Project description:Here, we developed a novel chromosome conformation capture (3C) method for capturing the 3D spatial contacts of endogenous genomic loci without a need for crosslinking. This method, i3C, was applied to multiple loci in two different human primary (ENCODE) cell lines, HUVEC and IMR90, and in the absence or presence of a proinflammatory stimulus (TNFalpha). Coupled to high throughput sequencing on an Illumina HiSeq200 platform, i3C generated aprrox. 8 million single-end reads per experiment.
Project description:We report the distribution of interactive sites with the sequence close from Meis2 promoter within the genome of mouse embryonic forebrain. We prepared the chromatin from 11 dpc embryonic forebrain and made 3C (chromosomal conformation capture) library. High-throughput sequencing applied for the 3C analysis revealed the distribution of modified interactive sites within developing forebrain. 4C-seq analysis of mouse 11 dpc embryonic forebrain with the sequence close from Meis2 promoter. Forebrain isolated and disected from 11 dpc embryos are fixed by 1% formaldehyde. After conventional 3C reaction, 3C library for highthroughput sequence is prepared by combination of adaptor ligation and nesting PCR reactions.
Project description:Chromosome conformation capture (3C) provides an adaptable tool through which to study diverse biological questions. Currently, 3C techniques provide either low-resolution interaction profiles across the entire genome, e.g. HiC, or high-resolution interaction profiles at up to several hundred loci, e.g. NG Capture-C and 4C-seq. Generation of high-resolution, genome-wide interaction profiles can feasibly be achieved through efficiency improvements to current high-resolution methods. To this end we systematically tested and removed areas inefficiency in NG Capture-C to develop a new method Nuclear Capture-C, which provides a 300% increase in informative sequencing content. Using Nuclear Capture-C we target 8,026 erythroid promoters in triplicate, showing that this method can achieve high-resolution genome-wide 3C interaction profiles at scale.
Project description:Here we report that the spatial organization of yeast tRNA genes depends upon both locus position and tRNA identity; supporting the idea that the genomic organization of tRNA loci utilizes tRNA dependent signals within the nucleoprotein-tRNA complexes that form into clusters. We use high-throughput sequencing coupled to Circular Chromosome Conformation Capture to detect interactions with two wild type tRNAs and these same positions replaced with suppressor tRNAs (SUP4-1). Detect DNA-DNA interactions (Circular chromosome conformation capture; 4C) with two wild type tRNAs and these same positions replaced with suppressor tRNAs (SUP4-1) Supplementary files: Alignment files generated by Topography v1.19 software.
Project description:We report the distribution of interactive sites with the sequence close from Meis2 promoter within the genome of mouse embryonic forebrain. We prepared the chromatin from 11 dpc embryonic forebrain and made 3C (chromosomal conformation capture) library. High-throughput sequencing applied for the 3C analysis revealed the distribution of modified interactive sites within developing forebrain.
Project description:Mammalian genomes are folded in a hierarchy of compartments, topologically associating domains (TADs), subTADs and looping interactions. Currently, there is a great need to evaluate the link between chromatin topology and genome function across many biological conditions and genetic perturbations. Hi-C generates high quality, high resolution maps of looping interactions genome-wide, but is intractable for high-throughput screening of loops across conditions due to the requirement of an enormous number of reads (>6 Billion) per library. Here, we describe 5C-ID, an updated version of Chromosome-Conformation-Capture-Carbon-Copy (5C) with restriction digest and ligation performed in the nucleus (in situ ChromosomeConformation-Capture (3C)) and ligation-mediated amplification performed with a new double alternating design. 5C-ID reduces spatial noise and enables higher resolution 3D genome folding maps than canonical 5C, allowing for a marked improvement in sensitivity and specificity of loop detection. 5C-ID enables the creation of high-resolution, high-coverage maps of chromatin loops in up to a 30 Megabase subset of the genome at a fraction of the cost of Hi-C.
Project description:Background: DNA in the nucleus of a living cell carries out its functions in the context of a complex, three-dimensional chromatin architecture. Several recently developed methods, each an extension of the chromatin conformation capture (3C) assay, have enabled the genome-wide profiling of chromatin contacts between pairs of loci in yeast, fruit fly, human and mouse. 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. Here we describe a novel assay to map genome-wide chromatin contacts, tethered multiple 3C (TM3C), that involves a simple protocol of restriction enzyme digestion and religation of fragments upon agarose gel beads followed by deep DNA paired-end sequencing. In addition to identifying contacts between pairs of loci, TM3C enables identification of contacts among more than two loci simultaneously. Results: 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, as well as a low noise-to-signal ratio between inter- and intrachromosomal contacts. We also confirm that TM3C captures several known cell type-specific contacts, ploidy shifts and translocations, such as Ph+ formation in KBM7. Furthermore, we develop a two-phase mapping strategy that separately maps chimeric subsequences within a single read, allowing us to identify contacts involving three or four loci simultaneously, potentially corresponding to combinatorial regulation events. This mapping strategy also greatly increases the number of distinct binary contacts identified and, therefore, the coverage obtained for a fixed number of mapped reads. We confirm a subset of the triplet contacts involving the IGF2-H19 imprinting control region (ICR) using PCR analysis for KBM7 cells. Assaying the genome architecture of a near-haploid cell line allows us to create 3D models of a human cell line without averaging signal from two homologous copies of a chromosome. Our 3D models of KBM7 show 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 complex chromatin loops that may play a role in regulating reciprocal expression of the IGF2 and H19 genes. Analysis of the spatial organization of two human cell lines (KBM7, a near-haploid chronic leukemia cell line, and NHEK, a normal diploid human epidermal keratinocyte cell line) using tethered multiple 3C (TM3C), a novel and simple protocol for ascertaining genome architecture which can be used to identify simultaneous contacts among three or four loci in addition to binary contacts that can be identified using traditional chromosome conformation capture coupled with next generation sequencing (Hi-C).
Project description:The spatiotemporal control of 3D chromatin structure is fundamental for gene regulation, yet it remains challenging to obtain high-resolution chromatin interacting profiles at cis-regulatory elements (CREs) by chromatin conformation capture (3C)-based methods. Here, we describe the redesigned dCas9-based CAPTURE method for multiplexed, high-throughput and high-resolution analysis of locus-specific chromatin interactions. Using C-terminally biotinylated dCas9, endogenous biotin ligase and pooled sgRNAs, the new system enables quantitative analysis of the spatial configuration of a few to hundreds of enhancers or promoters in a single experiment, enabling systematic comparisons across CREs within and between gene clusters. We reveal the hierarchical structure of super-enhancers (SEs) and distinct modes of SE-gene interactions. Multiplexed capture of temporal dynamics of promoter-centric interactions establishes the instructive function of enhancer-promoter looping in transcriptional regulation during lineage differentiation. These applications illustrate the ability of multiplexed CAPTURE for decoding the organizational principles of genome structure and function.