The shape of chromatin: insights from computational recognition of geometric patterns in Hi-C data.

The three-dimensional organization of chromatin plays a crucial role in gene regulation and cellular processes like deoxyribonucleic acid (DNA) transcription, replication and repair. Hi-C and related techniques provide detailed views of spatial proximities within the nucleus. However, data analysis is challenging partially due to a lack of well-defined, underpinning mathematical frameworks. Recently, recognizing and analyzing geometric patterns in Hi-C data has emerged as a powerful approach. This review provides a summary of algorithms for automatic recognition and analysis of geometric patterns in Hi-C data and their correspondence with chromatin structure. We classify existing algorithms on the basis of the data representation and pattern recognition paradigm they make use of. Finally, we outline some of the challenges ahead and promising future directions.

hictk: blazing fast toolkit to work with .hic and .cool files.

MOTIVATION: Hi-C is gaining prominence as a method for mapping genome organization. With declining sequencing costs and a growing demand for higher-resolution data, efficient tools for processing Hi-C datasets at different resolutions are crucial. Over the past decade, the .hic and Cooler file formats have become the de-facto standard to store interaction matrices produced by Hi-C experiments in binary format. Interoperability issues make it unnecessarily difficult to convert between the two formats and to develop applications that can process each format natively. RESULTS: We developed hictk, a toolkit that can transparently operate on .hic and .cool files with excellent performance. The toolkit is written in C++ and consists of a C++ library with Python and R bindings as well as CLI tools to perform common operations directly from the shell, including converting between .hic and .mcool formats. We benchmark the performance of hictk and compare it with other popular tools and libraries. We conclude that hictk significantly outperforms existing tools while providing the flexibility of natively working with both file formats without code duplication. AVAILABILITY AND IMPLEMENTATION: The hictk library, Python bindings and CLI tools are released under the MIT license as a multi-platform application available at github.com/paulsengroup/hictk. Pre-built binaries for Linux and macOS are available on bioconda. Python bindings for hictk are available on GitHub at github.com/paulsengroup/hictkpy, while R bindings are available on GitHub at github.com/paulsengroup/hictkR.

TAD cliques predict key features of chromatin organization.

BACKGROUND: Mechanisms underlying genome 3D organization and domain formation in the mammalian nucleus are not completely understood. Multiple processes such as transcriptional compartmentalization, DNA loop extrusion and interactions with the nuclear lamina dynamically act on chromatin at multiple levels. Here, we explore long-range interaction patterns between topologically associated domains (TADs) in several cell types. RESULTS: We find that TAD long-range interactions are connected to many key features of chromatin organization, including open and closed compartments, compaction and loop extrusion processes. Domains that form large TAD cliques tend to be repressive across cell types, when comparing gene expression, LINE/SINE repeat content and chromatin subcompartments. Further, TADs in large cliques are larger in genomic size, less dense and depleted of convergent CTCF motifs, in contrast to smaller and denser TADs formed by a loop extrusion process. CONCLUSIONS: Our results shed light on the organizational principles that govern repressive and active domains in the human genome.

Carcinogen susceptibility is regulated by genome architecture and predicts cancer mutagenesis.

The development of many sporadic cancers is directly initiated by carcinogen exposure. Carcinogens induce malignancies by creating DNA lesions (i.e., adducts) that can result in mutations if left unrepaired. Despite this knowledge, there has been remarkably little investigation into the regulation of susceptibility to acquire DNA lesions. In this study, we present the first quantitative human genome-wide map of DNA lesions induced by ultraviolet (UV) radiation, the ubiquitous carcinogen in sunlight that causes skin cancer. Remarkably, the pattern of carcinogen susceptibility across the genome of primary cells significantly reflects mutation frequency in malignant melanoma. Surprisingly, DNase-accessible euchromatin is protected from UV, while lamina-associated heterochromatin at the nuclear periphery is vulnerable. Many cancer driver genes have an intrinsic increase in carcinogen susceptibility, including the BRAF oncogene that has the highest mutation frequency in melanoma. These findings provide a genome-wide snapshot of DNA injuries at the earliest stage of carcinogenesis. Furthermore, they identify carcinogen susceptibility as an origin of genome instability that is regulated by nuclear architecture and mirrors mutagenesis in cancer.

The lipodystrophic hotspot lamin A p.R482W mutation deregulates the mesodermal inducer T/Brachyury and early vascular differentiation gene networks.

The p.R482W hotspot mutation in A-type nuclear lamins causes familial partial lipodystrophy of Dunnigan-type (FPLD2), a lipodystrophic syndrome complicated by early onset atherosclerosis. Molecular mechanisms underlying endothelial cell dysfunction conferred by the lamin A mutation remain elusive. However, lamin A regulates epigenetic developmental pathways and mutations could perturb these functions. Here, we demonstrate that lamin A R482W elicits endothelial differentiation defects in a developmental model of FPLD2. Genome modeling in fibroblasts from patients with FPLD2 caused by the lamin A R482W mutation reveals repositioning of the mesodermal regulator T/Brachyury locus towards the nuclear center relative to normal fibroblasts, suggesting enhanced activation propensity of the locus in a developmental model of FPLD2. Addressing this issue, we report phenotypic and transcriptional alterations in mesodermal and endothelial differentiation of induced pluripotent stem cells we generated from a patient with R482W-associated FPLD2. Correction of the LMNA mutation ameliorates R482W-associated phenotypes and gene expression. Transcriptomics links endothelial differentiation defects to decreased Polycomb-mediated repression of the T/Brachyury locus and over-activation of T target genes. Binding of the Polycomb repressor complex 2 to T/Brachyury is impaired by the mutated lamin A network, which is unable to properly associate with the locus. This leads to a deregulation of vascular gene expression over time. By connecting a lipodystrophic hotspot lamin A mutation to a disruption of early mesodermal gene expression and defective endothelial differentiation, we propose that the mutation rewires the fate of several lineages, resulting in multi-tissue pathogenic phenotypes.

Galaxy Portal: interacting with the galaxy platform through mobile devices.

UNLABELLED: : We present Galaxy Portal app, an open source interface to the Galaxy system through smart phones and tablets. The Galaxy Portal provides convenient and efficient monitoring of job completion, as well as opportunities for inspection of results and execution history. In addition to being useful to the Galaxy community, we believe that the app also exemplifies a useful way of exploiting mobile interfaces for research/high-performance computing resources in general. AVAILABILITY AND IMPLEMENTATION: The source is freely available under a GPL license on GitHub, along with user documentation and pre-compiled binaries and instructions for several platforms: https://github.com/Tarostar/QMLGalaxyPortal It is available for iOS version 7 (and newer) through the Apple App Store, and for Android through Google Play for version 4.1 (API 16) or newer. CONTACT: geirksa@ifi.uio.no.

The genome sequence of Atlantic cod reveals a unique immune system.

Atlantic cod (Gadus morhua) is a large, cold-adapted teleost that sustains long-standing commercial fisheries and incipient aquaculture. Here we present the genome sequence of Atlantic cod, showing evidence for complex thermal adaptations in its haemoglobin gene cluster and an unusual immune architecture compared to other sequenced vertebrates. The genome assembly was obtained exclusively by 454 sequencing of shotgun and paired-end libraries, and automated annotation identified 22,154 genes. The major histocompatibility complex (MHC) II is a conserved feature of the adaptive immune system of jawed vertebrates, but we show that Atlantic cod has lost the genes for MHC II, CD4 and invariant chain (Ii) that are essential for the function of this pathway. Nevertheless, Atlantic cod is not exceptionally susceptible to disease under natural conditions. We find a highly expanded number of MHC I genes and a unique composition of its Toll-like receptor (TLR) families. This indicates how the Atlantic cod immune system has evolved compensatory mechanisms in both adaptive and innate immunity in the absence of MHC II. These observations affect fundamental assumptions about the evolution of the adaptive immune system and its components in vertebrates.

Manifold Based Optimization for Single-Cell 3D Genome Reconstruction.

The three-dimensional (3D) structure of the genome is important for orchestration of gene expression and cell differentiation. While mapping genomes in 3D has for a long time been elusive, recent adaptations of high-throughput sequencing to chromosome conformation capture (3C) techniques, allows for genome-wide structural characterization for the first time. However, reconstruction of "consensus" 3D genomes from 3C-based data is a challenging problem, since the data are aggregated over millions of cells. Recent single-cell adaptations to the 3C-technique, however, allow for non-aggregated structural assessment of genome structure, but data suffer from sparse and noisy interaction sampling. We present a manifold based optimization (MBO) approach for the reconstruction of 3D genome structure from chromosomal contact data. We show that MBO is able to reconstruct 3D structures based on the chromosomal contacts, imposing fewer structural violations than comparable methods. Additionally, MBO is suitable for efficient high-throughput reconstruction of large systems, such as entire genomes, allowing for comparative studies of genomic structure across cell-lines and different species.

A statistical model of ChIA-PET data for accurate detection of chromatin 3D interactions.

Identification of three-dimensional (3D) interactions between regulatory elements across the genome is crucial to unravel the complex regulatory machinery that orchestrates proliferation and differentiation of cells. ChIA-PET is a novel method to identify such interactions, where physical contacts between regions bound by a specific protein are quantified using next-generation sequencing. However, determining the significance of the observed interaction frequencies in such datasets is challenging, and few methods have been proposed. Despite the fact that regions that are close in linear genomic distance have a much higher tendency to interact by chance, no methods to date are capable of taking such dependency into account. Here, we propose a statistical model taking into account the genomic distance relationship, as well as the general propensity of anchors to be involved in contacts overall. Using both real and simulated data, we show that the previously proposed statistical test, based on Fisher's exact test, leads to invalid results when data are dependent on genomic distance. We also evaluate our method on previously validated cell-line specific and constitutive 3D interactions, and show that relevant interactions are significant, while avoiding over-estimating the significance of short nearby interactions.

HiBrowse: multi-purpose statistical analysis of genome-wide chromatin 3D organization.

UNLABELLED: Recently developed methods that couple next-generation sequencing with chromosome conformation capture-based techniques, such as Hi-C and ChIA-PET, allow for characterization of genome-wide chromatin 3D structure. Understanding the organization of chromatin in three dimensions is a crucial next step in the unraveling of global gene regulation, and methods for analyzing such data are needed. We have developed HiBrowse, a user-friendly web-tool consisting of a range of hypothesis-based and descriptive statistics, using realistic assumptions in null-models. AVAILABILITY AND IMPLEMENTATION: HiBrowse is supported by all major browsers, and is freely available at http://hyperbrowser.uio.no/3d. Software is implemented in Python, and source code is available for download by following instructions on the main site.

The regulatory landscape of osteogenic differentiation.

Differentiation of osteoblasts from mesenchymal stem cells (MSCs) is an integral part of bone development and homeostasis, and may when improperly regulated cause disease such as bone cancer or osteoporosis. Using unbiased high-throughput methods we here characterize the landscape of global changes in gene expression, histone modifications, and DNA methylation upon differentiation of human MSCs to the osteogenic lineage. Furthermore, we provide a first genome-wide characterization of DNA binding sites of the bone master regulatory transcription factor Runt-related transcription factor 2 (RUNX2) in human osteoblasts, revealing target genes associated with regulation of proliferation, migration, apoptosis, and with a significant overlap with p53 regulated genes. These findings expand on emerging evidence of a role for RUNX2 in cancer, including bone metastases, and the p53 regulatory network. We further demonstrate that RUNX2 binds to distant regulatory elements, promoters, and with high frequency to gene 3' ends. Finally, we identify TEAD2 and GTF2I as novel regulators of osteogenesis.

Handling realistic assumptions in hypothesis testing of 3D co-localization of genomic elements.

The study of chromatin 3D structure has recently gained much focus owing to novel techniques for detecting genome-wide chromatin contacts using next-generation sequencing. A deeper understanding of the architecture of the DNA inside the nucleus is crucial for gaining insight into fundamental processes such as transcriptional regulation, genome dynamics and genome stability. Chromatin conformation capture-based methods, such as Hi-C and ChIA-PET, are now paving the way for routine genome-wide studies of chromatin 3D structure in a range of organisms and tissues. However, appropriate methods for analyzing such data are lacking. Here, we propose a hypothesis test and an enrichment score of 3D co-localization of genomic elements that handles intra- or interchromosomal interactions, both separately and jointly, and that adjusts for biases caused by structural dependencies in the 3D data. We show that maintaining structural properties during resampling is essential to obtain valid estimation of P-values. We apply the method on chromatin states and a set of mutated regions in leukemia cells, and find significant co-localization of these elements, with varying enrichment scores, supporting the role of chromatin 3D structure in shaping the landscape of somatic mutations in cancer.

The Genomic HyperBrowser: an analysis web server for genome-scale data.

The immense increase in availability of genomic scale datasets, such as those provided by the ENCODE and Roadmap Epigenomics projects, presents unprecedented opportunities for individual researchers to pose novel falsifiable biological questions. With this opportunity, however, researchers are faced with the challenge of how to best analyze and interpret their genome-scale datasets. A powerful way of representing genome-scale data is as feature-specific coordinates relative to reference genome assemblies, i.e. as genomic tracks. The Genomic HyperBrowser (http://hyperbrowser.uio.no) is an open-ended web server for the analysis of genomic track data. Through the provision of several highly customizable components for processing and statistical analysis of genomic tracks, the HyperBrowser opens for a range of genomic investigations, related to, e.g., gene regulation, disease association or epigenetic modifications of the genome.

Alkbh1 and Tzfp repress a non-repeat piRNA cluster in pachytene spermatocytes.

Piwi proteins and Piwi-interacting small RNAs (piRNAs) have known functions in transposon silencing in the male germline of fetal and newborn mice. Both are also present in adult testes; however, their function here remains a mystery. Here, we confirm that most piRNAs in meiotic spermatocytes originate from clusters in non-repeat intergenic regions of DNA. The regulation of these piRNA clusters, including the processing of the precursor transcripts into individual piRNAs, is accomplished through mostly unknown processes. We present a possible regulatory mechanism for one such cluster, named cluster 1082B, located on chromosome 7 in the mouse genome. The 1082B precursor transcript and its 788 unique piRNAs are repressed by the Alkbh1 dioxygenase and the testis-specific transcription repressor Tzfp. We observe a remarkable >1000-fold upregulation of individual piRNAs in pachytene spermatocytes isolated from Alkbh1- and Tzfp-deficient murine testes. Repression of cluster 1082B is further supported by the identification of a 10-bp Tzfp recognition sequence contained within the precursor transcript. Downregulation of LINE1 and IAP transcripts in the Alkbh1- and Tzfp-deficient mice leads us to propose a potential role for the 1082B-encoded piRNAs in transposon control.

Multi-level 3D genome organization deteriorates during breast cancer progression.

Breast cancer entails intricate alterations in genome organization and expression. However, how three-dimensional (3D) chromatin structure changes in the progression from a normal to a breast cancer malignant state remains unknown. To address this, we conducted an analysis combining Hi-C data with lamina-associated domains (LADs), epigenomic marks, and gene expression in an in vitro model of breast cancer progression. Our results reveal that while the fundamental properties of topologically associating domains (TADs) remain largely stable, significant changes occur in the organization of compartments and subcompartments. These changes are closely correlated with alterations in the expression of oncogenic genes. We also observe a restructuring of TAD-TAD interactions, coinciding with a loss of spatial compartmentalization and radial positioning of the 3D genome. Notably, we identify a previously unrecognized interchromosomal insertion event, wherein a locus on chromosome 8 housing the MYC oncogene is inserted into a highly active subcompartment on chromosome 10. This insertion leads to the formation of de novo enhancer contacts and activation of the oncogene, illustrating how structural variants can interact with the 3D genome to drive oncogenic states. In summary, our findings provide evidence for the degradation of genome organization at multiple scales during breast cancer progression revealing novel relationships between genome 3D structure and oncogenic processes.

Modeling the 3D Genome Using Hi-C and Nuclear Lamin-Genome Contacts.

The three-dimensional (3D) organization of the genome is shaped by interactions with multiple structures within the nucleus, affecting gene expression outcomes. Technological breakthroughs in recent years have generated vast data reflecting various aspects of nuclear genome architecture in space and time. Integrating these datasets into comprehensive 3D genome models can reveal new insights into genome structure and regulation in normal and disease states. In this chapter, we provide a step-by-step guide on how to generate publication-ready integrated 3D genome models from (raw) Hi-C data and from lamin-genome (LAD) contacts.

MoDLE: high-performance stochastic modeling of DNA loop extrusion interactions.

DNA loop extrusion emerges as a key process establishing genome structure and function. We introduce MoDLE, a computational tool for fast, stochastic modeling of molecular contacts from DNA loop extrusion capable of simulating realistic contact patterns genome wide in a few minutes. MoDLE accurately simulates contact maps in concordance with existing molecular dynamics approaches and with Micro-C data and does so orders of magnitude faster than existing approaches. MoDLE runs efficiently on machines ranging from laptops to high performance computing clusters and opens up for exploratory and predictive modeling of 3D genome structure in a wide range of settings.

4D nucleome modeling.

The intrinsic dynamic nature of chromosomes is emerging as a fundamental component in regulating DNA transcription, replication, and damage-repair among other nuclear functions. With this increased awareness, reinforced over the last ten years, many new experimental techniques, mainly based on microscopy and chromosome conformation capture, have been introduced to study the genome in space and time. Owing to the increasing complexity of these cutting-edge techniques, computational approaches have become of paramount importance to interpret, contextualize, and complement such experiments with new insights. Hence, it is becoming crucial for experimental biologists to have a clear understanding of the diverse theoretical modeling approaches available and the biological information each of them can provide.

Long-range interactions between topologically associating domains shape the four-dimensional genome during differentiation.

Genomic information is selectively used to direct spatial and temporal gene expression during differentiation. Interactions between topologically associating domains (TADs) and between chromatin and the nuclear lamina organize and position chromosomes in the nucleus. However, how these genomic organizers together shape genome architecture is unclear. Here, using a dual-lineage differentiation system, we report long-range TAD-TAD interactions that form constitutive and variable TAD cliques. A differentiation-coupled relationship between TAD cliques and lamina-associated domains suggests that TAD cliques stabilize heterochromatin at the nuclear periphery. We also provide evidence of dynamic TAD cliques during mouse embryonic stem-cell differentiation and somatic cell reprogramming and of inter-TAD associations in single-cell high-resolution chromosome conformation capture (Hi-C) data. TAD cliques represent a level of four-dimensional genome conformation that reinforces the silencing of repressed developmental genes.

Rapid evolution of the intergenic T-P spacer in the mtDNA of Arctic cod Arctogadus glacialis.

A noncoding intergenic spacer has previously been reported in mtDNA of Gadiformes. Here we present sequence information from two other cod species and variation within three species to clarify the evolution of this region. A general feature of the T-P spacer is high variation and folding into two or three hairpins. The variation among species both in structure of the region and sequence variation reflects the phylogenetic relationship of the species. A unique pattern is found within Arctic cod, Arctogadus glacialis, in which tandem repeat motifs result in new stable secondary structures. There is large variation in size of the region both within (heteroplasmy) and among individuals. A duplicated insertion is found in Greenland cod, Gadus ogac, at the same position as a corresponding duplication in Atlantic cod, Gadus morhua.