Comparison of Hi-C results using in-solution versus in-nucleus ligation

BackgroundChromosome conformation capture and various derivative methods such as 4C, 5C and Hi-C have emerged as standard tools to analyze the three-dimensional organization of the genome in the nucleus. These methods employ ligation of diluted cross-linked chromatin complexes, intended to favor proximity-dependent, intra-complex ligation. During development of single-cell Hi-C, we devised an alternative Hi-C protocol with ligation in preserved nuclei rather than in solution. Here we directly compare Hi-C methods employing in-nucleus ligation with the standard in-solution ligation.ResultsWe show in-nucleus ligation results in consistently lower levels of inter-chromosomal contacts. Through chromatin mixing experiments we show that a significantly large fraction of inter-chromosomal contacts are the result of spurious ligation events formed during in-solution ligation. In-nucleus ligation significantly reduces this source of experimental noise, and results in improved reproducibility between replicates. We also find that in-nucleus ligation eliminates restriction fragment length bias found with in-solution ligation. These improvements result in greater reproducibility of long-range intra-chromosomal and inter-chromosomal contacts, as well as enhanced detection of structural features such as topologically associated domain boundaries.ConclusionsWe conclude that in-nucleus ligation captures chromatin interactions more consistently over a wider range of distances, and significantly reduces both experimental noise and bias. In-nucleus ligation creates higher quality Hi-C libraries while simplifying the experimental procedure. We suggest that the entire range of 3C applications are likely to show similar benefits from in-nucleus ligation.

Electronic supplementary material

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Three-Dimensional Genome Architecture Influences Partner Selection for Chromosomal Translocations in Human Disease

Chromosomal translocations are frequent features of cancer genomes that contribute to disease progression. These rearrangements result from formation and illegitimate repair of DNA double-strand breaks (DSBs), a process that requires spatial colocalization of chromosomal breakpoints. The “contact first” hypothesis suggests that translocation partners colocalize in the nuclei of normal cells, prior to rearrangement. It is unclear, however, the extent to which spatial interactions based on three-dimensional genome architecture contribute to chromosomal rearrangements in human disease. Here we intersect Hi-C maps of three-dimensional chromosome conformation with collections of 1,533 chromosomal translocations from cancer and germline genomes. We show that many translocation-prone pairs of regions genome-wide, including the cancer translocation partners BCR-ABL and MYC-IGH, display elevated Hi-C contact frequencies in normal human cells. Considering tissue specificity, we find that translocation breakpoints reported in human hematologic malignancies have higher Hi-C contact frequencies in lymphoid cells than those reported in sarcomas and epithelial tumors. However, translocations from multiple tissue types show significant correlation with Hi-C contact frequencies, suggesting that both tissue-specific and universal features of chromatin structure contribute to chromosomal alterations. Our results demonstrate that three-dimensional genome architecture shapes the landscape of rearrangements directly observed in human disease and establish Hi-C as a key method for dissecting these effects.     

Bipartite structure of the inactive mouse X chromosome

BackgroundIn mammals, one of the female X chromosomes and all imprinted genes are expressed exclusively from a single allele in somatic cells. To evaluate structural changes associated with allelic silencing, we have applied a recently developed Hi-C assay that uses DNase I for chromatin fragmentation to mouse F1 hybrid systems.ResultsWe find radically different conformations for the two female mouse X chromosomes. The inactive X has two superdomains of frequent intrachromosomal contacts separated by a boundary region. Comparison with the recently reported two-superdomain structure of the human inactive X shows that the genomic content of the superdomains differs between species, but part of the boundary region is conserved and located near the Dxz4/DXZ4 locus. In mouse, the boundary region also contains a minisatellite, Ds-TR, and both Dxz4 and Ds-TR appear to be anchored to the nucleolus. Genes that escape X inactivation do not cluster but are located near the periphery of the 3D structure, as are regions enriched in CTCF or RNA polymerase. Fewer short-range intrachromosomal contacts are detected for the inactive alleles of genes subject to X inactivation compared with the active alleles and with genes that escape X inactivation. This pattern is also evident for imprinted genes, in which more chromatin contacts are detected for the expressed allele.ConclusionsBy applying a novel Hi-C method to map allelic chromatin contacts, we discover a specific bipartite organization of the mouse inactive X chromosome that probably plays an important role in maintenance of gene silencing.

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Identifying multi-locus chromatin contacts in human cells using tethered multiple 3C

Background

Several recently developed experimental methods, each an extension of the chromatin conformation capture (3C) assay, have enabled the genome-wide profiling of chromatin contacts between pairs of genomic loci in 3D. Especially in complex eukaryotes, data generated by these methods, coupled with other genome-wide datasets, demonstrated that non-random chromatin folding correlates strongly with cellular processes such as gene expression and DNA replication.

Results

We describe a genome architecture assay, tethered multiple 3C (TM3C), that maps genome-wide chromatin contacts via a simple protocol of restriction enzyme digestion and religation of fragments upon agarose gel beads followed by paired-end sequencing. In addition to identifying contacts between pairs of loci, TM3C enables identification of contacts among more than two loci simultaneously. We use TM3C to assay the genome architectures of two human cell lines: KBM7, a near-haploid chronic leukemia cell line, and NHEK, a normal diploid human epidermal keratinocyte cell line. We confirm that the contact frequency maps produced by TM3C exhibit features characteristic of existing genome architecture datasets, including the expected scaling of contact probabilities with genomic distance, megabase scale chromosomal compartments and sub-megabase scale topological domains. We also confirm that TM3C captures several known cell type-specific contacts, ploidy shifts and translocations, such as Philadelphia chromosome formation (Ph+) in KBM7. We confirm a subset of the triple contacts involving the IGF2-H19 imprinting control region (ICR) using PCR analysis for KBM7 cells. Our genome-wide analysis of pairwise and triple contacts demonstrates their preference for linking open chromatin regions to each other and for linking regions with higher numbers of DNase hypersensitive sites (DHSs) to each other. For near-haploid KBM7 cells, we infer whole genome 3D models that exhibit clustering of small chromosomes with each other and large chromosomes with each other, consistent with previous studies of the genome architectures of other human cell lines.

Conclusion

TM3C is a simple protocol for ascertaining genome architecture and can be used to identify simultaneous contacts among three or four loci. Application of TM3C to a near-haploid human cell line revealed large-scale features of chromosomal organization and multi-way chromatin contacts that preferentially link regions of open chromatin.

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Unbiased chromatin accessibility profiling by RED-seq uncovers unique features of nucleosome variants in vivo

Background

Differential accessibility of DNA to nuclear proteins underlies the regulation of numerous cellular processes. Although DNA accessibility is primarily determined by the presence or absence of nucleosomes, differences in nucleosome composition or dynamics may also regulate accessibility. Methods for mapping nucleosome positions and occupancies genome-wide (MNase-seq) have uncovered the nucleosome landscapes of many different cell types and organisms. Conversely, methods specialized for the detection of large nucleosome-free regions of chromatin (DNase-seq, FAIRE-seq) have uncovered numerous gene regulatory elements. However, these methods are less successful in measuring the accessibility of DNA sequences within nucelosome arrays.

Results

Here we probe the genome-wide accessibility of multiple cell types in an unbiased manner using restriction endonuclease digestion of chromatin coupled to deep sequencing (RED-seq). Using this method, we identified differences in chromatin accessibility between populations of cells, not only in nucleosome-depleted regions of the genome (e.g., enhancers and promoters), but also within the majority of the genome that is packaged into nucleosome arrays. Furthermore, we identified both large differences in chromatin accessibility in distinct cell lineages and subtle but significant changes during differentiation of mouse embryonic stem cells (ESCs). Most significantly, using RED-seq, we identified differences in accessibility among nucleosomes harboring well-studied histone variants, and show that these differences depend on factors required for their deposition.

Conclusions

Using an unbiased method to probe chromatin accessibility genome-wide, we uncover unique features of chromatin structure that are not observed using more widely-utilized methods. We demonstrate that different types of nucleosomes within mammalian cells exhibit different degrees of accessibility. These findings provide significant insight into the regulation of DNA accessibility.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1104) contains supplementary material, which is available to authorized users.     

ASHIC: hierarchical Bayesian modeling of diploid chromatin contacts and structures

The recently developed Hi-C technique has been widely applied to map genome-wide chromatin interactions. However, current methods for analyzing diploid Hi-C data cannot fully distinguish between homologous chromosomes. Consequently, the existing diploid Hi-C analyses are based on sparse and inaccurate allele-specific contact matrices, which might lead to incorrect modeling of diploid genome architecture. Here we present ASHIC, a hierarchical Bayesian framework to model allele-specific chromatin organizations in diploid genomes. We developed two models under the Bayesian framework: the Poisson-multinomial (ASHIC-PM) model and the zero-inflated Poisson-multinomial (ASHIC-ZIPM) model. The proposed ASHIC methods impute allele-specific contact maps from diploid Hi-C data and simultaneously infer allelic 3D structures. Through simulation studies, we demonstrated that ASHIC methods outperformed existing approaches, especially under low coverage and low SNP density conditions. Additionally, in the analyses of diploid Hi-C datasets in mouse and human, our ASHIC-ZIPM method produced fine-resolution diploid chromatin maps and 3D structures and provided insights into the allelic chromatin organizations and functions. To summarize, our work provides a statistically rigorous framework for investigating fine-scale allele-specific chromatin conformations. The ASHIC software is publicly available at https://github.com/wmalab/ASHIC.     

Microrheology for Hi-C Data Reveals the Spectrum of the Dynamic 3D Genome Organization

The one-dimensional information of genomic DNA is hierarchically packed inside the eukaryotic cell nucleus and organized in a three-dimensional (3D) space. Genome-wide chromosome conformation capture (Hi-C) methods have uncovered the 3D genome organization and revealed multiscale chromatin domains of compartments and topologically associating domains (TADs). Moreover, single-nucleosome live-cell imaging experiments have revealed the dynamic organization of chromatin domains caused by stochastic thermal fluctuations. However, the mechanism underlying the dynamic regulation of such hierarchical and structural chromatin units within the microscale thermal medium remains unclear. Microrheology is a way to measure dynamic viscoelastic properties coupling between thermal microenvironment and mechanical response. Here, we propose a new, to our knowledge, microrheology for Hi-C data to analyze the dynamic compliance property as a measure of rigidness and flexibility of genomic regions along with the time evolution. Our method allows the conversion of an Hi-C matrix into the spectrum of the dynamic rheological property along the genomic coordinate of a single chromosome. To demonstrate the power of the technique, we analyzed Hi-C data during the neural differentiation of mouse embryonic stem cells. We found that TAD boundaries behave as more rigid nodes than the intra-TAD regions. The spectrum clearly shows the dynamic viscoelasticity of chromatin domain formation at different timescales. Furthermore, we characterized the appearance of synchronous and liquid-like intercompartment interactions in differentiated cells. Together, our microrheology data derived from Hi-C data provide physical insights into the dynamics of the 3D genome organization.     

Relaxed 3D genome conformation facilitates the pluripotent to totipotent-like state transition in embryonic stem cells

The 3D genome organization is crucial for gene regulation. Although recent studies have revealed a uniquely relaxed genome conformation in totipotent early blastomeres of both fertilized and cloned embryos, how weakened higher-order chromatin structure is functionally linked to totipotency acquisition remains elusive. Using low-input Hi-C, ATAC-seq and ChIP-seq, we systematically examined the dynamics of 3D genome and epigenome during pluripotent to totipotent-like state transition in mouse embryonic stem cells (ESCs). The spontaneously converted 2-cell-embryo-like cells (2CLCs) exhibited more relaxed chromatin architecture compared to ESCs, including global weakening of both enhancer-promoter interactions and TAD insulation. While the former correlated with inactivation of ESC enhancers and down-regulation of pluripotent genes, the latter might facilitate contacts between the putative new enhancers arising in 2CLCs and neighboring 2C genes. Importantly, disruption of chromatin organization by depleting CTCF or the cohesin complex promoted the ESC to 2CLC transition. Our results thus establish a critical role of 3D genome organization in totipotency acquisition.     

Large-scale 3D chromatin reconstruction from chromosomal contacts

Background

Recent advances in genome analysis have established that chromatin has preferred 3D conformations, which bring distant loci into contact. Identifying these contacts is important for us to understand possible interactions between these loci. This has motivated the creation of the Hi-C technology, which detects long-range chromosomal interactions. Distance geometry-based algorithms, such as ChromSDE and ShRec3D, have been able to utilize Hi-C data to infer 3D chromosomal structures. However, these algorithms, being matrix-based, are space- and time-consuming on very large datasets. A human genome of 100 kilobase resolution would involve ∼30,000 loci, requiring gigabytes just in storing the matrices.

Results

We propose a succinct representation of the distance matrices which tremendously reduces the space requirement. We give a complete solution, called SuperRec, for the inference of chromosomal structures from Hi-C data, through iterative solving the large-scale weighted multidimensional scaling problem.

Conclusions

SuperRec runs faster than earlier systems without compromising on result accuracy. The SuperRec package can be obtained from http://www.cs.cityu.edu.hk/~shuaicli/SuperRec.

     

HiCORE: Hi-C Analysis for Identification of Core Chromatin Looping Regions with Higher Resolution

Genome-wide chromosome conformation capture (3C)-based high-throughput sequencing (Hi-C) has enabled identification of genome-wide chromatin loops. Because the Hi-C map with restriction fragment resolution is intrinsically associated with sparsity and stochastic noise, Hi-C data are usually binned at particular intervals; however, the binning method has limited reliability, especially at high resolution. Here, we describe a new method called HiCORE, which provides simple pipelines and algorithms to overcome the limitations of single-layered binning and predict core chromatin regions with three-dimensional physical interactions. In this approach, multiple layers of binning with slightly shifted genome coverage are generated, and interacting bins at each layer are integrated to infer narrower regions of chromatin interactions. HiCORE predicts chromatin looping regions with higher resolution, both in human and Arabidopsis genomes, and contributes to the identification of the precise positions of potential genomic elements in an unbiased manner.     

Defective chromatin architectures in embryonic stem cells derived from somatic cell nuclear transfer impair their differentiation potentials

Nuclear transfer embryonic stem cells (ntESCs) hold enormous promise for individual-specific regenerative medicine. However, the chromatin states of ntESCs remain poorly characterized. In this study, we employed ATAC-seq and Hi-C techniques to explore the chromatin accessibility and three-dimensional (3D) genome organization of ntESCs. The results show that the chromatin accessibility and genome structures of somatic cells are re-arranged to ESC-like states overall in ntESCs, including compartments, topologically associating domains (TADs) and chromatin loops. However, compared to fertilized ESCs (fESCs), ntESCs show some abnormal openness and structures that have not been reprogrammed completely, which impair the differentiation potential of ntESCs. The histone modification H3K9me3 may be involved in abnormal structures in ntESCs, including incorrect compartment switches and incomplete TAD rebuilding. Moreover, ntESCs and iPSCs show high similarity in 3D genome structures, while a few differences are detected due to different somatic cell origins and reprogramming mechanisms. Through systematic analyses, our study provides a global view of chromatin accessibility and 3D genome organization in ntESCs, which can further facilitate the understanding of the similarities and differences between ntESCs and fESCs.Subject terms: Embryonic stem cells, Reprogramming, Stem-cell differentiation     

HPRep: Quantifying Reproducibility in HiChIP and PLAC-Seq Datasets

HiChIP and PLAC-Seq are emerging technologies for studying genome-wide long-range chromatin interactions mediated by the protein of interest, enabling more sensitive and cost-efficient interrogation of protein-centric chromatin conformation. However, due to the unbalanced read distribution introduced by protein immunoprecipitation, existing reproducibility measures developed for Hi-C data are not appropriate for the analysis of HiChIP and PLAC-Seq data. Here, we present HPRep, a stratified and weighted correlation metric derived from normalized contact counts, to quantify reproducibility in HiChIP and PLAC-Seq data. We applied HPRep to multiple real datasets and demonstrate that HPRep outperforms existing reproducibility measures developed for Hi-C data. Specifically, we applied HPRep to H3K4me3 PLAC-Seq data from mouse embryonic stem cells and mouse brain tissues as well as H3K27ac HiChIP data from human lymphoblastoid cell line GM12878 and leukemia cell line K562, showing that HPRep can more clearly separate among pseudo-replicates, real replicates, and non-replicates. Furthermore, in an H3K4me3 PLAC-Seq dataset consisting of 11 samples from four human brain cell types, HPRep demonstrated the expected clustering of data that could not be achieved by existing methods developed for Hi-C data, highlighting the need for a reproducibility metric tailored to HiChIP and PLAC-Seq data.     

Extracting multi-way chromatin contacts from Hi-C data

There is a growing realization that multi-way chromatin contacts formed in chromosome structures are fundamental units of gene regulation. However, due to the paucity and complexity of such contacts, it is challenging to detect and identify them using experiments. Based on an assumption that chromosome structures can be mapped onto a network of Gaussian polymer, here we derive analytic expressions for n-body contact probabilities (n > 2) among chromatin loci based on pairwise genomic contact frequencies available in Hi-C, and show that multi-way contact probability maps can in principle be extracted from Hi-C. The three-body (triplet) contact probabilities, calculated from our theory, are in good correlation with those from measurements including Tri-C, MC-4C and SPRITE. Maps of multi-way chromatin contacts calculated from our analytic expressions can not only complement experimental measurements, but also can offer better understanding of the related issues, such as cell-line dependent assemblies of multiple genes and enhancers to chromatin hubs, competition between long-range and short-range multi-way contacts, and condensates of multiple CTCF anchors.     

NucleoMap: A computational tool for identifying nucleosomes in ultra-high resolution contact maps

Although poorly positioned nucleosomes are ubiquitous in the eukaryotic genome, they are difficult to identify with existing nucleosome identification methods. Recently available enhanced high-throughput chromatin conformation capture techniques such as Micro-C, DNase Hi-C, and Hi-CO characterize nucleosome-level chromatin proximity, probing the positions of mono-nucleosomes and the spacing between nucleosome pairs at the same time, enabling nucleosome profiling in poorly positioned regions. Here we develop a novel computational approach, NucleoMap, to identify nucleosome positioning from ultra-high resolution chromatin contact maps. By integrating nucleosome read density, contact distances, and binding preferences, NucleoMap precisely locates nucleosomes in both prokaryotic and eukaryotic genomes and outperforms existing nucleosome identification methods in both precision and recall. We rigorously characterize genome-wide association in eukaryotes between the spatial organization of mono-nucleosomes and their corresponding histone modifications, protein binding activities, and higher-order chromatin functions. We also find evidence of two tetra-nucleosome folding structures in human embryonic stem cells and analyze their association with multiple structural and functional regions. Based on the identified nucleosomes, nucleosome contact maps are constructed, reflecting the inter-nucleosome distances and preserving the contact distance profiles in original contact maps.     

Stella Regulates the Development of Female Germline Stem Cells by Modulating Chromatin Structure and DNA Methylation

Female germline stem cells (FGSCs) have the ability to self-renew and differentiate into oocytes. Stella, encoded by a maternal effect gene, plays an important role in oogenesis and early embryonic development. However, its function in FGSCs remains unclear. In this study, we showed that CRISPR/Cas9-mediated knockout of Stella promoted FGSC proliferation and reduced the level of genome-wide DNA methylation of FGSCs. Conversely, Stella overexpression led to the opposite results, and enhanced FGSC differentiation. We also performed an integrative analysis of chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), high-throughput genome-wide chromosome conformation capture (Hi-C), and use of our published epigenetic data. Results indicated that the binding sites of STELLA and active histones H3K4me3 and H3K27ac were enriched near the TAD boundaries. Hi-C analysis showed that Stella overexpression attenuated the interaction within TADs, and interestingly enhanced the TAD boundary strength in STELLA-associated regions. Taking these findings together, our study not only reveals the role of Stella in regulating DNA methylation and chromatin structure, but also provides a better understanding of FGSC development.