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Results for “Chromatin conformation capture sequencing”

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At least 19 recordsLinked to original sources
PubMed2026

ONT-only genome assembly of a Korean male individual using a semen sample.

BACKGROUND: Long-read sequencing has enabled the generation of high-quality human genome assemblies, but many previous assemblies were based on blood-derived DNA and often relied on limited data types from a single sequencing strategy. OBJECTIVE: This study aimed to generate high-quality phased genome assemblies of a Korean individual using multiple independent long-read datasets produced from a single sequencing platform and to evaluate their utility for chromosome-scale assembly and variant detection. METHODS: Genomic DNA was extracted from a semen sample of a Korean male. Long-read, ultra-long-read, and chromatin conformation capture sequencing data were generated using Oxford Nanopore Technologies. These datasets were integrated to construct phased genome assemblies, followed by correction of noticeable phasing errors and assessment of assembly continuity, chromosomal representation, telomeric repeat recovery, and variant detection performance. RESULTS: The final phased assemblies spanned approximately 2.9 Gb and represented 23 pairs of chromosomes with an NG50 of 150 Mb. Telomeric repeats were detected at 36 and 37 of the 48 chromosomal ends in the two assemblies, indicating high end-to-end completeness. In addition, we successfully identified structural variants, including small variants. These results demonstrate that combining multiple Oxford Nanopore data types can produce highly continuous and informative phased human genome assemblies. CONCLUSIONS: We generated high-quality phased genome assemblies of a Korean individual using Oxford Nanopore long-read sequencing data derived from semen DNA. This publicly available genome resource will support broader applications of long-read sequencing in human genomics and variant analysis.

Humans↗
PubMed2026

Chromosome-level genome assembly of a cosmopolitan marine harmful algal bloom diatom species Chaetoceros socialis (Chaetocerotaceae).

Chaetoceros socialis is a cosmopolitan diatom species that is crucial for maintaining marine ecosystem structure and driving elemental cycles. C. socialis can form harmful algal blooms (HABs) that may cause a negative impact on the marine ecosystems. Whole-genome information for C. socialis is still unavailable, which may hinder more targeted studies on its ecological adaptive responses and evolutionary drivers. To address this gap, we employed cutting-edge genomic technologies including PacBio single-molecule real-time (SMRT) sequencing and high-throughput chromatin conformation capture (Hi-C) to achieve the first chromosome-level genome assembly of C. socialis. The assembled genome is 60.22 Mb in size with a scaffold N50 of 7.81 Mb and has been anchored to eight pseudochromosomes. A total of 13,378 protein-coding genes were predicted, of which 12,069 (90.22%) were functionally annotated. This high-quality genomic resource provides a fundamental data platform for systematically elucidating the ecological adaptation mechanisms of C. socialis.

Chromosomes↗
PubMed2024

Principles of bacterial genome organization, a conformational point of view.

Bacterial chromosomes are large molecules that need to be highly compacted to fit inside the cells. Chromosome compaction must facilitate and maintain key biological processes such as gene expression and DNA transactions (replication, recombination, repair, and segregation). Chromosome and chromatin 3D-organization in bacteria has been a puzzle for decades. Chromosome conformation capture coupled to deep sequencing (Hi-C) in combination with other "omics" approaches has allowed dissection of the structural layers that shape bacterial chromosome organization, from DNA topology to global chromosome architecture. Here we review the latest findings using Hi-C and discuss the main features of bacterial genome folding.

Genome, Bacterial↗
PubMed2025

Using Chromosome Conformation Capture Combined with Deep Sequencing (Hi-C) to Study Genome Organization in Bacteria.

Genome organization is fundamental to all living organisms. Long DNA molecules are organized in hierarchical orders to be accommodated into eukaryotic nuclei or bacterial cells, which are thousands of folds shorter. Over the past two decades, chromosome conformation capture (3C) techniques substantially advanced our understanding of genome folding inside cells. 3C involves crosslinking and proximity ligation, and quantifies the physical contacts between two DNA regions within the genome. Coupled with high-throughput sequencing, 3C-seq and Hi-C techniques detect genome-wide DNA interactions, providing a comprehensive view of global genome organization. Here, we describe a detailed method to prepare Hi-C libraries using Bacillus subtilis, which includes procedures of crosslinking chromatin, digesting the crosslinked genome, labeling DNA ends with biotin, ligating DNA, and preparing the DNA library for sequencing using an Illumina platform.

High-Throughput Nucleotide Sequencing↗
PubMed2026

Functional chimeric mRNAs encode proteins in mammalian immunity.

Individual mammalian mRNAs and proteins are typically believed to originate from single genomic loci, with isoform diversity arising through cis-splicing of pre-mRNA. Whether mRNA from distant genes can undergo trans-splicing to generate functionally relevant chimeric transcripts has remained unclear. Here we develop a pipeline combining long-read direct RNA sequencing with non-targeted and targeted validation to identify chimeric transcripts in macrophages. Chromatin conformation capture studies reveal that inflammation induces interchromosomal DNA interactions, positioning parent genes proximally to facilitate the formation of chimeric mRNA. Notably, we identify a protein-coding chimeric mRNA representing a fusion between the pore-forming protein gasdermin D (GSDMD)1,2 and a C-terminal domain translated out of frame from Tmem106a (Gsdmd-Tmem106a) in mice. We show that inflammasome priming upregulates Gsdmd-Tmem106a, with the protein localizing to the plasma membrane. After activation of the inflammasome, GSDMD-TMEM106A directly interacts with canonical GSDMD N termini to accelerate and enhance pore formation and IL-1β release. Finally, we show that GSDMD-TMEM106A balances host defence and immunopathology in vivo: its loss protects against lethal sepsis but compromises antibacterial defence, whereas overexpression enhances host protection while increasing sepsis lethality. We establish that protein-coding chimeric mRNAs formed by regulated transcript fusion events are operative during inflammation and immunity.

Journal Article↗
PubMed2006

CTCF binding at the H19 imprinting control region mediates maternally inherited higher-order chromatin conformation to restrict enhancer access to Igf2.

It is thought that the H19 imprinting control region (ICR) directs the silencing of the maternally inherited Igf2 allele through a CTCF-dependent chromatin insulator. The ICR has been shown to interact physically with a silencer region in Igf2, differentially methylated region (DMR)1, but the role of CTCF in this chromatin loop and whether it restricts the physical access of distal enhancers to Igf2 is not known. We performed systematic chromosome conformation capture analyses in the Igf2/H19 region over >160 kb, identifying sequences that interact physically with the distal enhancers and the ICR. We found that, on the paternal chromosome, enhancers interact with the Igf2 promoters but that, on the maternal allele, this is prevented by CTCF binding within the H19 ICR. CTCF binding in the maternal ICR regulates its interaction with matrix attachment region (MAR)3 and DMR1 at Igf2, thus forming a tight loop around the maternal Igf2 locus, which may contribute to its silencing. Mutation of CTCF binding sites in the H19 ICR leads to loss of CTCF binding and de novo methylation of a CTCF target site within Igf2 DMR1, showing that CTCF can coordinate regional epigenetic marks. This systematic chromosome conformation capture analysis of an imprinting cluster reveals that CTCF has a critical role in the epigenetic regulation of higher-order chromatin structure and gene silencing over considerable distances in the genome.

Animals↗
PubMed2005

Long-range interactions between three transcriptional enhancers, active Vkappa gene promoters, and a 3' boundary sequence spanning 46 kilobases.

The mouse immunoglobulin kappa (Igkappa) gene contains an intronic enhancer and two enhancers downstream of its transcription unit. Using chromosome conformation capture technology, we demonstrate that rearranged and actively transcribed Igkappa alleles in MPC-11 plasmacytoma cells exhibit mutual interactions over 22 kb between these three enhancers and Vkappa gene promoters. In addition, the 5' region of the active transcription unit exhibits a continuum of interactions with downstream chromatin segments. We also observe interactions between Ei and E3' with 3' boundary sequences 24 kb downstream of Ed, adjacent to a neighboring housekeeping gene. Very similar interactions between the enhancers are also exhibited by normal B cells isolated from mouse splenic tissue but not by germ line transcriptionally inactive alleles of T cells or P815 mastocytoma cells, which exhibit a seemingly linear chromatin organization. These results fit a looping mechanism for enhancer function like in the beta-globin locus and suggest a dynamic modulation of the spatial organization of the active Igkappa locus. Chromatin immunoprecipitation experiments reveal that the interacting Igkappa gene cis-acting sequences are associated with AP-4, E47, and p65NF-kappaB, potential protein candidates that may be responsible for initiating and/or maintaining the formation of these higher-order complexes. However, S107 plasmacytoma cells that lack NF-kappaB still exhibit mutual interactions between the Igkappa gene enhancers.

3' Flanking Region↗
PubMed2006

Chromosome Conformation Capture Carbon Copy (5C): a massively parallel solution for mapping interactions between genomic elements.

Physical interactions between genetic elements located throughout the genome play important roles in gene regulation and can be identified with the Chromosome Conformation Capture (3C) methodology. 3C converts physical chromatin interactions into specific ligation products, which are quantified individually by PCR. Here we present a high-throughput 3C approach, 3C-Carbon Copy (5C), that employs microarrays or quantitative DNA sequencing using 454-technology as detection methods. We applied 5C to analyze a 400-kb region containing the human beta-globin locus and a 100-kb conserved gene desert region. We validated 5C by detection of several previously identified looping interactions in the beta-globin locus. We also identified a new looping interaction in K562 cells between the beta-globin Locus Control Region and the gamma-beta-globin intergenic region. Interestingly, this region has been implicated in the control of developmental globin gene switching. 5C should be widely applicable for large-scale mapping of cis- and trans- interaction networks of genomic elements and for the study of higher-order chromosome structure.

Base Sequence↗
PubMed2026

3D chromatin remodeling during domestication defines novel targets for crop improvement.

Three-dimensional (3D) genome folding shapes gene regulation, yet the genetic underpinnings linking 3D genome evolution to phenotypic innovation during domestication remain elusive. Using population-scale Hi-C profiling of 34 semi-wild and 267 cultivated allotetraploid cottons, we generated a pan-3D genome atlas capturing extensive diversity in topologically associating domains (TADs) and chromatin loops. Chromatin interactome-wide association studies identified 105 TAD reconfigurations and 58 loop rewirings that were established as the 3D chromatin basis of fiber quality, boosting heritability estimates for fiber strength by 16% and fiber length by 20%. We reveal that domestication selection within sequence-defined sweeps fixed 57% of 3D conformation signatures, thereby decoupling sequence-level from chromatin-level selection and shifting the subgenome expression balance of 39 homoeologs in cultivated cotton. Sequence-based modeling and mutational analyses identified the C2H2 zinc-finger protein YY1 as a conserved mediator of 3D genome organization. This study provides a resource for redefining precision-breeding paradigms by harnessing cryptic 3D chromatin targets.

3D genome↗
PubMed2004

Rounding up active cis-elements in the triple C corral: combining conservation, cleavage and conformation capture for the analysis of regulatory gene domains.

Identification and functional analysis of potential cis-regulatory elements is a laborious process that often depends on removing putative elements from their natural context to study their activity. While such methods provide valuable information about the isolated element, they disregard the potential role of an element's interaction(s) with other regulatory sequences and the three-dimensional structure of an active gene locus. Here, two novel methods are discussed--chromosome conformation capture (3C) and RNA-TRAP--that can be used to detect interactions between distal regulatory sites and which thus indicate the chromosomal conformation that is adopted by a gene locus in various states of transcriptional activity. Combined with comparative genomics and traditional DNase I hypersensitive site mapping, these methods form a powerful approach for the study of the mechanisms of long-range transcriptional regulation.

Animals↗
PubMed2025

Genome topology analysis and transcriptomics of human osteoclasts reveals enhancer-promoter interactions at loci for bone traits and diseases.

Genome-wide association studies (GWAS) relevant to osteoporosis have identified hundreds of loci; however, understanding how these variants influence the phenotype is complicated because most reside in non-coding DNA sequence that serves as transcriptional enhancers and repressors. To advance knowledge on these regulatory elements in osteoclasts (OCs), we performed Micro-C analysis, which informs on the genome topology of these cells and integrated the results with transcriptome and GWAS data to further define loci linked to BMD. Using blood cells isolated from 4 healthy participants aged 31-61 yr, we cultured OC in vitro and generated a Micro-C chromatin conformation capture dataset. We characterized chromatin loops (CLs) in OC from among more than 69 million chromatin interactions identified in the genome. Of the CL identified in OC, >16 000 were unique compared to precursor cells. When sentinel single nucleotide polymorphisms from osteoporosis and bone-related GWAS and those in linkage disequilibrium at r 2 > 0.6 were mapped to CL for OC, 12 588 of these variants were observed within chromatin contact regions. Notable in differential gene ontology enrichment analyses of the topology data for OC and precursors were pathways regulating pluripotency of stem cells, Wnt signaling, nucleotide-binding oligomerization domain (NOD)-like receptor signaling and chemokine signaling. These data, in combination with other 3D genome architecture and epigenetic data (eg, histone modifications and chromatin accessibility), will be useful in modeling to predict genome-wide, which enhancers regulate which genes in OC. This data will therefore also be informative for resolving GWAS hits. In conclusion, we have generated a high-resolution genome topology dataset for human OC and have used this to identify CLs relevant to studies of the genetics of osteoporosis. This data will serve as a powerful resource to inform future functional studies of OC biology.

BMD↗
PubMed2025

HiChIP for Plant Tissues.

While most epigenomics studies are based on a linear view of genome organization, the necessity to take the three-dimensional chromatin folding into account to understand transcriptional regulation is now clearly recognized. In the past years, approaches combining proximity-based ligation with high-throughput sequencing have opened the way to study long/short-range chromatin interactions and, thus, to analyze 3D chromatin organization. Among them, HiChIP, a protein-based method to capture chromatin interactions, gave rise to the most comprehensive view of the chromatin contacts involving specific chromatin components in a given system. Here, we describe a detailed procedure to produce HiChIP libraries starting from plant tissues.

Chromatin↗
PubMed2006

Distinct functions of dispersed GATA factor complexes at an endogenous gene locus.

The reciprocal expression of GATA-1 and GATA-2 during hematopoiesis is an important determinant of red blood cell development. Whereas Gata2 is preferentially transcribed early in hematopoiesis, elevated GATA-1 levels result in GATA-1 occupancy at sites upstream of the Gata2 locus and transcriptional repression. GATA-2 occupies these sites in the transcriptionally active locus, suggesting that a "GATA switch" abrogates GATA-2-mediated positive autoregulation. Chromatin immunoprecipitation (ChIP) coupled with genomic microarray analysis and quantitative ChIP analysis with GATA-1-null cells expressing an estrogen receptor ligand binding domain fusion to GATA-1 revealed additional GATA switches 77 kb upstream of Gata2 and within intron 4 at +9.5 kb. Despite indistinguishable GATA-1 occupancy at -77 kb and +9.5 kb versus other GATA switch sites, GATA-1 functioned uniquely at the different regions. GATA-1 induced histone deacetylation at and near Gata2 but not at the -77 kb region. The -77 kb region, which was DNase I hypersensitive in both active and inactive states, conferred equivalent enhancer activities in GATA-1- and GATA-2-expressing cells. By contrast, the +9.5 kb region exhibited considerably stronger enhancer activity in GATA-2- than in GATA-1-expressing cells, and other GATA switch sites were active only in GATA-1- or GATA-2-expressing cells. Chromosome conformation capture analysis demonstrated higher-order interactions between the -77 kb region and Gata2 in the active and repressed states. These results indicate that dispersed GATA factor complexes function via long-range chromatin interactions and qualitatively distinct activities to regulate Gata2 transcription.

Acetylation↗
PubMed2006

Identification of cis-regulatory elements for MECP2 expression.

Rett syndrome (RTT) is an X-linked dominant disabling neurodevelopmental disorder caused by loss of function mutations in the MECP2 gene, located at Xq28, which encodes a multifunctional protein. MECP2 expression is regulated in a developmental stage and cell-type-specific manner. The need for tightly controlled MeCP2 levels in brain is strongly suggested by neurologically abnormal phenotypes of mouse models with mild overexpression and by mental retardation in human males with MECP2 duplication. We set out to identify long-range cis-regulatory sequences that differentially regulate MECP2 transcription and, when mutated, may contribute to the pathogenesis of RTT, autism or X-linked mental retardation. By inter-species sequence comparisons, we detected 27 highly conserved non-coding DNA sequences within a 210 kb region covering MECP2. We functionally confirmed four enhancer and two silencer elements by performing luciferase reporter assays in four different human cell lines. The transcription factor binding capability of the identified regulatory elements was tested by gel shift assays. To locate the human MECP2 core promoter, we dissected the promoter region by reporter assays with deletion constructs. We then used chromosome conformation capture methods to document long-range interactions of three enhancers and two silencers with the MECP2 promoter. Acting over distances of up to 130 kb, these elements may influence chromatin configurations and regulate MECP2 transcription. Our study has defined the "MECP2 functional expression module" and identified enhancer and silencer elements that are likely to be responsible for the tissue-specific, developmental stage-specific or splice-variant-specific control of MeCP2 protein expression.

Animals↗
PubMed2004

Interaction between differentially methylated regions partitions the imprinted genes Igf2 and H19 into parent-specific chromatin loops.

Imprinted genes are expressed from only one of the parental alleles and are marked epigenetically by DNA methylation and histone modifications. The paternally expressed gene insulin-like growth-factor 2 (Igf2) is separated by approximately 100 kb from the maternally expressed noncoding gene H19 on mouse distal chromosome 7. Differentially methylated regions in Igf2 and H19 contain chromatin boundaries, silencers and activators and regulate the reciprocal expression of the two genes in a methylation-sensitive manner by allowing them exclusive access to a shared set of enhancers. Various chromatin models have been proposed that separate Igf2 and H19 into active and silent domains. Here we used a GAL4 knock-in approach as well as the chromosome conformation capture technique to show that the differentially methylated regions in the imprinted genes Igf2 and H19 interact in mice. These interactions are epigenetically regulated and partition maternal and paternal chromatin into distinct loops. This generates a simple epigenetic switch for Igf2 through which it moves between an active and a silent chromatin domain.

Animals↗
PubMed2025

Forty new genomes shed light on sexual reproduction and the origin of tetraploidy in Microsporidia.

Microsporidia are single-celled, obligately intracellular parasites with growing public health, agricultural, and economic importance. Despite this, Microsporidia remain relatively enigmatic, with many aspects of their biology and evolution unexplored. Key questions include whether Microsporidia undergo sexual reproduction, and the nature of the relationship between tetraploid and diploid lineages. While few high-quality microsporidian genomes currently exist to help answer such questions, large-scale biodiversity genomics initiatives, such as the Darwin Tree of Life project, can generate high-quality genome assemblies for microsporidian parasites when sequencing infected host species. Here, we present 40 new microsporidian genome assemblies from infected arthropod hosts that were sequenced to create reference genomes. Out of the 40, 32 are complete genomes, eight of which are chromosome-level, and eight are partial microsporidian genomes. We characterized 14 of these as polyploid and five as diploid. We found that tetraploid genome haplotypes are consistent with autopolyploidy, in that they coalesce more recently than species, and that they likely recombine. Within some genomes, we found large-scale rearrangements between the homeologous genomes. We also observed a high rate of rearrangement between genomes from different microsporidian groups, and a striking tolerance for segmental duplications. Analysis of chromatin conformation capture (Hi-C) data indicated that tetraploid genomes are likely organized into two diploid units, similar to dikaryotic cells in fungi, with evidence of recombination within and between units. Together, our results provide evidence for the existence of a sexual cycle in Microsporidia, and suggest a model for the microsporidian lifecycle that mirrors fungal reproduction.

Genome, Fungal↗
PubMed1993

Recovery of transcriptionally active chromatin restriction fragments by binding to organomercurial-agarose magnetic beads. A rapid and sensitive method for monitoring changes in higher order chromatin structure during gene activation and repression.

The unfolding of nucleosomes along transcriptionally active DNA sequences uncovers previously shielded cysteinyl-thiol groups of histone H3 molecules located at the center of the nucleosome core. This change in conformation and SH reactivity of nucleosomes along transcribed DNA sequences makes it possible to separate active from inactive nucleosomes by mercury affinity chromatography. The binding of thiol-reactive nucleosomes to an organomercurial-agarose column has been shown previously to reflect, with accuracy, both the timing and extent of transcription of the associated DNA sequences (Chen, T. A., and Allfrey, V. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5252-5256). Here, we extend this experimental approach to the analysis of higher order chromatin structures. Large chromatin fragments released by treating isolated nuclei with restriction endonucleases are fractionated on mercurated agarose magnetic beads that capture nucleosomes with accessible histone H3 thiols, but do not react with the hidden H3 thiols of the compactly beaded nucleosomes of inactive genes. The SH-reactive domains of c-myc and other genes are rapidly separated from the non-SH-reactive restriction fragments by the magnetic bead technique. The new method also overcomes a major limitation of mercurated agarose column chromatography, which is not suitable for studies of higher order chromatin structure because large chromatin fragments occlude the mercury column; occlusion is not a problem in magnetic separations using suspended mercurated agarose beads. Here, we describe the synthesis of mercurated agarose magnetic beads with high capacity for SH groups and test their application to the recovery of chromatin restriction fragments of c-myc and the growth arrest gene gas1.

Animals↗
PubMed2006

Circular chromosome conformation capture (4C) uncovers extensive networks of epigenetically regulated intra- and interchromosomal interactions.

Accumulating evidence converges on the possibility that chromosomes interact with each other to regulate transcription in trans. To systematically explore the epigenetic dimension of such interactions, we devised a strategy termed circular chromosome conformation capture (4C). This approach involves a circularization step that enables high-throughput screening of physical interactions between chromosomes without a preconceived idea of the interacting partners. Here we identify 114 unique sequences from all autosomes, several of which interact primarily with the maternally inherited H19 imprinting control region. Imprinted domains were strongly overrepresented in the library of 4C sequences, further highlighting the epigenetic nature of these interactions. Moreover, we found that the direct interaction between differentially methylated regions was linked to epigenetic regulation of transcription in trans. Finally, the patterns of interactions specific to the maternal H19 imprinting control region underwent reprogramming during in vitro maturation of embryonic stem cells. These observations shed new light on development, cancer epigenetics and the evolution of imprinting.

Animals↗