Active RB causes visible changes in nuclear organization

RB restricts G1/S progression by inhibiting E2F. Here, we show that sustained expression of active RB, and prolonged G1 arrest, causes visible changes in chromosome architecture that are not directly associated with E2F inhibition. Using FISH probes against two euchromatin RB-associated regions, two heterochromatin domains that lack RB-bound loci, and two whole-chromosome probes, we found that constitutively active RB (ΔCDK-RB) promoted a more diffuse, dispersed, and scattered chromatin organization. These changes were RB dependent, were driven by specific isoforms of monophosphorylated RB, and required known RB-associated activities. ΔCDK-RB altered physical interactions between RB-bound genomic loci, but the RB-induced changes in chromosome architecture were unaffected by dominant-negative DP1. The RB-induced changes appeared to be widespread and influenced chromosome localization within nuclei. Gene expression profiles revealed that the dispersion phenotype was associated with an increased autophagy response. We infer that, after cell cycle arrest, RB acts through noncanonical mechanisms to significantly change nuclear organization, and this reorganization correlates with transitions in cellular state.

Lamin C is required to establish genome organization after mitosis

Background The dynamic 3D organization of the genome is central to gene regulation and development. The nuclear lamina influences genome organization through the tethering of lamina-associated domains (LADs) to the nuclear periphery. Evidence suggests that lamins A and C are the predominant lamins involved in the peripheral association of LADs, potentially serving different roles. Results Here, we examine chromosome architecture in mouse cells in which lamin A or lamin C are downregulated. We find that lamin C, and not lamin A, is required for the 3D organization of LADs and overall chromosome organization. Striking differences in localization are present as cells exit mitosis and persist through early G1 and are linked to differential phosphorylation. Whereas lamin A associates with the nascent nuclear envelope (NE) during telophase, lamin C remains in the interior, surrounding globular LAD aggregates enriched on euchromatic regions. Lamin C association with the NE is delayed until several hours into G1 and correlates temporally and spatially with the post-mitotic NE association of LADs. Post-mitotic LAD association with the NE, and global 3D genome organization, is perturbed only in cells depleted of lamin C, and not lamin A. Conclusions Lamin C regulates LAD dynamics during exit from mitosis and is a key regulator of genome organization in mammalian cells. This reveals an unexpectedly central role for lamin C in genome organization, including inter-chromosomal LAD-LAD segregation and LAD scaffolding at the NE, raising intriguing questions about the individual and overlapping roles of lamin A/C in cellular function and disease.

ATM-dependent formation of a novel chromatin compartment regulates the Response to DNA Double Strand Breaks and the biogenesis of translocations

DNA Double-Strand Breaks (DSBs) repair is essential to safeguard genome integrity but the contribution of chromosome folding into this process remains elusive. Here we unveiled basic principles of chromosome dynamics upon DSBs in mammalian cells, controlled by key kinases from the DNA Damage Response. We report that ATM is responsible for the reinforcement of topologically associating domains (TAD) that experience a DSB. ATM further drives the formation of a new chromatin sub-compartment (″D″ compartment) upon clustering of damaged TADs decorated with γH2AX and 53BP1. ″D″ compartment formation mostly occurs in G1, is independent of cohesin and is enhanced upon DNA-PK pharmacological inhibition. Importantly, a subset of DNA damage responsive genes that are upregulated following DSBs also physically localize in the D sub-compartment and this ensures their optimal activation, providing a function for DSB clustering in activating the DNA Damage Response. However, these DSB-induced changes in genome organization also come at the expense of an increased translocations rate, which we could also detect on cancer genomes. Overall, our work provides a function for DSB-induced compartmentalization in orchestrating the DNA Damage Response and highlights the critical impact of chromosome architecture in genomic instability.

The Cohesin Complex and Its Interplay with Non-Coding RNAs

The cohesin complex is a multi-subunit protein complex initially discovered for its role in sister chromatid cohesion. However, cohesin also has several other functions and plays important roles in transcriptional regulation, DNA double strand break repair, and chromosome architecture thereby influencing gene expression and development in organisms from yeast to man. While most of these functions rely on protein–protein interactions, post-translational protein, as well as DNA modifications, non-coding RNAs are emerging as additional players that facilitate and modulate the function or expression of cohesin and its individual components. This review provides a condensed overview about the architecture as well as the function of the cohesin complex and highlights its multifaceted interplay with both short and long non-coding RNAs.

The 4D Nucleome Data Portal: a resource for searching and visualizing curated nucleomics data

The 4D Nucleome (4DN) Network aims to elucidate the complex structure and organization of chromosomes in the nucleus and the impact of their disruption in disease biology. We present the 4DN Data Portal (https://data.4dnucleome.org/), a repository for datasets generated in the 4DN network and relevant external datasets. Datasets were generated with a wide range of experiments, including chromosome conformation capture assays such as Hi-C and other innovative sequencing and microscopy-based assays probing chromosome architecture. All together, the 4DN data portal hosts more than 1800 experiment sets and 34000 files. Results of sequencing-based assays from different laboratories are uniformly processed and quality-controlled. The portal interface allows easy browsing, filtering, and bulk downloads, and the integrated HiGlass genome browser allows interactive visualization and comparison of multiple datasets. The 4DN data portal represents a primary resource for chromosome contact and other nuclear architecture data for the scientific community.

A Proposed Unified Mitotic Chromosome Architecture

A molecular architecture is proposed for an example mitotic chromosome, human Chromosome 10. This architecture is built on a previously described interphase chromosome structure based on Cryo-EM cellular tomography (1), thus unifying chromosome structure throughout the complete mitotic cycle. The basic organizational principle, for mitotic chromosomes, is specific coiling of the 11-nm nucleosome fiber into large scale approximately 200 nm structures (a Slinky (2, motif cited in 3) in interphase, and then further modification and subsequent additional coiling for the final structure. The final mitotic chromosome architecture accounts for the dimensional values as well as the well known cytological configurations. In addition, proof is experimentally provided, by digital PCR technology, that G1 T-cell nuclei are diploid, thus one DNA molecule per chromosome. Many nucleosome linker DNA sequences, the promotors and enhancers, are suggestive of optimal exposure on the surfaces of the large-scale coils.

Four-dimensional chromosome reconstruction elucidates the spatiotemporal reorganization of the mammalian X chromosome

Chromosomes are segmented into domains and compartments, but how these structures are spatially related in three dimensions (3D) is unclear. Here, we developed tools that directly extract 3D information from Hi-C experiments and integrate the data across time. With our “4DHiC” method, we use X chromosome inactivation (XCI) as a model to examine the time evolution of 3D chromosome architecture during large-scale changes in gene expression. Our modeling resulted in several insights. Both A/B and S1/S2 compartments divide the X chromosome into hemisphere-like structures suggestive of a spatial phase-separation. During the XCI, the X chromosome transits through A/B, S1/S2, and megadomain structures by undergoing only partial mixing to assume new structures. Interestingly, when an active X chromosome (Xa) is reorganized into an inactive X chromosome (Xi), original underlying compartment structures are not fully eliminated within the Xi superstructure. Our study affirms slow mixing dynamics in the inner chromosome core and faster dynamics near the surface where escapees reside. Once established, the Xa and Xi resemble glassy polymers where mixing no longer occurs. Finally, Xist RNA molecules initially reside within the A compartment but transition to the interface between the A and B hemispheres and then spread between hemispheres via both surface and core to establish the Xi.

Meiotic Chromosome Dynamics in Zebrafish

Recent studies in zebrafish have revealed key features of meiotic chromosome dynamics, including clustering of telomeres in the bouquet configuration, biogenesis of chromosome axis structures, and the assembly and disassembly of the synaptonemal complex that aligns homologs end-to-end. The telomere bouquet stage is especially pronounced in zebrafish meiosis and sub-telomeric regions play key roles in mediating pairing and homologous recombination. In this review, we discuss the temporal progression of these events in meiosis prophase I and highlight the roles of proteins associated with meiotic chromosome architecture in homologous recombination. Finally, we discuss the interplay between meiotic mutants and gonadal sex differentiation and future research directions to study meiosis in living cells, including cell culture.

Dynamics of the compartmentalized Streptomyces chromosome during metabolic differentiation

Bacteria of the genus Streptomyces are prolific producers of specialized metabolites, including antibiotics. The linear chromosome includes a central region harboring core genes, as well as extremities enriched in specialized metabolite biosynthetic gene clusters. Here, we show that chromosome structure in Streptomyces ambofaciens correlates with genetic compartmentalization during exponential phase. Conserved, large and highly transcribed genes form boundaries that segment the central part of the chromosome into domains, whereas the terminal ends tend to be transcriptionally quiescent compartments with different structural features. The onset of metabolic differentiation is accompanied by a rearrangement of chromosome architecture, from a rather ‘open’ to a ‘closed’ conformation, in which highly expressed specialized metabolite biosynthetic genes form new boundaries. Thus, our results indicate that the linear chromosome of S. ambofaciens is partitioned into structurally distinct entities, suggesting a link between chromosome folding, gene expression and genome evolution.