Isolation of active regulatory elements from eukaryotic chromatin using FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements) - PubMed (original) (raw)

Isolation of active regulatory elements from eukaryotic chromatin using FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements)

Paul G Giresi et al. Methods. 2009 Jul.

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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Figures

Figure 1. FAIRE Procedure

(A) The FAIRE procedure described in the text is shown on the left, while preparation of the reference or input sample is shown on the right. The DNA recovered from he aqueous phase of each extraction can then be used to identify sites of open chromatin using qPCR, tiling microarrays, or high-throughput sequencing applications. (B) For qPCR, a series of primers, depicted as convergent arrows, are designed to span a genomic region of interest. Sites of open chromatin are highlighted in blue, with qPCR results depicted above. Amplicons that span or are near the boundaries of open chromatin often result in lower relative enrichment due to shearing of DNA fragments, as shown by asterisks. (C) Microarrays. Typically we use high-resolution microarrays that tile either regions of interest or the entire genome of an organism with 50 to 70 bp oligonucleotides. (D) High-throughput sequencing technologies can be used to map the DNA fragments back to the reference genome.

Figure 2. FAIRE data

DNA isolated by FAIRE in human lymphoblastoid cells was mapped to the genome using both the Illumina GAII (black) and NimbleGen tiling microarrays (red). A 60 kb region of Chromosome 19 is displayed in the UCSC genome browser. For sequencing data, the number of extended reads overlapping each base is plotted (see text). The FAIRE microarray data (red) is plotted as z-scores (see text). Also shown is DNaseI hypersensitivity (blue) [56], and H3K4 mono-, di-, and tri-methylation from human CD4+ cells [57]. Each of these datasets are represented as the density estimates from fseq. Black arrows represent the UCSC Known Genes [58], with arrowheads indicating the direction of transcription. The FAIRE data colocalizes with transcriptional start sites, DNaseI hypersensitive sites, and is adjacent to histone modifications indicative of active 5′ ends of genes.

Figure 3. Formaldehyde crosslinking efficiency as the basis for FAIRE

Crosslinking between histones and DNA (or between one histone and another) is likely to dominate the chromatin crosslinking profile. (A) Here are a representative set of features from eukaryotic chromatin, including nucleosomes (blue spheres), a DNA-binding protein (light blue oval), and DNA (black line). Crosslinking with formaldehyde (red X) for most genomics applications only captures a portion of the potential interactions. Given that histone-DNA interactions constitute the majority of crosslinkable interactions in the genome, in a population of cells (ten rows) all of these interactions are likely to be captured. Whereas only a small proportion of the interactions between other DNA-binding proteins and DNA is actually captured by formaldehyde crosslinking. (B) The plot represents the expected FAIRE signal, which is inversely correlated with the occurrence of crosslinkable protein-DNA interactions.

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Isolation of active regulatory elements from eukaryotic chromatin using FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements) - PubMed (original) (raw)