Project description:Chromosome conformation capture (3C) and derivative (4C, 5C and Hi-C) methods employ ligation of diluted cross-linked chromatin complexes, intended to favor proximity-dependent, intra-complex ligation. We previously described 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" and the standard "in-solution ligation". The results show that in-nucleus ligation captures chromatin interactions more consistently over a wider range of distances, and significantly reduces both experimental noise and bias. Thus in-nucleus ligation not only simplifies the experimental procedures, but also produces higher quality data with benefits for the entire range of 3C applications. We created Hi-C libraries by two different methods, in-solution ligation and in-nucleus ligation, from two biological replicates each of mouse foetal liver cells (mouse-1 and mouse-2) and human ES cells (human-1 and human-2) or the mixture of these two species. We also sequenced a random ligation library prepared by reversal of the cross-links and purification of the DNA prior to ligation.
Project description:Differential gene transcription enables development and homeostasis in all animals and is regulated by two major classes of distal cis-regulatory DNA elements (CREs), enhancers and silencers. While enhancers have been thoroughly characterized, the properties and mechansisms of silencers remain largely unknown. By an unbiased genome-wide functional screen in Drosophila melanogaster S2 cells, we discover a class of silencers that bind one of three transcription factors (TFs) and are generally not included in chromatin-defined CRE catalogs, as they mostly lack detectable DNA accessibility. The silencer-binding TF CG11247, which we term Saft, safeguards cell fate decisions in vivo and functions via a highly-conserved domain we term ZAC and the corepressor G9a, independently of G9a’s H3K9-methyltransferase activity. Overall, our identification of silencers with unexpected properties and mechanisms has important implications for the understanding and future study of repressive CREs, as well as the functional annotation of animal genomes.
Project description:HiCUP is a pipeline for processing sequence data generated by Hi-C, a technique used to investigate the three-dimensional organisation of a genome. The pipeline maps data to a specified reference genome and removes artefacts that would otherwise hinder subsequent analysis. HiCUP also provides an easy-to-interpret yet detailed quality control report that may be used by researchers to refine their experimental protocol for future studies. The software is freely available and has already been used for processing Hi-C data in several recently published peer-reviewed research articles. This experiment investigates the impact of using HiCUP to remove putative PCR amplification products in heavily duplicated Capture Hi-C libraries. Examination of three Capture Hi-C libraries
Project description:Capture Hi-C (CHi-C) is a state-of-the art method for profiling chromosomal interactions involving targeted regions of interest (such as gene promoters) globally and at high resolution. Signal detection in CHi-C data involves a number of statistical challenges that are not observed when using other Hi-C-like techniques. We present a background model, and algorithms for normalisation and multiple testing that are specifically adapted to CHi-C experiments, in which many spatially dispersed regions are captured, such as in Promoter CHi-C. We implement these procedures in CHiCAGO (http://regulatorygenomicsgroup.org/chicago), an open-source package for robust interaction detection in CHi-C. We validate CHiCAGO by showing that promoter-interacting regions detected with this method are enriched for regulatory features and disease-associated SNPs. Three human CHi-C biological replicates were generated (comprising 1, 2and 3 technical replicates). Two mouse CHi-C biological replicates were generated (both comprising three technical replicates) and a mouse Hi-C dataset. The publicly available HiCUP pipeline (doi: 10.12688/f1000research.7334.1) was used to process the raw sequencing reads. This pipeline was used to map the read pairs against the mouse (mm9) and human (hg19) genomes, to filter experimental artefacts (such as circularized reads and re-ligations), and to remove duplicate reads. For the CHi-C data, the resulting BAM files were processed into CHiCAGO input files, retaining only those read pairs that mapped, at least on one end, to a captured bait. CHiCAGO then identified Hi-C restriction fragments interacting, with statistical significant, to captured baits.
Project description:Two biological replicates Hi-C and HPV16-specific Region Capture Hi-C libraries were prepared for each of the W12 cell lines. Capture Hi-C was performed using HPV16-specific RNA baits. Hi-C libraries alone were prepared from normal cervix tissue (Ncx).
Project description:The enhancer/promoter of the vitellogenin II (VTG) gene has been extensively studied as a model system of vertebrate transcriptional control. While deletion mutagenesis and in vivo footprinting identified the transcription factor (TF) binding sites governing its tissue specificity, DNase hypersensitivity- and DNA methylation studies revealed the epigenetic changes accompanying its hormone-dependent activation. Moreover, upon induction with estrogen (E2), the region flanking the estrogen-responsive element (ERE) was reported to undergo active DNA demethylation. We now show that although the VTG ERE is methylated in embryonic chicken liver and in LMH/2A hepatocytes, its induction by E2 was not accompanied by extensive demethylation. In contrast, E2 failed to activate a VTG enhancer/promoter-controlled luciferase reporter gene methylated by SssI. Surprisingly, this inducibility difference could be traced not to the ERE, but rather to a single CpG in an E-box (CACGTG) sequence upstream of the VTG TATA box, which is unmethylated in vivo, but methylated by SssI. We demonstrate that this E-box binds the upstream stimulating factor USF1/2. Selective methylation of the CpG within this binding site with an E-box-specific DNA methyltranferase Eco72IM was sufficient to attenuate USF1/2 binding in vitro and abolish the hormone-induced transcription of the VTG gene in the reporter system.
Project description:During animal development, a fertilized egg is initially under the control of maternal products and only starts zygotic transcription after several cell divisions. In animals such as Xenopus, zebrafish and Drosophila, a massive increase in zygotic transcription occurs during the mid-blastula transition (MBT), when cells shift from rapid, synchronous cell divisions without gap phases to prolonged asynchronous divisions. Before MBT, only a few so-called pre-MBT genes are expressed. How transcription is set up during these early stages is poorly understood. For example, paused RNA Polymerase (Pol II) is frequently found at developmental control genes in mammalian embryonic stem cells and Drosophila embryos but when Pol II pausing is first established in the embryo is unknown. We have analyzed the genome-wide Pol II occupancy during the maternal-to-zygotic transition in hand-staged Drosophila embryos. The results show that massive Pol II recruitment and pausing is established during MBT. The ~100 genes that are transcribed before MBT are particularly short, consistent with a need for rapid transcription during these early cell divisions. Remarkably, most of these genes are transcribed without Pol II pausing and this correlates with a TATA-enriched promoter type. This suggests that distinct strategies are used for activation in the early Drosophila embryo and this may reflect general dynamic properties of promoters used throughout development. ChIP-seq for Pol II, TBP, H3K4me3, H3K27me3 and H3Ac in Drosophila embryos
Project description:Chromatin organisation of trophoblast stem cells (TSC) were compared with that of embryonic stem cells (ESC). The method enriches Hi-C libraries, to detect promoter interactions at restriction fragment level. We prepared Hi-C libraries from TSC and ESC (serum grown) samples and enriched them with a promoter capture bait system that captures ~22.000 promoters. Promoter interactions were then analysed using the GOTHiC pipeline.
Project description:Here we report that the histone variant macroH2A acts as a barrier to induced pluripotency. Using fibroblasts isolated from macroH2A double knockout mice, we observed enhanced reprogramming efficiency compared to fibroblasts from wild type animals. We further show that macroH2A isoforms act synergistically in this process. Genomic analysis in wild type fibroblasts reveals that macroH2A1 and H3K27me3 domains co-localize and occupy pluripotency genes. While the absence of macroH2A does not affect H3K27me3 in fibroblasts, macroH2A1 is highly enriched at a set of Utx target genes that are reactivated early during iPS reprogramming. Mononucleosomes from Dermal Fibroblasts (from wt and macroH2A1 and macroH2A2 double knockout mice) were isolated and ChIP'd with mH2A1, H3K27me3 and H3K27ac antibodies. DNA from Input and ChIP samples was purified and sequenced on Illumina's Hiseq.
Project description:HPV integrated site capture (HISC) protocol used to detect HPV16 integration breakpoints in the genomes of W12 cell lines. Biotinylated HPV16-specific RNA baits were used to capture HPV16-human breakpoint junctions in genomic DNA.