Opportunities and Challenges in Building a Spatiotemporal Multi-scale Model of the Human Pancreatic ß Cell.

The construction of a predictive model of an entire eukaryotic cell that describes its dynamic structure from atomic to cellular scales is a grand challenge at the intersection of biology, chemistry, physics, and computer science. Having such a model will open new dimensions in biological research and accelerate healthcare advancements. Developing the necessary experimental and modeling methods presents abundant opportunities for a community effort to realize this goal. Here, we present a vision for creation of a spatiotemporal multi-scale model of the pancreatic ß-cell, a relevant target for understanding and modulating the pathogenesis of diabetes.

Spatial and temporal organization of the genome: Current state and future aims of the 4D nucleome project.

The four-dimensional nucleome (4DN) consortium studies the architecture of the genome and the nucleus in space and time. We summarize progress by the consortium and highlight the development of technologies for (1) mapping genome folding and identifying roles of nuclear components and bodies, proteins, and RNA, (2) characterizing nuclear organization with time or single-cell resolution, and (3) imaging of nuclear organization. With these tools, the consortium has provided over 2,000 public datasets. Integrative computational models based on these data are starting to reveal connections between genome structure and function. We then present a forward-looking perspective and outline current aims to (1) delineate dynamics of nuclear architecture at different timescales, from minutes to weeks as cells differentiate, in populations and in single cells, (2) characterize cis-determinants and trans-modulators of genome organization, (3) test functional consequences of changes in cis- and trans-regulators, and (4) develop predictive models of genome structure and function.

Computational methods for analysing multiscale 3D genome organization.

Recent progress in whole-genome mapping and imaging technologies has enabled the characterization of the spatial organization and folding of the genome in the nucleus. In parallel, advanced computational methods have been developed to leverage these mapping data to reveal multiscale three-dimensional (3D) genome features and to provide a more complete view of genome structure and its connections to genome functions such as transcription. Here, we discuss how recently developed computational tools, including machine-learning-based methods and integrative structure-modelling frameworks, have led to a systematic, multiscale delineation of the connections among different scales of 3D genome organization, genomic and epigenomic features, functional nuclear components and genome function. However, approaches that more comprehensively integrate a wide variety of genomic and imaging datasets are still needed to uncover the functional role of 3D genome structure in defining cellular phenotypes in health and disease.

Integrative genome modeling platform reveals essentiality of rare contact events in 3D genome organizations.

A multitude of sequencing-based and microscopy technologies provide the means to unravel the relationship between the three-dimensional organization of genomes and key regulatory processes of genome function. Here, we develop a multimodal data integration approach to produce populations of single-cell genome structures that are highly predictive for nuclear locations of genes and nuclear bodies, local chromatin compaction and spatial segregation of functionally related chromatin. We demonstrate that multimodal data integration can compensate for systematic errors in some of the data and can greatly increase accuracy and coverage of genome structure models. We also show that alternative combinations of different orthogonal data sources can converge to models with similar predictive power. Moreover, our study reveals the key contributions of low-frequency ('rare') interchromosomal contacts to accurately predicting the global nuclear architecture, including the positioning of genes and chromosomes. Overall, our results highlight the benefits of multimodal data integration for genome structure analysis, available through the Integrative Genome Modeling software package.

Comprehensive characterization of neutrophil genome topology.

Neutrophils are responsible for the first line of defense against invading pathogens. Their nuclei are uniquely structured as multiple lobes that establish a highly constrained nuclear environment. Here we found that neutrophil differentiation was not associated with large-scale changes in the number and sizes of topologically associating domains (TADs). However, neutrophil genomes were enriched for long-range genomic interactions that spanned multiple TADs. Population-based simulation of spherical and toroid genomes revealed declining radii of gyration for neutrophil chromosomes. We found that neutrophil genomes were highly enriched for heterochromatic genomic interactions across vast genomic distances, a process named supercontraction. Supercontraction involved genomic regions located in the heterochromatic compartment in both progenitors and neutrophils or genomic regions that switched from the euchromatic to the heterochromatic compartment during neutrophil differentiation. Supercontraction was accompanied by the repositioning of centromeres, pericentromeres, and long interspersed nuclear elements (LINEs) to the neutrophil nuclear lamina. We found that Lamin B receptor expression was required to attach centromeric and pericentromeric repeats but not LINE-1 elements to the lamina. Differentiating neutrophils also repositioned ribosomal DNA and mininucleoli to the lamina-a process that was closely associated with sharply reduced ribosomal RNA expression. We propose that large-scale chromatin reorganization involving supercontraction and recruitment of heterochromatin and nucleoli to the nuclear lamina facilitates the folding of the neutrophil genome into a confined geometry imposed by a multilobed nuclear architecture.

Assessment of scoring functions to rank the quality of 3D subtomogram clusters from cryo-electron tomography.

Cryo-electron tomography provides the opportunity for unsupervised discovery of endogenous complexes in situ. This process usually requires particle picking, clustering and alignment of subtomograms to produce an average structure of the complex. When applied to heterogeneous samples, template-free clustering and alignment of subtomograms can potentially lead to the discovery of structures for unknown endogenous complexes. However, such methods require scoring functions to measure and accurately rank the quality of aligned subtomogram clusters, which can be compromised by contaminations from misclassified complexes and alignment errors. Here, we provide the first study to assess the effectiveness of more than 15 scoring functions for evaluating the quality of subtomogram clusters, which differ in the amount of structural misalignments and contaminations due to misclassified complexes. We assessed both experimental and simulated subtomograms as ground truth data sets. Our analysis showed that the robustness of scoring functions varies largely. Most scores were sensitive to the signal-to-noise ratio of subtomograms and often required Gaussian filtering as preprocessing for improved performance. Two scoring functions, Spectral SNR-based Fourier Shell Correlation and Pearson Correlation in the Fourier domain with missing wedge correction, showed a robust ranking of subtomogram clusters without any preprocessing and irrespective of SNR levels of subtomograms. Of these two scoring functions, Spectral SNR-based Fourier Shell Correlation was fastest to compute and is a better choice for handling large numbers of subtomograms. Our results provide a guidance for choosing an accurate scoring function for template-free approaches to detect complexes from heterogeneous samples.

CancerDetector: ultrasensitive and non-invasive cancer detection at the resolution of individual reads using cell-free DNA methylation sequencing data.

The detection of tumor-derived cell-free DNA in plasma is one of the most promising directions in cancer diagnosis. The major challenge in such an approach is how to identify the tiny amount of tumor DNAs out of total cell-free DNAs in blood. Here we propose an ultrasensitive cancer detection method, termed 'CancerDetector', using the DNA methylation profiles of cell-free DNAs. The key of our method is to probabilistically model the joint methylation states of multiple adjacent CpG sites on an individual sequencing read, in order to exploit the pervasive nature of DNA methylation for signal amplification. Therefore, CancerDetector can sensitively identify a trace amount of tumor cfDNAs in plasma, at the level of individual reads. We evaluated CancerDetector on the simulated data, and showed a high concordance of the predicted and true tumor fraction. Testing CancerDetector on real plasma data demonstrated its high sensitivity and specificity in detecting tumor cfDNAs. In addition, the predicted tumor fraction showed great consistency with tumor size and survival outcome. Note that all of those testing were performed on sequencing data at low to medium coverage (1× to 10×). Therefore, CancerDetector holds the great potential to detect cancer early and cost-effectively.

Population-based 3D genome structure analysis reveals driving forces in spatial genome organization.

Conformation capture technologies (e.g., Hi-C) chart physical interactions between chromatin regions on a genome-wide scale. However, the structural variability of the genome between cells poses a great challenge to interpreting ensemble-averaged Hi-C data, particularly for long-range and interchromosomal interactions. Here, we present a probabilistic approach for deconvoluting Hi-C data into a model population of distinct diploid 3D genome structures, which facilitates the detection of chromatin interactions likely to co-occur in individual cells. Our approach incorporates the stochastic nature of chromosome conformations and allows a detailed analysis of alternative chromatin structure states. For example, we predict and experimentally confirm the presence of large centromere clusters with distinct chromosome compositions varying between individual cells. The stability of these clusters varies greatly with their chromosome identities. We show that these chromosome-specific clusters can play a key role in the overall chromosome positioning in the nucleus and stabilizing specific chromatin interactions. By explicitly considering genome structural variability, our population-based method provides an important tool for revealing novel insights into the key factors shaping the spatial genome organization.

TopDom: an efficient and deterministic method for identifying topological domains in genomes.

Genome-wide proximity ligation assays allow the identification of chromatin contacts at unprecedented resolution. Several studies reveal that mammalian chromosomes are composed of topological domains (TDs) in sub-mega base resolution, which appear to be conserved across cell types and to some extent even between organisms. Identifying topological domains is now an important step toward understanding the structure and functions of spatial genome organization. However, current methods for TD identification demand extensive computational resources, require careful tuning and/or encounter inconsistencies in results. In this work, we propose an efficient and deterministic method, TopDom, to identify TDs, along with a set of statistical methods for evaluating their quality. TopDom is much more efficient than existing methods and depends on just one intuitive parameter, a window size, for which we provide easy-to-implement optimization guidelines. TopDom also identifies more and higher quality TDs than the popular directional index algorithm. The TDs identified by TopDom provide strong support for the cross-tissue TD conservation. Finally, our analysis reveals that the locations of housekeeping genes are closely associated with cross-tissue conserved TDs. The software package and source codes of TopDom are available athttp://zhoulab.usc.edu/TopDom/.

Simulating cryo electron tomograms of crowded cell cytoplasm for assessment of automated particle picking.

BACKGROUND: Cryo-electron tomography is an important tool to study structures of macromolecular complexes in close to native states. A whole cell cryo electron tomogram contains structural information of all its macromolecular complexes. However, extracting this information remains challenging, and relies on sophisticated image processing, in particular for template-free particle extraction, classification and averaging. To develop these methods it is crucial to realistically simulate tomograms of crowded cellular environments, which can then serve as ground truth models for assessing and optimizing methods for detection of complexes in cell tomograms. RESULTS: We present a framework to generate crowded mixtures of macromolecular complexes for realistically simulating cryo electron tomograms including noise and image distortions due to the missing-wedge effects. Simulated tomograms are then used for assessing the template-free Difference-of-Gaussian (DoG) particle-picking method to detect complexes of different shapes and sizes under various crowding and noise levels. We identified DoG parameter settings that maximize precision and recall for detecting particles over a wide range of sizes and shapes. We observed that medium sized DoG scaling factors showed the overall best performance. To further improve performance, we propose a combination strategy for integrating results from multiple parameter settings. With increasing macromolecular crowding levels, the precision of particle picking remained relatively high, while the recall was dramatically reduced, which limits the detection of sufficient copy numbers of complexes in a crowded environment. Over a wide range of increasing noise levels, the DoG particle picking performance remained stable, but dramatically reduced beyond a specific noise threshold. CONCLUSIONS: Automatic and reference-free particle picking is an important first step in a visual proteomics analysis of cell tomograms. However, cell cytoplasm is highly crowded, which makes particle detection challenging. It is therefore important to test particle-picking methods in a realistic crowded setting. Here, we present a framework for simulating tomograms of cellular environments at high crowding levels and assess the DoG particle picking method. We determined optimal parameter settings to maximize the performance of the DoG particle-picking method.

Hi-Corrector: a fast, scalable and memory-efficient package for normalizing large-scale Hi-C data.

UNLABELLED: Genome-wide proximity ligation assays, e.g. Hi-C and its variant TCC, have recently become important tools to study spatial genome organization. Removing biases from chromatin contact matrices generated by such techniques is a critical preprocessing step of subsequent analyses. The continuing decline of sequencing costs has led to an ever-improving resolution of the Hi-C data, resulting in very large matrices of chromatin contacts. Such large-size matrices, however, pose a great challenge on the memory usage and speed of its normalization. Therefore, there is an urgent need for fast and memory-efficient methods for normalization of Hi-C data. We developed Hi-Corrector, an easy-to-use, open source implementation of the Hi-C data normalization algorithm. Its salient features are (i) scalability-the software is capable of normalizing Hi-C data of any size in reasonable times; (ii) memory efficiency-the sequential version can run on any single computer with very limited memory, no matter how little; (iii) fast speed-the parallel version can run very fast on multiple computing nodes with limited local memory. AVAILABILITY AND IMPLEMENTATION: The sequential version is implemented in ANSI C and can be easily compiled on any system; the parallel version is implemented in ANSI C with the MPI library (a standardized and portable parallel environment designed for solving large-scale scientific problems). The package is freely available at http://zhoulab.usc.edu/Hi-Corrector/.

Physical tethering and volume exclusion determine higher-order genome organization in budding yeast.

In this paper we show that tethering of heterochromatic regions to nuclear landmarks and random encounters of chromosomes in the confined nuclear volume are sufficient to explain the higher-order organization of the budding yeast genome. We have quantitatively characterized the contact patterns and nuclear territories that emerge when chromosomes are allowed to behave as constrained but otherwise randomly configured flexible polymer chains in the nucleus. Remarkably, this constrained random encounter model explains in a statistical manner the experimental hallmarks of the S. cerevisiae genome organization, including (1) the folding patterns of individual chromosomes; (2) the highly enriched interactions between specific chromatin regions and chromosomes; (3) the emergence, shape, and position of gene territories; (4) the mean distances between pairs of telomeres; and (5) even the co-location of functionally related gene loci, including early replication start sites and tRNA genes. Therefore, most aspects of the yeast genome organization can be explained without calling on biochemically mediated chromatin interactions. Such interactions may modulate the pre-existing propensity for co-localization but seem not to be the cause for the observed higher-order organization. The fact that geometrical constraints alone yield a highly organized genome structure, on which different functional elements are specifically distributed, has strong implications for the folding principles of the genome and the evolution of its function.

Automated target segmentation and real space fast alignment methods for high-throughput classification and averaging of crowded cryo-electron subtomograms.

MOTIVATION: Cryo-electron tomography allows the imaging of macromolecular complexes in near living conditions. To enhance the nominal resolution of a structure it is necessary to align and average individual subtomograms each containing identical complexes. However, if the sample of complexes is heterogeneous, it is necessary to first classify subtomograms into groups of identical complexes. This task becomes challenging when tomograms contain mixtures of unknown complexes extracted from a crowded environment. Two main challenges must be overcomed: First, classification of subtomograms must be performed without knowledge of template structures. However, most alignment methods are too slow to perform reference-free classification of a large number of (e.g. tens of thousands) of subtomograms. Second, subtomograms extracted from crowded cellular environments, contain often fragments of other structures besides the target complex. However, alignment methods generally assume that each subtomogram only contains one complex. Automatic methods are needed to identify the target complexes in a subtomogram even when its shape is unknown. RESULTS: In this article, we propose an automatic and systematic method for the isolation and masking of target complexes in subtomograms extracted from crowded environments. Moreover, we also propose a fast alignment method using fast rotational matching in real space. Our experiments show that, compared with our previously proposed fast alignment method in reciprocal space, our new method significantly improves the alignment accuracy for highly distorted and especially crowded subtomograms. Such improvements are important for achieving successful and unbiased high-throughput reference-free structural classification of complexes inside whole-cell tomograms. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Harmonizing the Generation and Pre-publication Stewardship of FAIR Image data.

Together with the molecular knowledge of genes and proteins, biological images promise to significantly enhance the scientific understanding of complex cellular systems and to advance predictive and personalized therapeutic products for human health. For this potential to be realized, quality-assured image data must be shared among labs at a global scale to be compared, pooled, and reanalyzed, thus unleashing untold potential beyond the original purpose for which the data was generated. There are two broad sets of requirements to enable image data sharing in the life sciences. One set of requirements is articulated in the companion White Paper entitled "Enabling Global Image Data Sharing in the Life Sciences," which is published in parallel and addresses the need to build the cyberinfrastructure for sharing the digital array data (arXiv:2401.13023 [q-bio.OT], https://doi.org/10.48550/arXiv.2401.13023). In this White Paper, we detail a broad set of requirements, which involves collecting, managing, presenting, and propagating contextual information essential to assess the quality, understand the content, interpret the scientific implications, and reuse image data in the context of the experimental details. We start by providing an overview of the main lessons learned to date through international community activities, which have recently made considerable progress toward generating community standard practices for imaging Quality Control (QC) and metadata. We then provide a clear set of recommendations for amplifying this work. The driving goal is to address remaining challenges, and democratize access to common practices and tools for a spectrum of biomedical researchers, regardless of their expertise, access to resources, and geographical location.

Determining the architectures of macromolecular assemblies.

To understand the workings of a living cell, we need to know the architectures of its macromolecular assemblies. Here we show how proteomic data can be used to determine such structures. The process involves the collection of sufficient and diverse high-quality data, translation of these data into spatial restraints, and an optimization that uses the restraints to generate an ensemble of structures consistent with the data. Analysis of the ensemble produces a detailed architectural map of the assembly. We developed our approach on a challenging model system, the nuclear pore complex (NPC). The NPC acts as a dynamic barrier, controlling access to and from the nucleus, and in yeast is a 50 MDa assembly of 456 proteins. The resulting structure, presented in an accompanying paper, reveals the configuration of the proteins in the NPC, providing insights into its evolution and architectural principles. The present approach should be applicable to many other macromolecular assemblies.

The molecular architecture of the nuclear pore complex.

Nuclear pore complexes (NPCs) are proteinaceous assemblies of approximately 50 MDa that selectively transport cargoes across the nuclear envelope. To determine the molecular architecture of the yeast NPC, we collected a diverse set of biophysical and proteomic data, and developed a method for using these data to localize the NPC's 456 constituent proteins (see the accompanying paper). Our structure reveals that half of the NPC is made up of a core scaffold, which is structurally analogous to vesicle-coating complexes. This scaffold forms an interlaced network that coats the entire curved surface of the nuclear envelope membrane within which the NPC is embedded. The selective barrier for transport is formed by large numbers of proteins with disordered regions that line the inner face of the scaffold. The NPC consists of only a few structural modules that resemble each other in terms of the configuration of their homologous constituents, the most striking of these being a 16-fold repetition of 'columns'. These findings provide clues to the evolutionary origins of the NPC.

Conformational analysis of chromosome structures reveals vital role of chromosome morphology in gene function.

The 3D conformations of chromosomes are highly variant and stochastic between single cells. Recent progress in multiplexed 3D FISH imaging, single cell Hi-C and genome structure modeling allows a closer analysis of the structural variations of chromosomes between cells to infer the functional implications of structural heterogeneity. Here, we introduce a two-step dimensionality reduction method to classify a population of single cell 3D chromosome structures, either from simulation or imaging experiment, into dominant conformational clusters with distinct chromosome morphologies. We found that almost half of all structures for each chromosome can be described by 5-10 dominant chromosome morphologies, which play a fundamental role in establishing conformational variation of chromosomes. These morphologies are conserved in different cell types, but vary in their relative proportion of structures. Chromosome morphologies are distinguished by the presence or absence of characteristic chromosome territory domains, which expose some chromosomal regions to varying nuclear environments in different morphologies, such as nuclear positions and associations to nuclear speckles, lamina, and nucleoli. These observations point to distinct functional variations for the same chromosomal region in different chromosome morphologies. We validated chromosome conformational clusters and their associated subnuclear locations with data from DNA-MERFISH imaging and single cell sci-HiC data. Our method provides an important approach to assess the variation of chromosome structures between cells and link differences in conformational states with distinct gene functions.

Evaluating the role of the nuclear microenvironment in gene function by population-based modeling.

The nuclear folding of chromosomes relative to nuclear bodies is an integral part of gene function. Here, we demonstrate that population-based modeling-from ensemble Hi-C data-provides a detailed description of the nuclear microenvironment of genes and its role in gene function. We define the microenvironment by the subnuclear positions of genomic regions with respect to nuclear bodies, local chromatin compaction, and preferences in chromatin compartmentalization. These structural descriptors are determined in single-cell models, thereby revealing the structural variability between cells. We demonstrate that the microenvironment of a genomic region is linked to its functional potential in gene transcription, replication, and chromatin compartmentalization. Some chromatin regions feature a strong preference for a single microenvironment, due to association with specific nuclear bodies in most cells. Other chromatin shows high structural variability, which is a strong indicator of functional heterogeneity. Moreover, we identify specialized nuclear microenvironments, which distinguish chromatin in different functional states and reveal a key role of nuclear speckles in chromosome organization. We demonstrate that our method produces highly predictive three-dimensional genome structures, which accurately reproduce data from a variety of orthogonal experiments, thus considerably expanding the range of Hi-C data analysis.

Mega-Enhancer Bodies Organize Neuronal Long Genes in the Cerebellum.

Dynamic regulation of gene expression plays a key role in establishing the diverse neuronal cell types in the brain. Recent findings in genome biology suggest that three-dimensional (3D) genome organization has important, but mechanistically poorly understood functions in gene transcription. Beyond local genomic interactions between promoters and enhancers, we find that cerebellar granule neurons undergoing differentiation in vivo exhibit striking increases in long-distance genomic interactions between transcriptionally active genomic loci, which are separated by tens of megabases within a chromosome or located on different chromosomes. Among these interactions, we identify a nuclear subcompartment enriched for near-megabase long enhancers and their associated neuronal long genes encoding synaptic or signaling proteins. Neuronal long genes are differentially recruited to this enhancer-dense subcompartment to help shape the transcriptional identities of granule neuron subtypes in the cerebellum. SPRITE analyses of higher-order genomic interactions, together with IGM-based 3D genome modeling and imaging approaches, reveal that the enhancer-dense subcompartment forms prominent nuclear structures, which we term mega-enhancer bodies. These novel nuclear bodies reside in the nuclear periphery, away from other transcriptionally active structures, including nuclear speckles located in the nuclear interior. Together, our findings define additional layers of higher-order 3D genome organization closely linked to neuronal maturation and identity in the brain.

High-throughput subtomogram alignment and classification by Fourier space constrained fast volumetric matching.

Cryo-electron tomography allows the visualization of macromolecular complexes in their cellular environments in close-to-live conditions. The nominal resolution of subtomograms can be significantly increased when individual subtomograms of the same kind are aligned and averaged. A vital step for such a procedure are algorithms that speedup subtomogram alignment and improve its accuracy to allow reference-free subtomogram classifications. Such methods will facilitate automation of tomography analysis and overall high throughput in the data processing. Building on previous work, here we propose a fast rotational alignment method that uses the Fourier equivalent form of a popular constrained correlation measure that considers missing wedge corrections and density variances in the subtomograms. The fast rotational search is based on 3D volumetric matching, which improves the rotational alignment accuracy in particular for highly distorted subtomograms with low SNR and tilt angle ranges in comparison to fast rotational matching of projected 2D spherical images. We further integrate our fast rotational alignment method in a reference-free iterative subtomogram classification scheme, and propose a local feature enhancement strategy in the classification process. As a proof of principle, we can demonstrate that the automatic method can successfully classify a large number of experimental subtomograms without the need of a reference structure.