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While colocalization within a bacterial operon enables coexpression of the constituent genes, the mechanistic logic of clustering of nonhomologous monocistronic genes in eukaryotes is not immediately obvious. Biosynthetic gene clusters that encode pathways for specialized metabolites are an exception to the classical eukaryote rule of random gene location and provide paradigmatic exemplars with which to understand eukaryotic cluster dynamics and regulation. Here, using 3C, Hi-C, and Capture Hi-C (CHi-C) organ-specific chromosome conformation capture techniques along with high-resolution microscopy, we investigate how chromosome topology relates to transcriptional activity of clustered biosynthetic pathway genes in Arabidopsis thaliana. Our analyses reveal that biosynthetic gene clusters are embedded in local hot spots of 3D contacts that segregate cluster regions from the surrounding chromosome environment. The spatial conformation of these cluster-associated domains differs between transcriptionally active and silenced clusters. We further show that silenced clusters associate with heterochromatic chromosomal domains toward the periphery of the nucleus, while transcriptionally active clusters relocate away from the nuclear periphery. Examination of chromosome structure at unrelated clusters in maize, rice, and tomato indicates that integration of clustered pathway genes into distinct topological domains is a common feature in plant genomes. Our results shed light on the potential mechanisms that constrain coexpression within clusters of nonhomologous eukaryotic genes and suggest that gene clustering in the one-dimensional chromosome is accompanied by compartmentalization of the 3D chromosome.

SMC complexes, such as condensin or cohesin, organize chromatin throughout the cell cycle by a process known as loop extrusion. SMC complexes reel in DNA, extruding and progressively growing DNA loops. Modeling assuming two-sided loop extrusion reproduces key features of chromatin organization across different organisms. In vitro single-molecule experiments confirmed that yeast condensins extrude loops, however, they remain anchored to their loading sites and extrude loops in a ‘one-sided’ manner. We therefore simulate one-sided loop extrusion to investigate whether ‘one-sided’ complexes can compact mitotic chromosomes, organize interphase domains, and juxtapose bacterial chromosomal arms, as can be done by ‘two-sided’ loop extruders. While one-sided loop extrusion cannot reproduce these phenomena, variants can recapitulate in vivo observations. We predict that SMC complexes in vivo constitute effectively two-sided motors or exhibit biased loading and propose relevant experiments. Our work suggests that loop extrusion is a viable general mechanism of chromatin organization. The different molecules of DNA in a cell are called chromosomes, and they change shape dramatically when cells divide. Ordinarily, chromosomes are packaged by proteins called histones to make thick fibres called chromatin. Chromatin fibres are further folded into a sparse collection of loops. These loops are important not only to make genetic material fit inside a cell, but also to make distant regions of the chromosomes interact with each other, which is important to regulate gene activities. The fibres compact to prepare for cell division: they fold into a much denser series of loops. This is a remarkable physical feat in which tiny protein machines wrangle lengthy strands of DNA. A process called loop extrusion could explain how chromatin folding works. In this process, ring-like protein complexes known as SMC complexes would act as motors that can form loops. SMC complexes could bind a chromatin fibre and reel it in to form the loops, with the density of loops increasing before cell division to further compact the chromosomes. Looping by SMC complexes has been observed in a variety of cell types, including mammalian and bacterial cells. From these studies, loop extrusion is generally assumed to be ‘two-sided’. This means that each SMC complex reels in the chromatin on both sides of it, thus growing the chromatin loop. However, imaging individual SMC complexes bound to single molecules of DNA showed that extrusion can be asymmetric, or ‘one-sided’. These observations show the SMC complex remains anchored in place and the chromatin is reeled in and extruded by only one side of the complex. So Banigan, van den Berg, Brandão et al. created a computer model to test whether the mechanism of one-sided extrusion could produce chromosomes that are organised, compact, and ready for cell division, like two-sided extrusion can. To answer this question, Banigan, van den Berg, Brandão et al. analysed imaging experiments and data that had been collected using a technique that captures how chromatin fibres are arranged inside cells. This was paired with computer simulations of chromosomes bound by SMC protein complexes. The simulations and analysis found that the simplest one-sided loop extrusion complexes generally cannot reproduce the same patterns of chromatin loops as two-sided complexes. However, a few specific variations of one-sided extrusion can actually recapitulate correct chromatin folding and organisation. These results show that some aspects of chromosome organization can be attained by one-sided extrusion, but many require two-sided extrusion. Banigan, van den Berg, Brandão et al. explain how the simulated mechanisms of loop extrusion could be consistent with seemingly contradictory observations from different sets of experiments. Altogether, they demonstrate that loop extrusion is a viable general mechanism to explain chromatin organisation, and that it likely possesses physical capabilities that have yet to be observed experimentally.

Abstract Bdellovibrio bacteriovorus, an obligate predatory Gram-negative bacterium that proliferates inside and kills other Gram-negative bacteria, was discovered more than 60 years ago. However, we have only recently begun to understand the detailed cell biology of this proficient bacterial killer. Bdellovibrio bacteriovorus exhibits a peculiar life cycle and bimodal proliferation, and thus represents an attractive model for studying novel aspects of bacterial cell biology. The life cycle of B. bacteriovorus consists of two phases: a free-living nonreplicative attack phase and an intracellular reproductive phase. During the reproductive phase, B. bacteriovorus grows as an elongated cell and undergoes binary or nonbinary fission, depending on the prey size. In this review, we discuss: (1) how the chromosome structure of B. bacteriovorus is remodeled during its life cycle; (2) how its chromosome replication dynamics depends on the proliferation mode; (3) how the initiation of chromosome replication is controlled during the life cycle, and (4) how chromosome replication is spatiotemporally coordinated with the proliferation program. A predatory bacterium, Bdellovibrio bacteriovorus possesses a tightly packed chromosome and exhibits a unique “asynchronous mode” of replication (re)initiation.

To ensure proper gene regulation within constrained nuclear space, chromosomes facilitate access to transcribed regions, while compactly packaging all other information. Recent studies revealed that chromosomes are organized into megabase‐scale domains that demarcate active and inactive genetic elements, suggesting that compartmentalization is important for genome function. Here, we show that very specific long‐range interactions are anchored by cohesin/CTCF sites, but not cohesin‐only or CTCF‐only sites, to form a hierarchy of chromosomal loops. These loops demarcate topological domains and form intricate internal structures within them. Post‐mitotic nuclei deficient for functional cohesin exhibit global architectural changes associated with loss of cohesin/CTCF contacts and relaxation of topological domains. Transcriptional analysis shows that this cohesin‐dependent perturbation of domain organization leads to widespread gene deregulation of both cohesin‐bound and non‐bound genes. Our data thereby support a role for cohesin in the global organization of domain structure and suggest that domains function to stabilize the transcriptional programmes within them. Chromosomal compartmentalization has been recognized as important for genome function. High‐resolution techniques such as Hi‐C, ChIP‐ and 4C‐seq offer novel insights into cohesin's dynamic role in shaping the nuclear architecture.

ABSTRACT Chromosomes are dynamic entities, whose organization and structure depend on the concerted activity of DNA-binding proteins and DNA-processing enzymes. In bacteria, chromosome replication, segregation, compaction and transcription are all occurring simultaneously, and to ensure that these processes are appropriately coordinated, all bacteria employ a mix of well-conserved and species-specific proteins. Unusually, Streptomyces bacteria have large, linear chromosomes and life cycle stages that include multigenomic filamentous hyphae and unigenomic spores. Moreover, their prolific secondary metabolism yields a wealth of bioactive natural products. These different life cycle stages are associated with profound changes in nucleoid structure and chromosome compaction, and require distinct repertoires of architectural—and regulatory—proteins. To date, chromosome organization is best understood during Streptomyces sporulation, when chromosome segregation and condensation are most evident, and these processes are coordinated with synchronous rounds of cell division. Advances are, however, now being made in understanding how chromosome organization is achieved in multigenomic hyphal compartments, in defining the functional and regulatory interplay between different architectural elements, and in appreciating the transcriptional control exerted by these ‘structural’ proteins. Streptomyces employ distinct repertoires of proteins to organize their chromosomes throughout their complex life cycle.

In all living organisms, it is essential to transmit genetic information faithfully to the next generation. The SMC-ParAB-parS system is widely employed for chromosome segregation in bacteria. A DNA-binding protein ParB nucleates on parS sites and must associate with neighboring DNA, a process known as spreading, to enable efficient chromosome segregation. Despite its importance, how the initial few ParB molecules nucleating at parS sites recruit hundreds of further ParB to spread is not fully understood. Here, we reconstitute a parS-dependent ParB spreading event using purified proteins from Caulobacter crescentus and show that CTP is required for spreading. We further show that ParB spreading requires a closed DNA substrate, and a DNA-binding transcriptional regulator can act as a roadblock to attenuate spreading unidirectionally in vitro. Our biochemical reconstitutions recapitulate many observed in vivo properties of ParB and opens up avenues to investigate the interactions between ParB-parS with ParA and SMC.

Bacterial chromosomes are organized by condensins, such as Structural Maintenance of Chromosomes (SMC) proteins. In Streptomyces , a genus of sporulating bacteria, SMC proteins align chromosomal arms and promote efficient compaction of chromosomal DNA during spore formation. We hypothesized that disrupting nucleoid architecture by deleting the smc gene would affect the positioning of the origin of replication ( oriC ) or the process of chromosome replication during spore germination. To test this hypothesis, we conducted marker frequency analyses and microscopy studies to observe the positioning of labelled oriC and replisomes in both wild type and Δ smc backgrounds. Additionally, we investigated the positioning of three chromosomal loci in early vegetative cells. Our results indicate that the deletion of smc impairs chromosome replication and hinders germ tube development. Furthermore, detailed analysis of chromosome organization revealed that, in the absence of SMC, the oriC region becomes mispositioned within the nucleoid. These findings underscore the important role of SMC in maintaining nucleoid architecture during the early growth stages of Streptomyces .

Chromosome conformation capture methods, such as Hi-C, have been used to profile chromosome organization from a wide variety of biosamples and conditions; however, existing methods for analyzing such datasets have disadvantages for large-scale integrative studies of long-range interactions. To address this shortcoming, we introduce an analytical framework, jointly-hic, that computes harmonized projections across arbitrarily many contact frequency matrices, suitable for integrative studies of compartmentalization and long-range interactions. Our approach produces robust and directly comparable first and higher-order principal component scores that collectively capture biologically meaningful information beyond traditional A/B compartment scores.

SMC complexes are widely conserved ATP-powered DNA-loop-extrusion motors indispensable for organizing and faithfully segregating chromosomes. How SMC complexes translocate along DNA for loop extrusion and what happens when two complexes meet on the same DNA molecule is largely unknown. Revealing the origins and the consequences of SMC encounters is crucial for understanding the folding process not only of bacterial, but also of eukaryotic chromosomes. Here, we uncover several factors that influence bacterial chromosome organization by modulating the probability of such clashes. These factors include the number, the strength, and the distribution of Smc loading sites, the residency time on the chromosome, the translocation rate, and the cellular abundance of Smc complexes. By studying various mutants, we show that these parameters are fine-tuned to reduce the frequency of encounters between Smc complexes, presumably as a risk mitigation strategy. Mild perturbations hamper chromosome organization by causing Smc collisions, implying that the cellular capacity to resolve them is limited. Altogether, we identify mechanisms that help to avoid Smc collisions and their resolution by Smc traversal or other potentially risky molecular transactions.

Synthetic nanobodies—also called sybodies—have proven valuable for stabilizing conformations of purified proteins, advancing structural and functional studies for example of transmembrane proteins. However, their utility in modulating protein function in living cells has remained less well explored. Structural Maintenance of Chromosomes (SMC) complexes facilitate chromosome organization, a fundamental process in all domains of life. In this study, we target the bacterial SMC complex, Smc-ScpAB, in Bacillus subtilis with synthetic nanobodies, aiming to identify key functional regions of the protein complex in a largely unbiased manner. We first isolate sybodies that specifically bind purified Smc-ScpAB and then express them in B. subtilis to select binders capable of disrupting Smc-ScpAB function, leading to chromosome segregation defects and cell death. Mapping and biochemical characterization show that the 14 disruptive sybodies belong to one of three library designs, target the Smc subunit near the same coiled coil arm interface and modulate its ATPase activity in two principal ways, highlighting the mid-region of the Smc coiled coil as critical feature of the SMC-DNA folding process. These findings underscore the potential of sybodies—and, by extension, designed binders—as versatile tools for probing dynamic protein function in living cells.