Chromatin Interaction Analysis using Paired-End Tag sequencing (ChIA-PET) is a technique developed for large-scale, de novo analysis of higher-order chromatin structures. Cells are treated with formaldehyde to cross-link chromatin interactions, DNA segments bound by protein factors are enriched by chromatin immunoprecipitation, and interacting DNA fragments are then captured by proximity ligation. The Paired-End Tag (PET) strategy is applied to the construction of ChIA-PET libraries, which are sequenced by high-throughput next-generation sequencing technologies. Finally, raw PET sequences are subjected to bioinformatics analysis, resulting in a genome-wide map of binding sites and chromatin interactions mediated by the protein factor under study. This unit describes ChIA-PET for genome-wide analysis of chromatin interactions in mammalian cells, with the application of Roche/454 and Illumina sequencing technologies.
"In our chosen models we will need to show a dynamic interdependence between AR binding, the recruitment of active RNA polymerase to a transcription start site for example, the opening of chromatin as inferred from histone marks and transcript production plus the reversal of all or some of these events by knocking down the AR or inhibiting its activity. In addition we will need to consider methods for mapping the true associations between AR complexes and sites elsewhere such as chromosome conformation capture protocols (Vassetzky et al., 2009) or chromatin interaction analysis with paired-end tag sequencing (ChIA-PET) (Fullwood et al., 2010). Table 1 Selected AR ChIP-on-chip and ChIP-seq studies (multi-parameter AR ChIP publications highlighted in bold). "
[Show abstract][Hide abstract] ABSTRACT: Alterations in transcriptional programs are fundamental to the development of cancers. The androgen receptor is central to the normal development of the prostate gland and to the development of prostate cancer. To a large extent this is believed to be due to the control of gene expression through the interaction of the androgen receptor with chromatin and subsequently with coregulators and the transcriptional machinery. Unbiased genome-wide studies have recently uncovered the recruitment sites that are gene-distal and intragenic rather than associated with proximal promoter regions. Whilst expression profiles from AR-positive primary prostate tumours and cell lines can directly relate to the AR cistrome in prostate cancer cells, this distribution raises significant challenges in making direct mechanistic connections. Furthermore, extrapolating from datasets assembled in one model to other model systems or clinical samples poses challenges if we are to use the AR-directed transcriptome to guide the development of novel biomarkers or treatment decisions. This review will provide an overview of the androgen receptor before addressing the challenges and opportunities created by whole-genome studies of the interplay between the androgen receptor and chromatin.
[Show abstract][Hide abstract] ABSTRACT: Structure is incorporated into the everyday objects, social and genomic networks that surround us providing a delicate balance of flexibility and stability. This chapter will discuss the methods that are currently used to untangle genome structure and the networks of interactions that occur between chromosomes to provide genomic stability. We will concentrate on the recently developed proximity-based ligation approaches which are opening new avenues of investigation into genome architecture. Finally, we will discuss a range of pitfalls that must be avoided when designing and performing these deceptively simple experiments. Although these methods are still in the early stages of development and application, they promise to bring about an explosion in our understanding of genome organization and stability.
Advances in Genetics Research, Volume 5 edited by Kevin V. Urbano, 01/2011: chapter 4: pages 111-130; NOVA Science Publishers, Inc.., ISBN: 978-1-61122-072-8
[Show abstract][Hide abstract] ABSTRACT: Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14.
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8.
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.
Journal of Visualized Experiments 04/2012; DOI:10.3791/3770 · 1.33 Impact Factor
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