary homeostasis. Thus, our study identifies perinuclear anchors in Drosophila and demonstrates the importance of gene repression at the nuclear periphery for cell fate.

Monoallelic expression of olfactory receptors.

The sense of smell collects vital information about the environment by detecting a multitude of chemical odorants. Breadth and sensitivity are provided by a huge number of chemosensory receptor proteins, including more than 1,400 olfactory receptors (ORs). Organizing the sensory information generated by these receptors so that it can be processed and evaluated by the central nervous system is a major challenge. This challenge is overcome by monogenic and monoallelic expression of OR genes. The single OR expressed by each olfactory sensory neuron determines the neuron's odor sensitivity and the axonal connections it will make to downstream neurons in the olfactory bulb. The expression of a single OR per neuron is accomplished by coupling a slow chromatin-mediated activation process to a fast negative-feedback signal that prevents activation of additional ORs. Singular OR activation is likely orchestrated by a network of interchromosomal enhancer interactions and large-scale changes in nuclear architecture.

Context-Dependent Gene Regulation by Homeodomain Transcription Factor Complexes Revealed by Shape-Readout Deficient Proteins.

Eukaryotic transcription factors (TFs) form complexes with various partner proteins to recognize their genomic target sites. Yet, how the DNA sequence determines which TF complex forms at any given site is poorly understood. Here, we demonstrate that high-throughput in vitro DNA binding assays coupled with unbiased computational analysis provide unprecedented insight into how different DNA sequences select distinct compositions and configurations of homeodomain TF complexes. Using inferred knowledge about minor groove width readout, we design targeted protein mutations that destabilize homeodomain binding both in vitro and in vivo in a complex-specific manner. By performing parallel systematic evolution of ligands by exponential enrichment sequencing (SELEX-seq), chromatin immunoprecipitation sequencing (ChIP-seq), RNA sequencing (RNA-seq), and Hi-C assays, we not only classify the majority of in vivo binding events in terms of complex composition but also infer complex-specific functions by perturbing the gene regulatory network controlled by a single complex.

Upon microbial challenge, human neutrophils undergo rapid changes in nuclear architecture and chromatin folding to orchestrate an immediate inflammatory gene program.

Differentiating neutrophils undergo large-scale changes in nuclear morphology. How such alterations in structure are established and modulated upon exposure to microbial agents is largely unknown. Here, we found that prior to encounter with bacteria, an armamentarium of inflammatory genes was positioned in a transcriptionally passive environment suppressing premature transcriptional activation. Upon microbial exposure, however, human neutrophils rapidly (<3 h) repositioned the ensemble of proinflammatory genes toward the transcriptionally permissive compartment. We show that the repositioning of genes was closely associated with the swift recruitment of cohesin across the inflammatory enhancer landscape, permitting an immediate transcriptional response upon bacterial exposure. We found that activated enhancers, marked by increased deposition of H3K27Ac, were highly enriched for cistromic elements associated with PU.1, CEBPB, TFE3, JUN, and FOSL2 occupancy. These data reveal how upon microbial challenge the cohesin machinery is recruited to an activated enhancer repertoire to instruct changes in chromatin folding, nuclear architecture, and to activate an inflammatory gene program.

LncRNAs: the missing link to senescence nuclear architecture.

During cellular senescence and organismal aging, cells display various molecular and morphological changes. Although many aging-related long noncoding RNAs (lncRNAs) are highly associated with senescence-associated secretory phenotype, the roles of lncRNAs in senescence-associated nuclear architecture and morphological changes are just starting to emerge. Here I review lncRNAs associated with nuclear structure establishment and maintenance, their aging-related changes, and then focus on the pervasive, yet underappreciated, role of RNA double-strand DNA triplexes for lncRNAs to recognize targeted genomic regions, making lncRNAs the nexus between DNA and proteins to regulate nuclear structural changes. Finally, I discuss the future of deciphering direct links of lncRNA changes to various nuclear morphology changes assisted by artificial intelligence and genetic perturbations.

Nuclear actin dynamics and functions at a glance.

Actin is well known for its cytoskeletal functions, where it helps to control and maintain cell shape and architecture, as well as regulating cell migration and intracellular cargo transport, among others. However, actin is also prevalent in the nucleus, where genome-regulating roles have been described, including it being part of chromatin-remodeling complexes. More recently, with the help of advances in microscopy techniques and specialized imaging probes, direct visualization of nuclear actin filament dynamics has helped elucidate new roles for nuclear actin, such as in cell cycle regulation, DNA replication and repair, chromatin organization and transcriptional condensate formation. In this Cell Science at a Glance article, we summarize the known signaling events driving the dynamic assembly of actin into filaments of various structures within the nuclear compartment for essential genome functions. Additionally, we highlight the physiological role of nuclear F-actin in meiosis and early embryonic development.

Competent for commitment: you've got to have heart!

The mature heart is composed primarily of four different cell types: cardiac myocytes, endothelium, smooth muscle, and fibroblasts. These cell types derive from pluripotent progenitors that become progressively restricted with regard to lineage potential, giving rise to multipotent cardiac progenitor cells and, ultimately, the differentiated cell types of the heart. Recent studies have begun to shed light on the defining characteristics of the intermediary cell types that exist transiently during this developmental process and the extrinsic and cell-autonomous factors that influence cardiac lineage decisions and cellular competence. This information will shape our understanding of congenital and adult cardiac disease and guide regenerative therapeutic approaches. In addition, cardiac progenitor specification can serve as a model for understanding basic mechanisms regulating the acquisition of cellular identity. In this review, we present the concept of "chromatin competence" that describes the potential for three-dimensional chromatin organization to function as the molecular underpinning of the ability of a progenitor cell to respond to inductive lineage cues and summarize recent studies advancing our understanding of cardiac cell specification, gene regulation, and chromatin organization and how they impact cardiac development.

DNA replication and replication stress response in the context of nuclear architecture.

The DNA replication process needs to be coordinated with other DNA metabolism transactions and must eventually extend to the full genome, regardless of chromatin status, gene expression, secondary structures and DNA lesions. Completeness and accuracy of DNA replication are crucial to maintain genome integrity, limiting transformation in normal cells and offering targeting opportunities for proliferating cancer cells. DNA replication is thus tightly coordinated with chromatin dynamics and 3D genome architecture, and we are only beginning to understand the underlying molecular mechanisms. While much has recently been discovered on how DNA replication initiation is organised and modulated in different genomic regions and nuclear territories-the so-called "DNA replication program"-we know much less on how the elongation of ongoing replication forks and particularly the response to replication obstacles is affected by the local nuclear organisation. Also, it is still elusive how specific components of nuclear architecture participate in the replication stress response. Here, we review known mechanisms and factors orchestrating replication initiation, and replication fork progression upon stress, focusing on recent evidence linking genome organisation and nuclear architecture with the cellular responses to replication interference, and highlighting open questions and future challenges to explore this exciting new avenue of research.

Myc Regulates Chromatin Decompaction and Nuclear Architecture during B Cell Activation.

50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long range to predominantly short range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells.

Biochemical Deconstruction and Reconstruction of Nuclear Matrix Reveals the Layers of Nuclear Organization.

Nuclear matrix (NuMat) is the fraction of the eukaryotic nucleus insoluble to detergents and high-salt extractions that manifests as a pan-nuclear fiber-granule network. NuMat consists of ribonucleoprotein complexes, members of crucial nuclear functional modules, and DNA fragments. Although NuMat captures the organization of nonchromatin nuclear space, very little is known about components organization within NuMat. To understand the organization of NuMat components, we subfractionated it with increasing concentrations of the chaotrope guanidinium hydrochloride (GdnHCl) and analyzed the proteomic makeup of the fractions. We observe that the solubilization of proteins at different concentrations of GdnHCl is finite and independent of the broad biophysical properties of the protein sequences. Looking at the extraction pattern of the nuclear envelope and nuclear pore complex, we surmise that this fractionation represents easily solubilized/loosely bound and difficultly solubilized/tightly bound components of NuMat. Microscopic analyses of the localization of key NuMat proteins across sequential GdnHCl extractions of in situ NuMat further elaborate on the divergent extraction patterns. Furthermore, we solubilized NuMat in 8M GdnHCl and upon removal of GdnHCl through dialysis, en masse renaturation leads to RNA-dependent self-assembly of fibrous structures. The major proteome component of the self-assembled fibers comes from the difficultly solubilized, tightly bound component. This fractionation of the NuMat reveals different organizational levels within it which may reflect the structural and functional organization of nuclear architecture.

Maintenance and dynamic reprogramming of chromatin organization during development.

Controlled transcription of genes is critical for cell differentiation and development. Gene expression regulation therefore involves a multilayered control from nucleosome composition in histone variants and their post-translational modifications to higher-order folding of chromatin fibers and chromatin interactions in nuclear space. Recent technological advances have allowed gaining insight into these mechanisms, the interplay between local and higher-order chromatin organization, and the dynamic changes that occur during stress response and developmental transitions. In this review, we will discuss chromatin organization from the nucleosome to its three-dimensional structure in the nucleus, and consider how these different layers of organization are maintained during the cell cycle or rapidly reprogrammed during development.