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1. Generating Chromosome Geometries in a Minimal Cell From Cryo-Electron Tomograms and Chromosome Conformation Capture Maps

   https://doi.org/10.3389/fmolb.2021.644133  

   Gilbert, Benjamin R. ; Thornburg, Zane R. ; Lam, Vinson ; Rashid, Fatema-Zahra M. ; Glass, John I. ; Villa, Elizabeth ; Dame, Remus T. ; Luthey-Schulten, Zaida ( July 2021 , Frontiers in Molecular Biosciences)

   null (Ed.)

   JCVI-syn3A is a genetically minimal bacterial cell, consisting of 493 genes and only a single 543 kbp circular chromosome. Syn3A’s genome and physical size are approximately one-tenth those of the model bacterial organism Escherichia coli ’s, and the corresponding reduction in complexity and scale provides a unique opportunity for whole-cell modeling. Previous work established genome-scale gene essentiality and proteomics data along with its essential metabolic network and a kinetic model of genetic information processing. In addition to that information, whole-cell, spatially-resolved kinetic models require cellular architecture, including spatial distributions of ribosomes and the circular chromosome’s configuration. We reconstruct cellular architectures of Syn3A cells at the single-cell level directly from cryo-electron tomograms, including the ribosome distributions. We present a method of generating self-avoiding circular chromosome configurations in a lattice model with a resolution of 11.8 bp per monomer on a 4 nm cubic lattice. Realizations of the chromosome configurations are constrained by the ribosomes and geometry reconstructed from the tomograms and include DNA loops suggested by experimental chromosome conformation capture (3C) maps. Using ensembles of simulated chromosome configurations we predict chromosome contact maps for Syn3A cells at resolutions of 250 bp and greater and compare them to the experimental maps. Additionally, the spatial distributions of ribosomes and the DNA-crowding resulting from the individual chromosome configurations can be used to identify macromolecular structures formed from ribosomes and DNA, such as polysomes and expressomes. 

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2. Braiding topology and the energy landscape of chromosome organization proteins

   https://doi.org/10.1073/pnas.1917750117  

   Krepel, Dana ; Davtyan, Aram ; Schafer, Nicholas P. ; Wolynes, Peter G. ; Onuchic, José N. ( December 2019 , Proceedings of the National Academy of Sciences)

   Assemblies of structural maintenance of chromosomes (SMC) proteins and kleisin subunits are essential to chromosome organization and segregation across all kingdoms of life. While structural data exist for parts of the SMC−kleisin complexes, complete structures of the entire complexes have yet to be determined, making mechanistic studies difficult. Using an integrative approach that combines crystallographic structural information about the globular subdomains, along with coevolutionary information and an energy landscape optimized force field (AWSEM), we predict atomic-scale structures for several tripartite SMC−kleisin complexes, including prokaryotic condensin, eukaryotic cohesin, and eukaryotic condensin. The molecular dynamics simulations of the SMC−kleisin protein complexes suggest that these complexes exist as a broad conformational ensemble that is made up of different topological isomers. The simulations suggest a critical role for the SMC coiled-coil regions, where the coils intertwine with various linking numbers. The twist and writhe of these braided coils are coupled with the motion of the SMC head domains, suggesting that the complexes may function as topological motors. Opening, closing, and translation along the DNA of the SMC−kleisin protein complexes would allow these motors to couple to the topology of DNA when DNA is entwined with the braided coils.

    

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3. From telomere to telomere: The transcriptional and epigenetic state of human repeat elements

   https://doi.org/10.1126/science.abk3112  

   Hoyt, Savannah J. ; Storer, Jessica M. ; Hartley, Gabrielle A. ; Grady, Patrick G. ; Gershman, Ariel ; de Lima, Leonardo G. ; Limouse, Charles ; Halabian, Reza ; Wojenski, Luke ; Rodriguez, Matias ; et al ( April 2022 , Science)

   INTRODUCTION Transposable elements (TEs), repeat expansions, and repeat-mediated structural rearrangements play key roles in chromosome structure and species evolution, contribute to human genetic variation, and substantially influence human health through copy number variants, structural variants, insertions, deletions, and alterations to gene transcription and splicing. Despite their formative role in genome stability, repetitive regions have been relegated to gaps and collapsed regions in human genome reference GRCh38 owing to the technological limitations during its development. The lack of linear sequence in these regions, particularly in centromeres, resulted in the inability to fully explore the repeat content of the human genome in the context of both local and regional chromosomal environments. RATIONALE Long-read sequencing supported the complete, telomere-to-telomere (T2T) assembly of the pseudo-haploid human cell line CHM13. This resource affords a genome-scale assessment of all human repetitive sequences, including TEs and previously unknown repeats and satellites, both within and outside of gaps and collapsed regions. Additionally, a complete genome enables the opportunity to explore the epigenetic and transcriptional profiles of these elements that are fundamental to our understanding of chromosome structure, function, and evolution. Comparative analyses reveal modes of repeat divergence, evolution, and expansion or contraction with locus-level resolution. RESULTS We implemented a comprehensive repeat annotation workflow using previously known human repeats and de novo repeat modeling followed by manual curation, including assessing overlaps with gene annotations, segmental duplications, tandem repeats, and annotated repeats. Using this method, we developed an updated catalog of human repetitive sequences and refined previous repeat annotations. We discovered 43 previously unknown repeats and repeat variants and characterized 19 complex, composite repetitive structures, which often carry genes, across T2T-CHM13. Using precision nuclear run-on sequencing (PRO-seq) and CpG methylated sites generated from Oxford Nanopore Technologies long-read sequencing data, we assessed RNA polymerase engagement across retroelements genome-wide, revealing correlations between nascent transcription, sequence divergence, CpG density, and methylation. These analyses were extended to evaluate RNA polymerase occupancy for all repeats, including high-density satellite repeats that reside in previously inaccessible centromeric regions of all human chromosomes. Moreover, using both mapping-dependent and mapping-independent approaches across early developmental stages and a complete cell cycle time series, we found that engaged RNA polymerase across satellites is low; in contrast, TE transcription is abundant and serves as a boundary for changes in CpG methylation and centromere substructure. Together, these data reveal the dynamic relationship between transcriptionally active retroelement subclasses and DNA methylation, as well as potential mechanisms for the derivation and evolution of new repeat families and composite elements. Focusing on the emerging T2T-level assembly of the HG002 X chromosome, we reveal that a high level of repeat variation likely exists across the human population, including composite element copy numbers that affect gene copy number. Additionally, we highlight the impact of repeats on the structural diversity of the genome, revealing repeat expansions with extreme copy number differences between humans and primates while also providing high-confidence annotations of retroelement transduction events. CONCLUSION The comprehensive repeat annotations and updated repeat models described herein serve as a resource for expanding the compendium of human genome sequences and reveal the impact of specific repeats on the human genome. In developing this resource, we provide a methodological framework for assessing repeat variation within and between human genomes. The exhaustive assessment of the transcriptional landscape of repeats, at both the genome scale and locally, such as within centromeres, sets the stage for functional studies to disentangle the role transcription plays in the mechanisms essential for genome stability and chromosome segregation. Finally, our work demonstrates the need to increase efforts toward achieving T2T-level assemblies for nonhuman primates and other species to fully understand the complexity and impact of repeat-derived genomic innovations that define primate lineages, including humans. Telomere-to-telomere assembly of CHM13 supports repeat annotations and discoveries. The human reference T2T-CHM13 filled gaps and corrected collapsed regions (triangles) in GRCh38. Combining long read–based methylation calls, PRO-seq, and multilevel computational methods, we provide a compendium of human repeats, define retroelement expression and methylation profiles, and delineate locus-specific sites of nascent transcription genome-wide, including previously inaccessible centromeres. SINE, short interspersed element; SVA, SINE–variable number tandem repeat– Alu ; LINE, long interspersed element; LTR, long terminal repeat; TSS, transcription start site; pA, xxxxxxxxxxxxxxxx. 

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4. Loops and the activity of loop extrusion factors constrain chromatin dynamics

   https://doi.org/10.1091/mbc.E23-04-0119  

   Bailey, Mary Lou ; Surovtsev, Ivan ; Williams, Jessica F. ; Yan, Hao ; Yuan, Tianyu ; Li, Kevin ; Duseau, Katherine ; Mochrie, Simon G. ; King, Megan C. ( July 2023 , Molecular Biology of the Cell)

   Bloom, Kerry (Ed.)

   The chromosomes—DNA polymers and their binding proteins—are compacted into a spatially organized, yet dynamic, three-dimensional structure. Recent genome-wide chromatin conformation capture experiments reveal a hierarchical organization of the DNA structure that is imposed, at least in part, by looping interactions arising from the activity of loop extrusion factors. The dynamics of chromatin reflects the response of the polymer to a combination of thermal fluctuations and active processes. However, how chromosome structure and enzymes acting on chromatin together define its dynamics remains poorly understood. To gain insight into the structure-dynamics relationship of chromatin, we combine high-precision microscopy in living Schizosaccharomyces pombe cells with systematic genetic perturbations and Rouse model polymer simulations. We first investigated how the activity of two loop extrusion factors, the cohesin and condensin complexes, influences chromatin dynamics. We observed that deactivating cohesin, or to a lesser extent condensin, increased chromatin mobility, suggesting that loop extrusion constrains rather than agitates chromatin motion. Our corresponding simulations reveal that the introduction of loops is sufficient to explain the constraining activity of loop extrusion factors, highlighting that the conformation adopted by the polymer plays a key role in defining its dynamics. Moreover, we find that the number of loops or residence times of loop extrusion factors influence the dynamic behavior of the chromatin polymer. Last, we observe that the activity of the INO80 chromatin remodeler, but not the SWI/SNF or RSC complexes, is critical for ATP-dependent chromatin mobility in fission yeast. Taking the data together, we suggest that thermal and INO80-dependent activities exert forces that drive chromatin fluctuations, which are constrained by the organization of the chromosome into loops. 

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5. Ribosome biogenesis in replicating cells: Integration of experiment and theory

   https://doi.org/10.1002/bip.22892  

   Earnest, Tyler M. ; Cole, John A. ; Peterson, Joseph R. ; Hallock, Michael J. ; Kuhlman, Thomas E. ; Luthey‐Schulten, Zaida ( July 2016 , Biopolymers)

   Ribosomes—the primary macromolecular machines responsible for translating the genetic code into proteins—are complexes of precisely folded RNA and proteins. The ways in which their production and assembly are managed by the living cell is of deep biological importance. Here we extend a recent spatially resolved whole‐cell model of ribosome biogenesis in a fixed volume [Earnest et al., Biophys J 2015, 109, 1117–1135] to include the effects of growth, DNA replication, and cell division. All biological processes are described in terms of reaction‐diffusion master equations and solved stochastically using the Lattice Microbes simulation software. In order to determine the replication parameters, we construct and analyze a series ofEscherichia colistrains with fluorescently labeled genes distributed evenly throughout their chromosomes. By measuring these cells’ lengths and number of gene copies at the single‐cell level, we could fit a statistical model of the initiation and duration of chromosome replication. We found that for our slow‐growing (120 min doubling time)E. colicells, replication was initiated 42 min into the cell cycle and completed after an additional 42 min. While simulations of the biogenesis model produce the correct ribosome and mRNA counts over the cell cycle, the kinetic parameters for transcription and degradation are lower than anticipated from a recent analytical time dependent model of in vivo mRNA production. Describing expression in terms of a simple chemical master equation, we show that the discrepancies are due to the lack of nonribosomal genes in the extended biogenesis model which effects the competition of mRNA for ribosome binding, and suggest corrections to parameters to be used in the whole‐cell model when modeling expression of the entire transcriptome. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 735–751, 2016.

    

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