Chromosome structure is a crucial regulatory factor for a wide range of nuclear processes. Chromosome conformation capture (3C)-based experiments combined with computational modelling are pivotal for ...unveiling 3D chromosome structure. Here, we introduce TADdyn, a tool that integrates time-course 3C data, restraint-based modelling, and molecular dynamics to simulate the structural rearrangements of genomic loci in a completely data-driven way. We apply TADdyn on in situ Hi-C time-course experiments studying the reprogramming of murine B cells to pluripotent cells, and characterize the structural rearrangements that take place upon changes in the transcriptional state of 21 genomic loci of diverse expression dynamics. By measuring various structural and dynamical properties, we find that during gene activation, the transcription starting site contacts with open and active regions in 3D chromatin domains. We propose that these 3D hubs of open and active chromatin may constitute a general feature to trigger and maintain gene transcription.
To investigate the three-dimensional (3D) genome architecture across normal B cell differentiation and in neoplastic cells from different subtypes of chronic lymphocytic leukemia and mantle cell ...lymphoma patients, here we integrate in situ Hi-C and nine additional omics layers. Beyond conventional active (A) and inactive (B) compartments, we uncover a highly-dynamic intermediate compartment enriched in poised and polycomb-repressed chromatin. During B cell development, 28% of the compartments change, mostly involving a widespread chromatin activation from naive to germinal center B cells and a reversal to the naive state upon further maturation into memory B cells. B cell neoplasms are characterized by both entity and subtype-specific alterations in 3D genome organization, including large chromatin blocks spanning key disease-specific genes. This study indicates that 3D genome interactions are extensively modulated during normal B cell differentiation and that the genome of B cell neoplasias acquires a tumor-specific 3D genome architecture.
An increasing number of long noncoding RNAs (lncRNAs) have been proposed to act as nuclear organization factors during interphase. Direct RNA-DNA interactions can be achieved by the formation of ...triplex helix structures where a single-stranded RNA molecule hybridizes by complementarity into the major groove of double-stranded DNA. However, whether and how these direct RNA-DNA associations influence genome structure in interphase chromosomes remain poorly understood. Here we theorize that RNA organizes the genome in space via a triplex-forming mechanism. To test this theory, we apply a computational modeling approach of chromosomes that combines restraint-based modeling with polymer physics. Our models suggest that colocalization of triplex hotspots targeted by lncRNAs could contribute to large-scale chromosome compartmentalization cooperating, rather than competing, with architectural transcription factors such as CTCF.
Three‐dimensional electron microscopy is currently one of the most promising techniques used to study macromolecular assemblies. Rigid and flexible fitting of atomic models into density maps is often ...essential to gain further insights into the assemblies they represent. Currently, tools that facilitate the assessment of fitted atomic models and maps are needed. TEMPy (template and electron microscopy comparison using Python) is a toolkit designed for this purpose. The library includes a set of methods to assess density fits in intermediate‐to‐low resolution maps, both globally and locally. It also provides procedures for single‐fit assessment, ensemble generation of fits, clustering, and multiple and consensus scoring, as well as plots and output files for visualization purposes to help the user in analysing rigid and flexible fits. The modular nature of TEMPy helps the integration of scoring and assessment of fits into large pipelines, making it a tool suitable for both novice and expert structural biologists.
Membrane attack complex/perforin/cholesterol-dependent cytolysin (MACPF/CDC) proteins constitute a major superfamily of pore-forming proteins that act as bacterial virulence factors and effectors in ...immune defence. Upon binding to the membrane, they convert from the soluble monomeric form to oligomeric, membrane-inserted pores. Using real-time atomic force microscopy (AFM), electron microscopy (EM), and atomic structure fitting, we have mapped the structure and assembly pathways of a bacterial CDC in unprecedented detail and accuracy, focussing on suilysin from Streptococcus suis. We show that suilysin assembly is a noncooperative process that is terminated before the protein inserts into the membrane. The resulting ring-shaped pores and kinetically trapped arc-shaped assemblies are all seen to perforate the membrane, as also visible by the ejection of its lipids. Membrane insertion requires a concerted conformational change of the monomeric subunits, with a marked expansion in pore diameter due to large changes in subunit structure and packing.
Abstract
The rapid development of Chromosome Conformation Capture (3C-based techniques), as well as imaging together with bioinformatics analyses, has been fundamental for unveiling that chromosomes ...are organized into the so-called topologically associating domains or TADs. While TADs appear as nested patterns in the 3C-based interaction matrices, the vast majority of available TAD callers are based on the hypothesis that TADs are individual and unrelated chromatin structures. Here we introduce TADpole, a computational tool designed to identify and analyze the entire hierarchy of TADs in intra-chromosomal interaction matrices. TADpole combines principal component analysis and constrained hierarchical clustering to provide a set of significant hierarchical chromatin levels in a genomic region of interest. TADpole is robust to data resolution, normalization strategy and sequencing depth. Domain borders defined by TADpole are enriched in main architectural proteins (CTCF and cohesin complex subunits) and in the histone mark H3K4me3, while their domain bodies, depending on their activation-state, are enriched in either H3K36me3 or H3K27me3, highlighting that TADpole is able to distinguish functional TAD units. Additionally, we demonstrate that TADpole's hierarchical annotation, together with the new DiffT score, allows for detecting significant topological differences on Capture Hi-C maps between wild-type and genetically engineered mouse.
Kinesins are a superfamily of microtubule-based ATP-powered motors, important for multiple, essential cellular functions. How microtubule binding stimulates their ATPase and controls force generation ...is not understood. To address this fundamental question, we visualized microtubule-bound kinesin-1 and kinesin-3 motor domains at multiple steps in their ATPase cycles--including their nucleotide-free states--at ∼ 7 Å resolution using cryo-electron microscopy. In both motors, microtubule binding promotes ordered conformations of conserved loops that stimulate ADP release, enhance microtubule affinity and prime the catalytic site for ATP binding. ATP binding causes only small shifts of these nucleotide-coordinating loops but induces large conformational changes elsewhere that allow force generation and neck linker docking towards the microtubule plus end. Family-specific differences across the kinesin-microtubule interface account for the distinctive properties of each motor. Our data thus provide evidence for a conserved ATP-driven mechanism for kinesins and reveal the critical mechanistic contribution of the microtubule interface.
PapC ushers are outer-membrane proteins enabling assembly and secretion of P pili in uropathogenic E. coli. Their translocation domain is a large β-barrel occluded by a plug domain, which is ...displaced to allow the translocation of pilus subunits across the membrane. Previous studies suggested that this gating mechanism is controlled by a β-hairpin and an α-helix. To investigate the role of these elements in allosteric signal communication, we developed a method combining evolutionary and molecular dynamics studies of the native translocation domain and mutants lacking the β-hairpin and/or the α-helix. Analysis of a hybrid residue interaction network suggests distinct regions (residue 'communities') within the translocation domain (especially around β12-β14) linking these elements, thereby modulating PapC gating. Antibiotic sensitivity and electrophysiology experiments on a set of alanine-substitution mutants confirmed functional roles for four of these communities. This study illuminates the gating mechanism of PapC ushers and its importance in maintaining outer-membrane permeability.
Allosteric signalling in the outer membrane translocation domain of PapC usher. eLife 3:e03532. doi: 10.7554/eLife.03532. Published October 01, 2014 In the published article there was an error in the ...schematic representation of residues involved in the allosteric signalling to control PapC gating (Figure 7), showing the wrong shape (corresponding to the cellular phenotype) for two residues (C327 and K427).
Nuclear organization impacts gene expression activity and cell phenotype. Our current understanding is mainly derived from ensemble-level sequencing studies that reflect the 3D genome structure of ...millions of cells. These approaches have provided invaluable details on the 3D organizations of the genome and their relation to other nuclear landmarks. However, they mostly lack the ability to provide multimodal information simultaneously at the single-cell level. In recent years, cutting-edge imaging technologies have risen to the challenge of simultaneously describing multiple components of the nuclear space at the single-cell level, paving the way for a deeper understanding of the genome structure–function relationship. This review will focus on the development and utilization of such technologies to gain a multi-component view of the nucleus at single-cell resolution, dissecting the complexity and heterogeneity of nuclear organization.
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