Tag-Based Next Generation Sequencing

Department of Biology and Carolina Center for the Genome Sciences, University of North Carolina at Chapel Hill, CB #3280, 408 Fordham Hall, Chapel Hill, NC 27599-3280, USA
Methods (Impact Factor: 3.65). 07/2009; 48(3):233-239. DOI: 10.1016/j.ymeth.2009.03.003

ABSTRACT The binding of sequence-specific regulatory factors and the recruitment of chromatin remodeling activities cause nucleosomes to be evicted from chromatin in eukaryotic cells. Traditionally, these active sites have been identified experimentally through their sensitivity to nucleases. Here we describe the details of a simple procedure for the genome-wide isolation of nucleosome-depleted DNA from human chromatin, termed FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements). We also provide protocols for different methods of detecting FAIRE-enriched DNA, including use of PCR, DNA microarrays, and next-generation sequencing. FAIRE works on all eukaryotic chromatin tested to date. To perform FAIRE, chromatin is crosslinked with formaldehyde, sheared by sonication, and phenol–chloroform extracted. Most genomic DNA is crosslinked to nucleosomes and is sequestered to the interphase, whereas DNA recovered in the aqueous phase corresponds to nucleosome-depleted regions of the genome. The isolated regions are largely coincident with the location of DNaseI hypersensitive sites, transcriptional start sites, enhancers, insulators, and active promoters. Given its speed and simplicity, FAIRE has utility in establishing chromatin profiles of diverse cell types in health and disease, isolating DNA regulatory elements en masse for further characterization, and as a screening assay for the effects of small molecules on chromatin organization.

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    • "Like most other transcription factors, VDR competes with the intrinsic repressive nature of chromatin for access to its genomic binding sites [25] [26]. Open chromatin regions can be detected by the genome-wide method formaldehyde-assisted isolation of regulatory elements sequencing (FAIRE-seq) [27] [28] [29]. "
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    ABSTRACT: Vitamin D3 belongs to the few nutritional compounds that has, via the binding of its metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) to the transcription factor vitamin D receptor (VDR), a direct effect on gene regulation. The relation of thousands of genomic VDR-binding sites to a few hundred primary 1,25(OH)2D3 target genes is still largely unresolved. We studied chromatin domains containing genes for the adhesion molecules CD97 and LRRC8A, the glucose transporter SLC37A2 and the coactivator NRIP1. These domains vary significantly in size (7.3 to 956 kb) but contain each one major VDR-binding site. In monocytic cells these four sites are associated with open chromatin and occupied by VDR, while in macrophage-like cells only the sites of LRRC8A, SLC37A2 and NRIP1 are accessible and receptor bound. The VDR site of CD97 does, in contrast to the three other loci, not carry any DR3-type binding sequence. CD97, LRRC8A, SLC37A2 and NRIP1 are early responding 1,25(OH)2D3 target genes in monocytic cells, while in macrophage-like cells they respond less and, in part, delayed. In primary human peripheral blood mononuclear cells from 71 prediabetic subjects of a vitamin D3 intervention study (VitDmet) CD97, LRRC8A, SLC37A2 and NRIP1 can be used as transcriptomic biomarkers for classifying human individuals for their possible benefit from vitamin D3 supplementation. In particular, NRIP1 exceeds the potential of the previously identified marker CD14 by more than 40% and seems to be a well-suited molecular marker for the vitamin D3 status in the hematopoietic system.
    The Journal of nutritional biochemistry 04/2014; 25(8). DOI:10.1016/j.jnutbio.2014.04.002 · 3.79 Impact Factor
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    • "De Cecco et al. (2013) examined replicatively senescent cells using the FAIRE (formaldehyde-assisted isolation of regulatory elements) technique (Giresi and Lieb, 2009), which measures open (transcriptionally active) and closed (transcriptionally silent) chromatin states. The technique exploits the differential partitioning during phenol/chloroform extraction of DNA fragments containing densely packed histones (more protein, more likely to be in organic phase) or relatively sparse histones (more DNA, more likely to be in aqueous phase). "
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    ABSTRACT: Epigenetic regulatory mechanisms are increasingly appreciated as central to a diverse array of biological processes, including aging. An association between heterochromatic silencing and longevity has long been recognized in yeast, and in more recent years evidence has accumulated of age-related chromatin changes in Caenorhabditis elegans, Drosophila, and mouse model systems, as well as in the tissue culture-based replicative senescence model of cell aging. In addition, a number of studies have linked expression of transposable elements (TEs), as well as changes in the RNAi pathways that cells use to combat TEs, to the aging process. This review summarizes the recent evidence linking chromatin structure and function to aging, with a particular focus on the relationship of heterochromatin structure to organismal aging.
    Frontiers in Genetics 12/2013; 4:274. DOI:10.3389/fgene.2013.00274
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    • "EL4 cells were fixed in 1% formaldehyde for 15 min at RT. 10×106 nuclei (isolated as described for FAIRE in [15]) were resuspended in 350 µl Buffer 3 (21) and sonicated using a BioRuptor (Diagenode) until the average chromatin fragment size was ∼500 bp. Sonicated samples were centrifuged at 16,000 g for 10 min at RT and supernatants diluted 1 to 8 in dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM TrisHCl pH8, 1X protease inhibitor). "
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    ABSTRACT: Mina is an epigenetic gene regulatory protein known to function in multiple physiological and pathological contexts, including pulmonary inflammation, cell proliferation, cancer and immunity. We showed previously that the level of Mina gene expression is subject to natural genetic variation linked to 21 SNPs occurring in the Mina 5' region [1]. In order to explore the mechanisms regulating Mina gene expression, we set out to molecularly characterize the Mina promoter in the region encompassing these SNPs. We used three kinds of assays - reporter, gel shift and chromatin immunoprecipitation - to analyze a 2 kb genomic fragment spanning the upstream and intron 1 regions flanking exon 1. Here we discovered a pair of Mina promoters (P1 and P2) and a P1-specific enhancer element (E1). Pharmacologic inhibition and siRNA knockdown experiments suggested that Sp1/3 transcription factors trigger Mina expression through additive activity targeted to a cluster of four Sp1/3 binding sites forming the P1 promoter. These results set the stage for comprehensive analysis of Mina gene regulation from the context of tissue specificity, the impact of inherited genetic variation and the nature of upstream signaling pathways.
    PLoS ONE 12/2013; 8(12):e80638. DOI:10.1371/journal.pone.0080638 · 3.23 Impact Factor
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