Human chromosomes have a complex 3D spatial organization in the cell nucleus, which comprises a hierarchy of physical interactions across genomic scales. Such an architecture serves important functional roles, as genes and their regulators have to physically interact to control gene regulation. However, the molecular mechanisms underlying the formation of those contacts remain poorly understood. Here, we describe a polymer-physics-based approach to investigate the machinery shaping genome folding and function. In silico model predictions on DNA single-molecule 3D structures are validated against independent super-resolution single-cell microscopy data, supporting a scenario whereby chromosome architecture is controlled by thermodynamics mechanisms of phase separation. Finally, as an application of our methods, the validated single-polymer conformations of the theory are used to benchmark powerful technologies to probe genome structure, such as Hi-C, SPRITE, and GAM.

For a coherent response to environmental changes, bacterial evolution has formed a complex transcriptional regulatory system comprising classical DNA binding proteins sigma factors and modulation of DNA topology. In this study, we investigate replication-induced gene copy numbers - a regulatory concept that is unlike the others not based on modulation of promoter activity but on replication dynamics. We show that a large fraction of genes are predominantly affected by transient copy numbers and identify cellular functions and central pathways governed by this mechanism in Escherichia coli. Furthermore, we show quantitatively that the previously observed spatio-temporal expression pattern between different growth phases mainly emerges from transient chromosomal copy numbers. We extend the analysis to the plant pathogen Dickeya dadantii and the biotechnologically relevant organism Vibrio natriegens. The analysis reveals a connection between growth phase dependent gene expression and evolutionary gene migration in these species. A further extension to the bacterial kingdom indicates that chromosome evolution is governed by growth rate related transient copy numbers.

Gene expression and cell fate determination require precise and coordinated epigenetic regulation. The complex three-dimensional (3D) genome organization plays a critical role in transcription in myriad biological processes. A wide range of architectural features of the 3D genome, including chromatin loops, topologically associated domains (TADs), chromatin compartments, and phase separation, together regulate the chromatin state and transcriptional activity at multiple levels. With the help of 3D genome informatics, recent biochemistry and imaging approaches based on different strategies have revealed functional interactions among biomacromolecules, even at the single-cell level. Here, we review the occurrence, mechanistic basis, and functional implications of dynamic genome organization, and outline recent experimental and computational approaches for profiling multiscale genome architecture to provide robust tools for studying the 3D genome.

Actin is a highly conserved protein in mammals. The actin dynamics is regulated by actin-binding proteins and actin-related proteins. Nuclear actin and these regulatory proteins participate in multiple nuclear processes, including chromosome architecture organization, chromatin remodeling, transcription machinery regulation, and DNA repair. It is well known that the dysfunctions of these processes contribute to the development of cancer. Moreover, emerging evidence has shown that the deregulated actin dynamics is also related to cancer. This chapter discusses how the deregulation of nuclear actin dynamics contributes to tumorigenesis via such various nuclear events.

The eukaryotic genome is partitioned into distinct topological domains separated by boundary elements. Emerging data support the concept that several well-established nuclear compartments are ribonucleoprotein condensates assembled through the physical process of phase separation. Here, based on our demonstration that chemical disruption of nuclear condensate assembly weakens the insulation properties of a specific subset (∼20%) of topologically associated domain (TAD) boundaries, we report that the disrupted boundaries are characterized by a high level of transcription and striking spatial clustering. These topological boundary regions tend to be spatially associated, even interchromosomally, segregate with nuclear speckles, and harbor a specific subset of \"housekeeping\" genes widely expressed in diverse cell types. These observations reveal a previously unappreciated mode of genome organization mediated by conserved boundary elements harboring highly and widely expressed transcription units and associated transcriptional condensates.

In this interview, Professor Jane Skok speaks with Storm Johnson, commissioning editor for Epigenomics, on her work to date in the field of chromosome architecture and regulatory elements. Jane Skok\'s lab uses sophisticated microscopic techniques to visualize recombination in individual cells, tracing the dynamic changes in chromosome architecture and nuclear location at different stages of this complex process. This line of research unites two lifelong passions: science and art. After completing her PhD in immunology and genetics at the Imperial Cancer Research Fund in Lincoln\'s Inn Fields, Dr Skok took 12 years off and pursued training in art while caring for her young children. She then returned to science, joining David Gray\'s lab at Imperial College London as a postdoctoral fellow to study B cell biology and acquired expertise in Mandy Fisher\'s lab to understand how nuclear organization of the antigen receptor genes regulate V(D)J recombination and allelic exclusion. Dr Skok continued to pursue these questions in her own lab at University College London and elucidated the roles of Pax5, locus contraction and nuclear subcompartmentalization in maintaining allelic exclusion. In 2006, Dr Skok was recruited to New York University School of Medicine, where her lab has revealed the activities of several signaling factors in guiding B cell development and they made the surprising discovery that the RAG proteins and the DNA damage response factor ATM help ensure allelic exclusion at the immunoglobulin gene loci. More recently, those at the Skok lab have turned their attention to understanding how localized and long-range chromatin contacts impact gene regulation in health and disease settings.

Genome sequencing methods and assembly tools have improved dramatically since the 2013 publication of draft genome assemblies for the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae). We conducted proximity ligation library sequencing and scaffolding to improve contiguity, and then used linkage mapping and recent bioinformatic tools for correction and further improvement. The new assemblies have dramatically improved contiguity and gaps compared to the originals: N50 values increased 26- to 36-fold, and the number of gaps were reduced by half. Ninety per cent of the content of the assemblies is now contained in 12 and 11 scaffolds for the female and male assemblies, respectively. Based on linkage mapping information, the 12 largest scaffolds in both assemblies represent all 11 autosomal chromosomes and the neo-X chromosome. These assemblies now have nearly chromosome-sized scaffolds and will be instrumental for studying genomic architecture, chromosome evolution, population genomics, functional genomics, and adaptation in this and other pest insects. We also identified regions in two chromosomes, including the ancestral-X portion of the neo-X chromosome, with elevated differentiation between northern and southern Canadian populations.

Cyclins and CDKs (Cyclin Dependent Kinases) are key players in the biology of eukaryotic cells, representing hubs for the orchestration of physiological conditions with cell cycle progression. Furthermore, as in the case of meiosis, cyclins and CDKs have acquired novel functions unrelated to this primal role in driving the division cycle. Meiosis is a specialized developmental program that ensures proper propagation of the genetic information to the next generation by the production of gametes with accurate chromosome content, and meiosis-specific cyclins are widespread in evolution. We have explored the diversification of CDK functions studying the meiosis-specific Crs1 cyclin in fission yeast. In addition to the reported role in DSB (Double Strand Break) formation, this cyclin is required for meiotic S-phase progression, a canonical role, and to maintain the architecture of the meiotic chromosomes. Crs1 localizes at the SPB (Spindle Pole Body) and is required to stabilize the cluster of telomeres at this location (bouquet configuration), as well as for normal SPB motion. In addition, Crs1 exhibits CDK(Cdc2)-dependent kinase activity in a biphasic manner during meiosis, in contrast to a single wave of protein expression, suggesting a post-translational control of its activity. Thus, Crs1 displays multiple functions, acting both in cell cycle progression and in several key meiosis-specific events.

Mitotic spindle is a self-assembling macromolecular machine responsible for the faithful segregation of chromosomes during cell division. Assembly of the spindle is believed to be governed by the \'Search & Capture\' (S&C) principle in which dynamic microtubules explore space in search of kinetochores while the latter capture microtubules and thus connect chromosomes to the spindle. Due to the stochastic nature of the encounters between kinetochores and microtubules, the time required for incorporating all chromosomes into the spindle is profoundly affected by geometric constraints, such as the size and shape of kinetochores as well as their distribution in space at the onset of spindle assembly. In recent years, several molecular mechanisms that control these parameters have been discovered. It is now clear that stochastic S&C takes place in structured space, where components are optimally distributed and oriented to minimize steric hindrances. Nucleation of numerous non-centrosomal microtubules near kinetochores accelerates capture, while changes in the kinetochore architecture at various stages of spindle assembly promote proper connection of sister kinetochores to the opposite spindle poles. Here we discuss how the concerted action of multiple facilitating mechanisms ensure that the spindle assembles rapidly yet with a minimal number of errors.

Nuclear speckles are prominent nuclear bodies that contain proteins and RNA involved in gene expression. Although links between nuclear speckles and gene activation are emerging, the mechanisms regulating association of genes with speckles are unclear. We find that speckle association of p53 target genes is driven by the p53 transcription factor. Focusing on p21, a key p53 target, we demonstrate that speckle association boosts expression by elevating nascent RNA amounts. p53-regulated speckle association did not depend on p53 transactivation functions but required an intact proline-rich domain and direct DNA binding, providing mechanisms within p53 for regulating gene-speckle association. Beyond p21, a substantial subset of p53 targets have p53-regulated speckle association. Strikingly, speckle-associating p53 targets are more robustly activated and occupy a distinct niche of p53 biology compared with non-speckle-associating p53 targets. Together, our findings illuminate regulated speckle association as a mechanism used by a transcription factor to boost gene expression.