Ultrastructural Details of Mammalian Chromosome Architecture.

Over the past decade, 3C-related methods have provided remarkable insights into chromosome folding in vivo. To overcome the limited resolution of prior studies, we extend a recently developed Hi-C variant, Micro-C, to map chromosome architecture at nucleosome resolution in human ESCs and fibroblasts. Micro-C robustly captures known features of chromosome folding including compartment organization, topologically associating domains, and interactions between CTCF binding sites. In addition, Micro-C provides a detailed map of nucleosome positions and localizes contact domain boundaries with nucleosomal precision. Compared to Hi-C, Micro-C exhibits an order of magnitude greater dynamic range, allowing the identification of ∼20,000 additional loops in each cell type. Many newly identified peaks are localized along extrusion stripes and form transitive grids, consistent with their anchors being pause sites impeding cohesin-dependent loop extrusion. Our analyses comprise the highest-resolution maps of chromosome folding in human cells to date, providing a valuable resource for studies of chromosome organization.

Frozen-hydrated chromatin from metaphase chromosomes has an interdigitated multilayer structure.

Cryo-electron tomography and small-angle X-ray scattering were used to investigate the chromatin folding in metaphase chromosomes. The tomographic 3D reconstructions show that frozen-hydrated chromatin emanated from chromosomes is planar and forms multilayered plates. The layer thickness was measured accounting for the contrast transfer function fringes at the plate edges, yielding a width of ~ 7.5 nm, which is compatible with the dimensions of a monolayer of nucleosomes slightly tilted with respect to the layer surface. Individual nucleosomes are visible decorating distorted plates, but typical plates are very dense and nucleosomes are not identifiable as individual units, indicating that they are tightly packed. Two layers in contact are ~ 13 nm thick, which is thinner than the sum of two independent layers, suggesting that nucleosomes in the layers interdigitate. X-ray scattering of whole chromosomes shows a main scattering peak at ~ 6 nm, which can be correlated with the distance between layers and between interdigitating nucleosomes interacting through their faces. These observations support a model where compact chromosomes are composed of many chromatin layers stacked along the chromosome axis.

BHi-Cect: a top-down algorithm for identifying the multi-scale hierarchical structure of chromosomes.

High-throughput chromosome conformation capture (Hi-C) technology enables the investigation of genome-wide interactions among chromosome loci. Current algorithms focus on topologically associating domains (TADs), that are contiguous clusters along the genome coordinate, to describe the hierarchical structure of chromosomes. However, high resolution Hi-C displays a variety of interaction patterns beyond what current TAD detection methods can capture. Here, we present BHi-Cect, a novel top-down algorithm that finds clusters by considering every locus with no assumption of genomic contiguity using spectral clustering. Our results reveal that the hierarchical structure of chromosome is organized as 'enclaves', which are complex interwoven clusters at both local and global scales. We show that the nesting of local clusters within global clusters characterizing enclaves, is associated with the epigenomic activity found on the underlying DNA. Furthermore, we show that the hierarchical nesting that links different enclaves integrates their respective function. BHi-Cect provides means to uncover the general principles guiding chromatin architecture.

Evaluation and comparison of methods for recapitulation of 3D spatial chromatin structures.

How chromosomes fold and how distal genomic elements interact with one another at a genomic scale have been actively pursued in the past decade following the seminal work describing the Chromosome Conformation Capture (3C) assay. Essentially, 3C-based technologies produce two-dimensional (2D) contact maps that capture interactions between genomic fragments. Accordingly, a plethora of analytical methods have been proposed to take a 2D contact map as input to recapitulate the underlying whole genome three-dimensional (3D) structure of the chromatin. However, their performance in terms of several factors, including data resolution and ability to handle contact map features, have not been sufficiently evaluated. This task is taken up in this article, in which we consider several recent and/or well-regarded methods, both optimization-based and model-based, for their aptness of producing 3D structures using contact maps generated based on a population of cells. These methods are evaluated and compared using both simulated and real data. Several criteria have been used. For simulated data sets, the focus is on accurate recapitulation of the entire structure given the existence of the gold standard. For real data sets, comparison with distances measured by Florescence in situ Hybridization and consistency with several genomic features of known biological functions are examined.

Chromosomal analysis for embryos from balanced chromosomal rearrangement carriers using next generation sequencing.

We aimed to use next generation sequencing (NGS) to investigate chromosomal abnormalities in blastocyst trophectoderm (TE) samples, and reproductive outcomes with the different types of chromosomal rearrangements (CR) and for each sex of CR carrier. A total of 1189 blastocyst TE samples were evaluated using NGS to detect chromosomal unbalanced translocations as well as aneuploidy, including blastocytes from 637 blastocysts from carriers of balanced CR and 552 blastocysts from carriers of normal chromosomes. The optimal embryos had lower chromosomal abnormality rates compared to the poor-quality embryos. The experimental group had significantly reduced rates of normal embryos and euploidy, and higher rates of total abnormalities, aneuploidy and unbalanced chromosomal aberrations. Carriers of reciprocal translocations had a reduced rate of normal embryos and an increased percentage of embryos with total abnormalities and unbalanced chromosomal aberrations compared with carriers of Robertsonian translocations. Couples with female carriers of chromosomal abnormalities had significantly reduced rates of normal embryos and euploidy, and a higher percentage of embryos with total abnormalities, aneuploidy, and unbalanced chromosomal aberrations compared with couples of male carriers. Our preimplantation genetic testing (PGT) study identified higher rates of chromosomal abnormalities, including chromosomal unbalanced translocations and aneuploidy, in blastocysts from CR carriers, especially from the female carriers, in a Chinese population. The PGT cycles successfully improved clinical outcomes by increasing the fertilization rate and reducing the early spontaneous abortion rate compared with the in vitro fertilization and intracytoplasmic sperm injection cycles, especially for CR carriers.

Higher-Order Structure of Human Chromosomes Observed by Electron Diffraction and Electron Tomography.

It is well known that two DNA molecules are wrapped around histone octamers and folded together to form a single chromosome. However, the nucleosome fiber folding within a chromosome remains an enigma, and the higher-order structure of chromosomes also is not understood. In this study, we employed electron diffraction which provides a noninvasive analysis to characterize the internal structure of chromosomes. The results revealed the presence of structures with 100­200 nm periodic features directionally perpendicular to the chromosome axis in unlabeled isolated human chromosomes. We also visualized the 100­200 nm periodic features perpendicular to the chromosome axis in an isolated chromosome whose DNA molecules were specifically labeled with OsO4 using electron tomography in 300 keV and 1 MeV transmission electron microscopes.

Reconstructing spatial organizations of chromosomes through manifold learning.

Decoding the spatial organizations of chromosomes has crucial implications for studying eukaryotic gene regulation. Recently, chromosomal conformation capture based technologies, such as Hi-C, have been widely used to uncover the interaction frequencies of genomic loci in a high-throughput and genome-wide manner and provide new insights into the folding of three-dimensional (3D) genome structure. In this paper, we develop a novel manifold learning based framework, called GEM (Genomic organization reconstructor based on conformational Energy and Manifold learning), to reconstruct the three-dimensional organizations of chromosomes by integrating Hi-C data with biophysical feasibility. Unlike previous methods, which explicitly assume specific relationships between Hi-C interaction frequencies and spatial distances, our model directly embeds the neighboring affinities from Hi-C space into 3D Euclidean space. Extensive validations demonstrated that GEM not only greatly outperformed other state-of-art modeling methods but also provided a physically and physiologically valid 3D representations of the organizations of chromosomes. Furthermore, we for the first time apply the modeled chromatin structures to recover long-range genomic interactions missing from original Hi-C data.

De novo prediction of human chromosome structures: Epigenetic marking patterns encode genome architecture.

Inside the cell nucleus, genomes fold into organized structures that are characteristic of cell type. Here, we show that this chromatin architecture can be predicted de novo using epigenetic data derived from chromatin immunoprecipitation-sequencing (ChIP-Seq). We exploit the idea that chromosomes encode a 1D sequence of chromatin structural types. Interactions between these chromatin types determine the 3D structural ensemble of chromosomes through a process similar to phase separation. First, a neural network is used to infer the relation between the epigenetic marks present at a locus, as assayed by ChIP-Seq, and the genomic compartment in which those loci reside, as measured by DNA-DNA proximity ligation (Hi-C). Next, types inferred from this neural network are used as an input to an energy landscape model for chromatin organization [Minimal Chromatin Model (MiChroM)] to generate an ensemble of 3D chromosome conformations at a resolution of 50 kilobases (kb). After training the model, dubbed Maximum Entropy Genomic Annotation from Biomarkers Associated to Structural Ensembles (MEGABASE), on odd-numbered chromosomes, we predict the sequences of chromatin types and the subsequent 3D conformational ensembles for the even chromosomes. We validate these structural ensembles by using ChIP-Seq tracks alone to predict Hi-C maps, as well as distances measured using 3D fluorescence in situ hybridization (FISH) experiments. Both sets of experiments support the hypothesis of phase separation being the driving process behind compartmentalization. These findings strongly suggest that epigenetic marking patterns encode sufficient information to determine the global architecture of chromosomes and that de novo structure prediction for whole genomes may be increasingly possible.

Predicting CTCF-mediated chromatin loops using CTCF-MP.

Motivation: The three dimensional organization of chromosomes within the cell nucleus is highly regulated. It is known that CCCTC-binding factor (CTCF) is an important architectural protein to mediate long-range chromatin loops. Recent studies have shown that the majority of CTCF binding motif pairs at chromatin loop anchor regions are in convergent orientation. However, it remains unknown whether the genomic context at the sequence level can determine if a convergent CTCF motif pair is able to form a chromatin loop. Results: In this article, we directly ask whether and what sequence-based features (other than the motif itself) may be important to establish CTCF-mediated chromatin loops. We found that motif conservation measured by 'branch-of-origin' that accounts for motif turn-over in evolution is an important feature. We developed a new machine learning algorithm called CTCF-MP based on word2vec to demonstrate that sequence-based features alone have the capability to predict if a pair of convergent CTCF motifs would form a loop. Together with functional genomic signals from CTCF ChIP-seq and DNase-seq, CTCF-MP is able to make highly accurate predictions on whether a convergent CTCF motif pair would form a loop in a single cell type and also across different cell types. Our work represents an important step further to understand the sequence determinants that may guide the formation of complex chromatin architectures. Availability and implementation: The source code of CTCF-MP can be accessed at: https://github.com/ma-compbio/CTCF-MP. Supplementary information: Supplementary data are available at Bioinformatics online.

Quantifying the similarity of topological domains across normal and cancer human cell types.

Motivation: Three-dimensional chromosome structure has been increasingly shown to influence various levels of cellular and genomic functions. Through Hi-C data, which maps contact frequency on chromosomes, it has been found that structural elements termed topologically associating domains (TADs) are involved in many regulatory mechanisms. However, we have little understanding of the level of similarity or variability of chromosome structure across cell types and disease states. In this study, we present a method to quantify resemblance and identify structurally similar regions between any two sets of TADs. Results: We present an analysis of 23 human Hi-C samples representing various tissue types in normal and cancer cell lines. We quantify global and chromosome-level structural similarity, and compare the relative similarity between cancer and non-cancer cells. We find that cancer cells show higher structural variability around commonly mutated pan-cancer genes than normal cells at these same locations. Availability and implementation: Software for the methods and analysis can be found at https://github.com/Kingsford-Group/localtadsim.

Mammalian 5' C-rich telomeric overhangs are a mark of recombination-dependent telomere maintenance.

Recent evidence for 5'-cytosine (C)-rich overhangs at the telomeres of the nematode Caenorhabditis elegans provided the impetus to re-examine the end structure of mammalian telomeres. Two-dimensional (2D) gel electrophoresis, single telomere-length analysis (STELA), and strand-specific exonuclease assays revealed the presence of a 5'-C-rich overhang at the telomeres of human and mouse chromosomes. C-overhangs were prominent in G1/S arrested as well as terminally differentiated cells, indicating that they did not represent replication intermediates. C-rich overhangs were far more prevalent in tumor cells engaged in the alternative lengthening of telomeres (ALT) pathway of telomere maintenance, which relies on the homologous recombination (HR) machinery. Transient siRNA-based depletion of the HR-specific proteins RAD51, RAD52, and XRCC3 resulted in changes in C-overhang levels, implicating the involvement of 5'-C-overhangs in the HR-dependent pathway of telomere maintenance.

3D genome structure modeling by Lorentzian objective function.

The 3D structure of the genome plays a vital role in biological processes such as gene interaction, gene regulation, DNA replication and genome methylation. Advanced chromosomal conformation capture techniques, such as Hi-C and tethered conformation capture, can generate chromosomal contact data that can be used to computationally reconstruct 3D structures of the genome. We developed a novel restraint-based method that is capable of reconstructing 3D genome structures utilizing both intra-and inter-chromosomal contact data. Our method was robust to noise and performed well in comparison with a panel of existing methods on a controlled simulated data set. On a real Hi-C data set of the human genome, our method produced chromosome and genome structures that are consistent with 3D FISH data and known knowledge about the human chromosome and genome, such as, chromosome territories and the cluster of small chromosomes in the nucleus center with the exception of the chromosome 18. The tool and experimental data are available at https://missouri.box.com/v/LorDG.

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.

Large-scale probabilistic 3D organization of human chromosome territories.

There is growing evidence that chromosome territories (CT) have a probabilistic non-random arrangement within the cell nucleus of mammalian cells including radial positioning and preferred patterns of interchromosomal interactions that are cell-type specific. While it is generally assumed that the three-dimensional (3D) arrangement of genes within the CT is linked to genomic regulation, the degree of non-random organization of individual CT remains unclear. As a first step to elucidating the global 3D organization (topology) of individual CT, we performed multi-color fluorescence in situ hybridization using six probes extending across each chromosome in human WI38 lung fibroblasts. Six CT were selected ranging in size and gene density (1, 4, 12, 17, 18 and X). In-house computational geometric algorithms were applied to measure the 3D distances between every combination of probes and to elucidate data-mined structural patterns. Our findings demonstrate a high degree of non-random arrangement of individual CT that vary from chromosome to chromosome and display distinct changes during the cell cycle. Application of a classic, well-defined data mining and pattern recognition approach termed the 'k-means' generated 3D models for the best fit arrangement of each chromosome. These predicted models correlated well with the detailed distance measurements and analysis. We propose that the unique 3D topology of each CT and characteristic changes during the cell cycle provide the structural framework for the global gene expression programs of the individual chromosomes.

Foundations of identifying individual chromosomes by imaging flow cytometry with applications in radiation biodosimetry.

Biodosimetry is an important tool for triage in the case of large-scale radiological or nuclear emergencies, but traditional microscope-based methods can be tedious and prone to scorer fatigue. While the dicentric chromosome assay (DCA) has been adapted for use in triage situations, it is still time-consuming to create and score slides. Recent adaptations of traditional biodosimetry assays to imaging flow cytometry (IFC) methods have dramatically increased throughput. Additionally, recent improvements in image analysis algorithms in the IFC software have resulted in improved specificity for spot counting of small events. In the IFC method for the dicentric chromosome analysis (FDCA), lymphocytes isolated from whole blood samples are cultured with PHA and Colcemid. After incubation, lymphocytes are treated with a hypotonic solution and chromosomes are isolated in suspension, labelled with a centromere marker and stained for DNA content with DRAQ5. Stained individual chromosomes are analyzed on the ImageStream®X (EMD-Millipore, Billerica, MA) and mono- and dicentric chromosome populations are identified and enumerated using advanced image processing techniques. Both the preparation of the isolated chromosome suspensions as well as the image analysis methods were fine-tuned in order to optimize the FDCA. In this paper we describe the method to identify and score centromeres in individual chromosomes by IFC and show that the FDCA method may further improve throughput for triage biodosimetry in the case of large-scale radiological or nuclear emergencies.

Mining the topography and dynamics of the 4D Nucleome to identify novel CNS drug pathways.

The pharmacoepigenome can be defined as the active, noncoding province of the genome including canonical spatial and temporal regulatory mechanisms of gene regulation that respond to xenobiotic stimuli. Many psychotropic drugs that have been in clinical use for decades have ill-defined mechanisms of action that are beginning to be resolved as we understand the transcriptional hierarchy and dynamics of the nucleus. In this review, we describe spatial, temporal and biomechanical mechanisms mediated by psychotropic medications. Focus is placed on a bioinformatics pipeline that can be used both for detection of pharmacoepigenomic variants that discretize drug response and adverse events to improve pharmacogenomic testing, and for the discovery of novel CNS therapeutics. This approach integrates the functional topology and dynamics of the transcriptional hierarchy of the pharmacoepigenome, gene variant-driven identification of pharmacogenomic regulatory domains, and mesoscale mapping for the discovery of novel CNS pharmacodynamic pathways in human brain. Examples of the application of this pipeline are provided, including the discovery of valproic acid (VPA) mediated transcriptional reprogramming of neuronal cell fate following injury, and mapping of a CNS pathway glutamatergic pathway for the mood stabilizer lithium. These examples in regulatory pharmacoepigenomics illustrate how ongoing research using the 4D nucleome provides a foundation to further insight into previously unrecognized psychotropic drug pharmacodynamic pathways in the human CNS.

Simulated binding of transcription factors to active and inactive regions folds human chromosomes into loops, rosettes and topological domains.

Biophysicists are modeling conformations of interphase chromosomes, often basing the strengths of interactions between segments distant on the genetic map on contact frequencies determined experimentally. Here, instead, we develop a fitting-free, minimal model: bivalent or multivalent red and green 'transcription factors' bind to cognate sites in strings of beads ('chromatin') to form molecular bridges stabilizing loops. In the absence of additional explicit forces, molecular dynamic simulations reveal that bound factors spontaneously cluster-red with red, green with green, but rarely red with green-to give structures reminiscent of transcription factories. Binding of just two transcription factors (or proteins) to active and inactive regions of human chromosomes yields rosettes, topological domains and contact maps much like those seen experimentally. This emergent 'bridging-induced attraction' proves to be a robust, simple and generic force able to organize interphase chromosomes at all scales.

Multi-scale structural community organisation of the human genome.

BACKGROUND: Structural interaction frequency matrices between all genome loci are now experimentally achievable thanks to high-throughput chromosome conformation capture technologies. This ensues a new methodological challenge for computational biology which consists in objectively extracting from these data the structural motifs characteristic of genome organisation. RESULTS: We deployed the fast multi-scale community mining algorithm based on spectral graph wavelets to characterise the networks of intra-chromosomal interactions in human cell lines. We observed that there exist structural domains of all sizes up to chromosome length and demonstrated that the set of structural communities forms a hierarchy of chromosome segments. Hence, at all scales, chromosome folding predominantly involves interactions between neighbouring sites rather than the formation of links between distant loci. CONCLUSIONS: Multi-scale structural decomposition of human chromosomes provides an original framework to question structural organisation and its relationship to functional regulation across the scales. By construction the proposed methodology is independent of the precise assembly of the reference genome and is thus directly applicable to genomes whose assembly is not fully determined.