Editorial overview: Drivers, droplets, and deeds of nuclear genome organization

The spatial or three-dimensional (3D) architecture of the genome is intimately linked to the regulation of gene expression, and the mechanisms driving it remain an area of intense investigation. In this issue, we are joined by a group of leading experts in the field of spatial genome architecture and gene expression, who each offer their perspectives on a particular aspect and future of this evolving field. We begin the issue with articles on mechanisms and genomic elements that drive structural looping and folding of the genome and include contributions on recent advances in biochemical, computational, and imaging methods that inform current models of genome architecture. These are followed by a set of reviews that focus on genome organization within the nuclear space and the roles of nuclear bodies, scaffolds, and microenvironments in functional compartmentalization of the genome and in promoting architectural structure. Finally, we conclude with contributions on the functions of 3D genome architecture in developmental regulation, epigenetic inheritance, and responses to environmental signals, highlighting our growing understanding of how genome architecture informs the regulation of gene expression.

How to make loops, topologically associated domains, and compartments — structural mechanisms of genome architecture

Molecular and imaging studies over the last 2 decades have led to the current predominant model of hierarchical genome organization that begins with individual DNA loops, which are further organized into self-interacting modules of topologically associated domains (TADs), which can in turn belong to one of two types of large-scale A and B compartments. The interplay between these layers of genome architecture is complex, and their relationship to epigenetic states and transcriptional activity is at the center of many current research efforts. Yet, as the basic unit of genome organization is a genomic loop, which results from a contact between two separated genomic loci, any force that promotes or opposes such contacts or that extends or restricts the resulting loop plays an important role in genome architecture. These diverse architectural forces are the subject of several reviews of this issue.

One of the main pathways for setting up loops and shaping mammalian genomes is mediated by Structural maintenance of chromosomes (SMC) or cohesin proteins in a dynamic process of loop extrusion. Current models of this process and additional mechanisms that lead to the formation of chromatin loops are discussed extensively in a review by Magnitov and de Witt. A key aspect of loop extrusion is knowing when to stop, and the review highlights the most prominent factors, most notably, insulator protein CCCTC-binding factor (CTCF), that have been implicated as barriers of the loop extrusion process, as well as the interplay of loop extrusion with transcriptional wiring and large-scale chromatin compartments.

Arising from looping and self-interacting forces, TADs are another fundamental unit of genome organization. Although the relative stability of TADs in a cell population remains under investigation, their existence is remarkably conserved across species. Borders of TADs are demarcated by insulator proteins, such as CTCF, which can regulate enhancer wiring and have been widely implicated in partitioning TADs. Current models of how insulator proteins, in cooperation with transcription, shape TADs are the subject of a review by Bhattacharya et al. This timely discussion includes an overview of the mechanisms employed by Drosophila, which relies on a large variety of insulator proteins and may lack classic loop extrusion, highlighting the different strategies that can produce the conserved TAD organization.

To partition TADs and mediate looping interactions, insulator proteins target specific DNA elements in the genome. Interestingly, an additional class of genome-organizing DNA elements, termed tethering elements, were recently found to regulate a subset of looping interactions in the fly genome and are profiled in a review by Li and Levine. Tethering elements appear to promote specific types of loops, such as enhancer–promoter interactions within Hox genes, promoter–promoter interactions of gene paralogs, and ultra-long loops between distant TADs. Mechanistically, such elements appear to utilize GAGA factor (GAF) and Polycomb repressive complexes and may represent an additional driving force for setting up genome architecture across species.

Our understanding of genome architecture is bound by the detection resolution of the methodologies used to analyze it. Recent increases in the sequencing coverage and the resulting resolution of chromosome capture methods have expanded our model of hierarchical genome organization, particularly the current view of A and B compartments — a subject discussed by Harris and Rowley. Their discussion covers the latest evidence for suggested drivers of genome compartmentalization, such as DNA sequence, nuclear lamina, and transcriptional activity, underscoring the complexity of compartment formation and the potential existence of not two but multiple compartments. Equally important to understanding genome architecture are microscopy-based methods, and exciting recent advances in these are profiled by Woodworth and Lakadamyali. This review focuses on the latest live, super-resolution, and high-throughput imaging approaches and the power of integrating structural, epigenetic, and transcriptional information to obtain the full functional significance of gene looping events. Such technologies and the computational analysis needed to integrate them will continue to make critical insights into physiological functions of genome organization.

Organizing it all in nuclear space — nuclear bodies and microenvironments

In addition to regulators of structural looping and folding, a fundamental force that directs genome architecture is nuclear microenvironments or bodies, which interact with chromatin and promote functional compartmentalization of the genome. Many such structures exist in the nucleus and vary from organelle-like nuclear bodies such as nucleoli and paraspeckles to locus-specific microenvironments such as transcriptional hubs. Their formation relies on both protein factors and RNA, and as many recent studies have shown, often involves phase separation, creating local high concentrations of key regulatory molecules. The connection of such nuclear structures to genome architecture and gene expression is of great interest to the field, and this issue includes discussions of their functional roles and dynamic mechanisms that drive their establishment.

Nuclear microenvironments and their increasingly appreciated role in genome architecture are the subject of an illuminating review by Hayward-Lara et al., which discusses how the transient and dynamic nature of protein–DNA or DNA–DNA interactions can give rise to stable functional outputs via the local formation of microenvironments. Enhancer–promoter interactions are one well-documented example of such a paradox that can be reconciled by the existence of a separated microenvironment, which promotes repeated contacts or frequent binding of transcriptional regulators. Highlighting such models and recent high-resolution time-lapse studies that inform them, this discussion underscores the importance of determining parameters and mechanisms that define nuclear microenvironments to understanding gene regulation.

The emerging importance of phase separation in the formation and maintenance of repressive chromatin environments is the topic reviewed by Akilli et al. Phase separation is a ubiquitous process via which molecules in a solution can separate into a compositionally distinct phase. In biological settings, it often involves liquid–liquid phase separation (LLPS), resulting in biomolecular condensates that are enriched for specific regulatory factors but may limit the nonspecific entry of others. Constituents of both types of heterochromatic domains, the constitutive HP1-defined heterochromatin and the facultative Polycomb-bound heterochromatin, have been found to undergo LLPS. This review highlights these findings and considers compelling evidence that phase separation can help explain the epigenetic inheritance of repressive domains beyond the ‘read and write’ models of histone modifications alone.

The intriguing roles of phase separation in various aspects of genome organization are highlighted by multiple reviews of this issue, and understanding this role fully is likely to impact many mechanistic models of the genome architecture field. Decades of research have also pointed to critical roles of noncoding RNAs in the formation of nuclear bodies and in the nuclear organization of chromatin. These roles, particularly the ones carried out by long noncoding RNAs (lincRNAs) and their connection to biomolecular condensates, are discussed in a review by Palihati and Saitoh. Their discussion centers on the ability of lincRNAs to seed many distinct nuclear bodies and nuclear environments via the recruitment of RNA-binding proteins, many of which carry intrinsically disordered regions with a high propensity for phase separation. The authors highlight this critical interplay between lincRNAs, phase separation, and genome architecture in multiple contexts, including disease states.

Although many nuclear bodies have been described over the years, perhaps the most discernable and biophysically stable structure in the nucleus is the nuclear envelope (NE) — a double membrane that surrounds nuclear chromatin and is associated with a large and diverse proteome of its own. The interactions of the genome with the NE and the regulatory potential of this unique nuclear environment are discussed by Czapiewski and Schirmer. In addition to a well-known role of lamina-associated domains in organizing repressive compartment B chromatin, their discussion highlights intriguing evidence that the NE can regulate enhancer–promoter loops and restrict inter-TAD contacts. Given that the protein composition of the NE can be highly cell type–specific, NE–genome interactions are likely to impact multiple key aspects of cell type–specific genome architecture.

A distinct environment within the NE is the nuclear pore complex (NPC), which is a massive protein complex that creates a transport channel through the NE. In addition to mediating nucleocytoplasmic transport, the NPC contacts the genome, which has been shown to regulate gene expression, 3D genome architecture, and genome stability. The evolutionarily conserved role of the NPC in genome stability, DNA damage repair, and DNA metabolism is reviewed in a contribution by Simon et al. Their discussion examines the widely observed phenomenon of many types of DNA damage sites re-localizing to the NPC, where NPC components have been found to promote specific DNA repair pathways. Perhaps most consequential for global genome architecture is the identified NPC recruitment of R-loops, which have been implicated in the regulation of genome stability, transcription, and genomic looping.

Translating into function — genome architecture in development and physiology

The overarching goal of the genome architecture field is to connect structure to function. Changes in genome architecture during development and cellular or organismal responses to external signals have offered a window into understanding the functional outcomes of architectural processes. Yet, the regulation of gene expression involves complex transcriptional and chromatin-associated mechanisms that are integrated with 3D genome architecture, and the challenge to causally and directly link architectural events to developmental or physiological outcomes remains. Our issue concludes with contributions discussing the proposed roles of genome architecture in developmental and epigenetic processes, as well as in responses to environmental cues.

Since the early observations by T. Boveri, it has been proposed that chromosomes occupy distinct territories in the interphase nucleus. With the development of molecular, imaging, and cell biological tools, we now know that the spatial and architectural organization of chromosomes influence gene regulation and genome function. Kathrin Plath and colleagues provide an overview on the functional relevance of X chromosome architecture in mammalian dosage compensation. X chromosome inactivation (XCI) is a phenomenon through which one of the two X chromosomes in females is randomly chosen to be inactivated, equalizing X-linked gene expression between females and males. The XIST lincRNA plays a key role during the initiation of XCI and the establishment of the future inactive X chromosome. In their review, the authors discuss recent advances on how this critical lincRNA participates in the architectural changes of the inactive X and how this process influences the establishment of chromatin reconfiguration and compaction, fine-tuning distinct levels of gene silencing of X-linked genes. Continuing with the topic of XCI, Martitz and Schulz discuss recent advances in the understanding of the spatial organization of the inactive X chromosome. Their discussion considers how architectural features and key architectural mechanisms such as loop extrusion contribute to the topological organization of the X-inactivation center (Xic) and to the establishment of the compact spatial organization of the inactive X that promotes gene silencing. They also highlight the establishment of micro chromatin compartments on the X chromosome and how this process is coupled with replication timing and changes in chromatin structure.

The germline represents a particularly important developmental and epigenetic state, being the earliest point in an organism’s lifecycle and connecting generations to allow for any transgenerational epigenetic inheritance. Kitamura and Namekawa in their review focus on the epigenetic reprogramming in the male germline and discuss molecular components of epigenetic priming — a phenomenon that presets chromatin states that allow for rapid induction of gene expression programs in response to differentiation cues. They describe how the regulation of bivalent histone modifications, super-enhancer activity, and 3D chromatin organization prime genes for future transcriptional states, raising interesting questions on whether features of genome architecture are passed on to the next generation and whether they contribute to gene regulation during embryogenesis.

In addition to development, environmental factors such as nutrient availability, temperature changes, or pathogens have been found to impact 3D genome organization, although it remains a difficult task to untangle the direct effect of these environmental cues on genome architecture versus transcriptional activity. In their review, Pudelko and Cabianca focus on the recent advances on how environmental signals influence spatial chromatin organization and impact genome function. Specifically, they discuss recent literature on how genome architecture, including TADs, compartments, and nuclear position of genes, is regulated in response to changes in nutrient availability, temperature stress, and exposure to pathogens, hypoxia, and osmotic stress across different organisms, offering insight into the emerging role of genome architecture in cellular responses to these signals.

Environmental stimuli can also trigger long-term changes in transcriptional responses, and epigenetic transcriptional memory underlies the stable alteration of the transcriptional program in response to developmental or environmental stimuli. In their review, Ge and Brickner discuss the modes of epigenetic inheritance and the molecular basis of epigenetic transcriptional memory, including DNA methylation, lncRNA-mediated regulation, specific transcription factors, nuclear positioning, and posttranslational histone modifications. At the molecular level, transcriptional memory is most often associated with H3K4 di-methylation and with binding components of the NPC in various organisms, including yeast, Drosophila, and mammals. The authors consider the complex interplay of these factors in several examples of transcriptional epigenetic memory, highlighting the importance of epigenetic inheritance of transcriptional responses to cell–environment interactions and organismal fitness.

Biographies

Maya Capelson is an Associate Professor of Biology at the Department of Biology and the Cell and Molecular Biology Program at San Diego State University. Her lab’s work is focused on understanding the basic principles of nuclear genome organization and the roles of the nuclear pore complex in genome architecture, transcriptional regulation, and developmental processes.

Eda Yildirim is an Assistant Professor of Molecular Biology and Genetics at the College of Sciences of the Koç University in Istanbul, Turkey. She also holds an adjunct Assistant Professor position at the Department of Cell Biology of the Duke University School of Medicine. Her group’s work is focused on understanding the epigenetic mechanisms of long noncoding RNA and nuclear structure mediated gene regulation and genome function.