Hi-C Method Research Articles

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0728-8) contains supplementary material, which is available to authorized users.

Hi-C in Budding Yeast.

Hi-C enables simultaneous detection of interaction frequencies between all possible pairs of restriction fragments in the genome. The Hi-C method is based on chromosome conformation capture (3C), which uses formaldehyde cross-linking to fix chromatin regions that interact in three-dimensional space, irrespective of their genomic locations. In the Hi-C protocol described here, cross-linked chromatin is digested with HindIII and the ends are filled in with a nucleotide mix containing biotinylated dCTP. These fragments are ligated together, and the resulting chimeric molecules are purified and sheared to reduce length. Finally, biotinylated ligation junctions are pulled down with streptavidin-coated beads, linked to high-throughput sequencing adaptors, and amplified via polymerase chain reaction (PCR). The resolution of the Hi-C data set will depend on the depth of sequencing and choice of restriction enzyme. When sufficient sequence reads are obtained, information on chromatin interactions and chromosome conformation can be derived at single restriction fragment resolution for complete genomes.

Investigation of the spatial genome organization of mouse sperm and fibroblasts by the Hi-C method

The spatial organization of eukaryotic genome plays an important role in the control of gene expression. The new Hi-C method allows investigation of chromosome contact in the three-dimensional architecture of the whole genome; however, it has not yet been applied to study the spatial configuration of the germ cells genome. Here we describe a protocol for production and quality control of Hi-C DNA libraries from mouse sperm cells and fibroblasts. Our results demonstrate that the Hi-C method can be used for studying the spatial organization of the densely packed sperm genome.

STABILITY INDICATING HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHIC METHOD FOR DETERMINATION OF ATAZANAVIR AND RITONAVIR IN COMBINED TABLET DOSAGE FORMS

A new simple, accurate, precise and selective stability - indicating high performance thin layer chromatograp hic (HPTLC) method has been developed and validated for simultaneous estimation of Atazanavir and Ritonavir in combined tablet dosage form. The mobil e phase selected was Toluene: Ethyl acetate: Methanol ( 6: 4.5: 0. 6 , v/v/v) with UV detection at 2 4 0 nm. The retention factor for Atazanavir and Ritonavir were found to be 0. 25 ± 0.0 04 and 0. 41 ± 0. 0 0 4 . The method was validated with respect to linearity, accuracy, precision and robustness . The drugs were subjected to stress condition of hydrolysis (acid, base), oxidation, photolysis and thermal degradation. Results found to be linear in the concentration range of 1 0 00 - 8 0 00 ng band - 1 for Atazanavir and 500 - 4 000 ng band - 1 for Ritonavir respectively. The method has been successfully applied for the analysis of drugs in pharmaceutical formulation. The % assay (Mean ± S.D.) was found to be 100 . 40 ± 0.96 4 for Ritonavir and 9 9 . 5 9 ± 1 . 103 for Atazanavir .

FisHiCal: an R package for iterative FISH-based calibration of Hi-C data.

The fluorescence in situ hybridization (FISH) method has been providing valuable information on physical distances between loci (via image analysis) for several decades. Recently, high-throughput data on nearby chemical contacts between and within chromosomes became available with the Hi-C method. Here, we present FisHiCal, an R package for an iterative FISH-based Hi-C calibration that exploits in full the information coming from these methods. We describe here our calibration model and present 3D inference methods that we have developed for increasing its usability, namely, 3D reconstruction through local stress minimization and detection of spatial inconsistencies. We next confirm our calibration across three human cell lines and explain how the output of our methods could inform our model, defining an iterative calibration pipeline, with applications for quality assessment and meta-analysis. FisHiCal v1.1 is available from http://cran.r-project.org/.

Species-level deconvolution of metagenome assemblies with Hi-C-based contact probability maps.

Microbial communities consist of mixed populations of organisms, including unknown species in unknown abundances. These communities are often studied through metagenomic shotgun sequencing, but standard library construction methods remove long-range contiguity information; thus, shotgun sequencing and de novo assembly of a metagenome typically yield a collection of contigs that cannot readily be grouped by species. Methods for generating chromatin-level contact probability maps, e.g., as generated by the Hi-C method, provide a signal of contiguity that is completely intracellular and contains both intrachromosomal and interchromosomal information. Here, we demonstrate how this signal can be exploited to reconstruct the individual genomes of microbial species present within a mixed sample. We apply this approach to two synthetic metagenome samples, successfully clustering the genome content of fungal, bacterial, and archaeal species with more than 99% agreement with published reference genomes. We also show that the Hi-C signal can secondarily be used to create scaffolded genome assemblies of individual eukaryotic species present within the microbial community, with higher levels of contiguity than some of the species’ published reference genomes.

Strain- and plasmid-level deconvolution of a synthetic metagenome by sequencing proximity ligation products.

Metagenomics is a valuable tool for the study of microbial communities but has been limited by the difficulty of “binning” the resulting sequences into groups corresponding to the individual species and strains that constitute the community. Moreover, there are presently no methods to track the flow of mobile DNA elements such as plasmids through communities or to determine which of these are co-localized within the same cell. We address these limitations by applying Hi-C, a technology originally designed for the study of three-dimensional genome structure in eukaryotes, to measure the cellular co-localization of DNA sequences. We leveraged Hi-C data generated from a simple synthetic metagenome sample to accurately cluster metagenome assembly contigs into groups that contain nearly complete genomes of each species. The Hi-C data also reliably associated plasmids with the chromosomes of their host and with each other. We further demonstrated that Hi-C data provides a long-range signal of strain-specific genotypes, indicating such data may be useful for high-resolution genotyping of microbial populations. Our work demonstrates that Hi-C sequencing data provide valuable information for metagenome analyses that are not currently obtainable by other methods. This metagenomic Hi-C method could facilitate future studies of the fine-scale population structure of microbes, as well as studies of how antibiotic resistance plasmids (or other genetic elements) mobilize in microbial communities. The method is not limited to microbiology; the genetic architecture of other heterogeneous populations of cells could also be studied with this technique.

HOLOGRAPHIC OPTICAL ELEMENTS FOR BEAUTIFICATION

In this research, holographic optical element appli ed for beautification recorded in holographic surfa ce relief photoresist. Various symmetric periodic structure with high diff raction efficiency in photosensitive material depen ds on various recording geometry parameters are explained. We used holograp hic dual beam multiple interference method for this pattern creation. A long range periodicity confirmed with the help of o ptical microscope, laser light diffraction and scan ning electron microscope. Combination of symmetric structured holographic opt ical elements, DPSS laser source and bi-directional RPM controller results the proto type HOLO-DRZZLER product. Our auspicious aim is to commercialize and our product is mainly meant for outdoor beautification application. Index Terms: Holography, photoresist, holographic decoration, l aser and holographic optical element

CytoHiC: a cytoscape plugin for visual comparison of Hi-C networks

With the introduction of the Hi-C method new and fundamental properties of the nuclear architecture are emerging. The ability to interpret data generated by this method, which aims to capture the physical proximity between and within chromosomes, is crucial for uncovering the three dimensional structure of the nucleus. Providing researchers with tools for interactive visualization of Hi-C data can help in gaining new and important insights. Specifically, visual comparison can pinpoint changes in spatial organization between Hi-C datasets, originating from different cell lines or different species, or normalized by different methods. Here, we present CytoHiC, a Cytsocape plugin, which allow users to view and compare spatial maps of genomic landmarks, based on normalized Hi-C datasets. CytoHiC was developed to support intuitive visual comparison of Hi-C data and integration of additional genomic annotations. The CytoHiC plugin, source code, user manual, example files and documentation are available at: http://apps.cytoscape.org/apps/cytohicplugin

The Properties of Genome Conformation and Spatial Gene Interaction and Regulation Networks of Normal and Malignant Human Cell Types

The spatial conformation of a genome plays an important role in the long-range regulation of genome-wide gene expression and methylation, but has not been extensively studied due to lack of genome conformation data. The recently developed chromosome conformation capturing techniques such as the Hi-C method empowered by next generation sequencing can generate unbiased, large-scale, high-resolution chromosomal interaction (contact) data, providing an unprecedented opportunity to investigate the spatial structure of a genome and its applications in gene regulation, genomics, epigenetics, and cell biology. In this work, we conducted a comprehensive, large-scale computational analysis of this new stream of genome conformation data generated for three different human leukemia cells or cell lines by the Hi-C technique. We developed and applied a set of bioinformatics methods to reliably generate spatial chromosomal contacts from high-throughput sequencing data and to effectively use them to study the properties of the genome structures in one-dimension (1D) and two-dimension (2D). Our analysis demonstrates that Hi-C data can be effectively applied to study tissue-specific genome conformation, chromosome-chromosome interaction, chromosomal translocations, and spatial gene-gene interaction and regulation in a three-dimensional genome of primary tumor cells. Particularly, for the first time, we constructed genome-scale spatial gene-gene interaction network, transcription factor binding site (TFBS) – TFBS interaction network, and TFBS-gene interaction network from chromosomal contact information. Remarkably, all these networks possess the properties of scale-free modular networks.

Unique for human centromeric regions of interphase chromatin homing (CENTRICH) govern dynamic features of chromatin fractal globules

AbstractWe report the results of the genome-wide alignment of inter- and intra-chromosomal chromatin interactions within the context of interphase chromatin binding to nuclear lamina and nucleolus. For all human chromosomes, a significant correlation exists between binding of chromosomal loci to nuclear lamina and segregation into spatially-defined distinct compartments of genome-wide chromatin interactions identified by Hi-C method. We report identification of near-centromeric intergenic regions on human chromosomes (chr2; chr10; chr17; chr1), which are highly enriched for interphase chromatin homing sites and function as attractors of long-range physical interactions (Centromeric Regions of Interphase Chromatin Homing, CENTRICH). CENTRICH are engaged in 397-1526 pair-wise interactions per 1 Kb distance, which represents 199 - 716-fold enrichment of interphase chromatin homing sites compared to genome-wide average (2-tail Fisher's exact test p values range 2.10E-101 - 1.08E-292). CENTRICH represent unique for human highly homologous DNA sequences of 3.9 - 22.4 Kb in size which are: 1) associated with nucleolus; 2) exhibit remarkably diverse regulatory protein contexts of chromatin state maps; 3) bind multiple intergenic disease-associated genomic loci (IDAGL) with documented long-range enhancer activities and established links to increased risk of developing epithelial malignancies and other common human disorders. For cancer, coronary artery disease, and type 2 diabetes, there is a statistically significant inverse correlation between the genome-wide association studies (GWAS)-defined odds ratios of increased risk of a disease and distances of SNP loci homing sites to the middle-point genomic coordinates of CENTRICH. We conclude that interactions with nucleolus and nuclear lamina may have a physiologically and pathologically significant global impact on 3D genome architecture by governing a dynamic transition of large segments of interphase human chromosomes from the open loop to folded fractal globule conformations. We propose that emergence of CENTRICH in genome's architecture during the evolution contributes to distinct phenotypic features of H.sapiens.

Chromosome Conformation Capture (3C) and 3C-based Methods

Chromosome conformation is thought to play an important role in gene expression regulation. Chromosome conformation capture (3C), circular chromosome conformation capture (4C), 3C-carbon copy (5C), ChIP-loop assay, ChIA-PET and Hi-C method were developed to study spatial organization of long genomic regions in living cells. All steps of 3C, those 3C-based methods and key procedures in experiment were also discussed. The aim of this paper is to introduce some new methods to study gene regulation in 3D.

Analysis of human α-thrombin by hydrophobic interaction high-performance liquid chromatography

The major active form of human thrombin, α-thrombin, was analyzed by hydrophobic interaction high-performance liquid chromatography (HIC-HPLC). The chromatographic system included a polymeric phenyl column and elution was performed by a gradient, 2–0 M sodium chloride (5–20 min). Total analysis time was 30 min per injection. By this method, a good resolution between α-thrombin and the proteolytically modified thrombin forms, β- and γ-thrombin, was obtained. In addition, the thrombin preforms, prothrombin, prethrombin 1, and prethrombin 2, were also resolved from α-thrombin in the system. The results from the HIC method were compared to those obtained from non-reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis. By this high-resolution chromatographic method, the rapid analysis of purified α-thrombin is possible.

Separation of Native and Latent Forms of Human Antithrombin by Hydrophobic Interaction High-Performance Liquid Chromatography

Hydrophobic interaction high-performance liquid chromatography (HIC-HPLC) was utilized for the separation of native human antithrombin (AT) and a partially denaturated form of AT, known as the latent form (L-AT). The AT used in this study is commercially available (Atenativ, Pharmacia & Upjohn, Sweden) and contains albumin as the main stabilizer. The AT was reconstituted and heat treated in order to generate L-AT. This latent form of AT has been shown to exhibit a strong antiangiogenic activity and also to suppress tumor growth. The HPLC system included a TSK Phenyl 5PW column and a segmented gradient, 4.5-0 mol/L sodium chloride. Antithrombin was eluted at about 13 min, and L-AT, at 30 min, corresponding to about 4.2 and 1.6 mol/L sodium chloride, respectively. A reference sample gave 42% L-AT when analyzed by the HIC method and 41% L-AT when analyzed by the heparin affinity chromatography method. The resolution between AT and L-AT was higher with the HIC method than with the heparin affinity method. Incubation of Atenativ at 45 degrees C for 15 h gave about 18% L-AT and was shown by native polyacrylamide gel electrophoresis to contain only monomeric AT. A good resolution between AT and L-AT, but not between albumin and L-AT, was also achieved by a linear gradient of 2-0 mol/L ammonium sulfate, in 25 mmol/L Tris/HCl, pH 8.0.

La distribution des émetteurs α peu solubles déposés après inhalation dans les tissus pulmonaires : nouveaux abords méthodologiques

We have developed an autoradiograp hic method to provide representative images of dose rate and dose distribution in the lungs after actinide oxide inhalation. This was based on the study of 30 um thick dry sections obtained from 200 um thick cryostat sections. In this case, solid track detector had a recording yield similar to that obtain for 5 um thin sections and the resolution was less than 90 um. Using this method, we visualized a heterogeneous particle distribution as soon as 3 months after exposure of rats to (U, Pu)0 2 aerosols. Key-words - autoradiography, lungs, inhalation, actini.de oxides, dosimetry.