Elsevier

Methods

Volume 48, Issue 3, July 2009, Pages 233-239
Methods

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

https://doi.org/10.1016/j.ymeth.2009.03.003Get rights and content

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.

Introduction

In eukaryotes, packaging of DNA into chromatin reduces the accessibility of genetic information to the set of proteins involved in regulating DNA-templated processes such as transcription. Successful orchestration of DNA-dependent processes is achieved in part by regulating the stability of nucleosomes at these sites [1], [2], [3]. Here “stability” refers to the probability of an intact nucleosome at a given nucleotide position, versus a nucleosome in an absent or disrupted state at that position. Several mechanisms exist to modulate nucleosome stability, including competition with sequence-specific factors [4], [5], [6], [7], ATP-dependent nucleosome remodeling complexes [8], [9], [10] and post-translational modifications of the histone tails [11], [12], [13], [14]. Nucleosome stability at any given locus is governed by a combination of factors acting in concert, which results in a context-specific set of DNA elements bound by regulatory factors for each cell type.

Traditionally, active regulatory elements have been identified by their increased sensitivity to nuclease digestion, such as DNase I [15], [16], [17], [18], [19], [20]. Typically this involves subjecting isolated nuclei to a mild nuclease treatment, followed by detection using Southern blots to identify nuclease hypersensitive sites. Several groups have recently adapted the procedure for genome-wide detection with DNA microarrays or next-generation sequencing [21], [22], [23], [24]. However, requirements for a clean nuclei preparation from a single-cell suspension, and the need for laborious enzyme titrations means that it is difficult to perform DNase hypersensitivity assays on solid tissues, on a limited number of cells, or in parallel on many different samples.

Here we describe an alternative strategy for genome-wide isolation of active regulatory elements termed FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements). It is a simple, high-throughput procedure to isolate and map genomic regions depleted of nucleosomes. The procedure involves crosslinking proteins to DNA using formaldehyde, shearing the chromatin, and performing a phenol–chloroform extraction. The genomic regions preferentially segregated into the aqueous phase are then mapped back to the genome by hybridization to tiling microarrays or are read directly using next-generation DNA sequencing (Fig. 1). Quantitative PCR can be used to assay individual loci, which is useful when screening many cell or tissue types. The relatively straightforward nature and tractability of FAIRE has broad utility for the genome-wide detection of active regulatory elements across all eukaryotic species, in clinical samples, and for high-throughout screens.

FAIRE was first demonstrated in Saccharomyces cerevisiae [25]. In yeast, the genomic regions immediately upstream of genes were preferentially segregated into the aqueous phase, in a manner that was strongly negatively correlated with nucleosome occupancy [26], [27], [28], [29]. Subsequent studies demonstrated that FAIRE efficiently isolated nucleosome-depleted regions of the Homo sapiens genome, which included both transcription start sites and distal regulatory elements such as enhancers and silencers [30] (Fig. 2). Results from both yeast and human found that enrichment of the upstream regions of genes was positively correlated with transcription of the downstream gene. However, in human cells the vast majority of sites identified were far from any annotated gene. For the majority of these distal sites, it is not yet possible to ascribe a function, identify what factors might be bound, or determine the genes being regulated by each regulatory element.

The enrichment of regulatory regions in the aqueous phase is thought to result from the very high crosslinking efficiency of histone proteins to DNA, versus the lower efficiency of crosslinking sequence-specific proteins to DNA. This difference in crosslinking efficiency is likely due in part to formaldehyde’s very short crosslinking distance. Formaldehyde is a small molecule (HCHO) and crosslinks are only formed between proteins and DNA in direct contact. There are approximately 10–15 histone–DNA interactions within a nucleosome that serve as potential crosslinking sites [31]. However, for most DNA-binding proteins there are far fewer potential crosslinking sites. The average binding sites are 5–15 bp [32], with only a few of the bases close enough to the protein contacts to be crosslinked [33]. In addition, formaldehyde requires a ε-amino group such as occurs on lysine, to form a crosslink [34], [35]. Approximately 10% of the amino-acid composition of histones are lysine, a much higher proportion than a typical protein. Due to both of these factors nucleosomes are much more readily crosslinkable to DNA, and are likely to dominate the crosslinking profile (Fig. 3).

Section snippets

FAIRE procedure

The following provides a general framework for performing FAIRE, which specifically emphasizes performing FAIRE on cells grown in culture. The final methods section provides the modifications required to perform FAIRE on tissue samples. The protocols for cells and tissues are also included as one-page Supplementary files for easier use at the bench.

Crosslinking

For cells grown in culture, add 37% formaldehyde directly to the growth media to a final concentration of 1% and incubate at room temperature on an

Concluding remarks

Several aspects of FAIRE make it a powerful genome-wide approach for detecting functional in vivo regulatory elements in eukaryotes. It requires little treatment of cells prior to the addition of formaldehyde and involves only a few reagents: formaldehyde, phenol, chloroform, and ethanol. The successful application of FAIRE on a limited numbers of cells expands its utility beyond what other DNA accessibility assays can accomplish. This provides an opportunity to perform genome-wide assays of

Acknowledgments

We thank members of the Lieb lab for discussions. Support for this work has been provided by Grants from the NHGRI.

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