Accessibility to soluble nuclear factors regulating transcription, repair and recombination isclearly of utmost importance. DNase I and MNase sensitivity studies provide suchinformation, but they destroy accessible DNA fragments, and thus do not allow theirisolation for further characterization. FAIRE is a method designed to identify and isolatespecific genomic DNA sequences that are not readily trapped by formaldehyde crosslinkingof chromatin [7,8]. Understanding the role of such genomic regions should provide insightinto the organizational principles of chromatin.
NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptThe discovery of FAIRE
FAIRE is based on the fact that all regions of chromosomal DNA do not crosslink to
chromosomal proteins equally well with formaldehyde. DNA segments that are trapped bycrosslinked DNA binding proteins are retained in the interphase during phenol-chloroformextraction, while those DNA segments that are not protein associated accumulate in theaqueous phase. The method involves the following steps: 1) Formaldehyde crosslinking ofthe cells of interest. 2) Sonication to obtain DNA fragments a few hundred nucleotides long.3) Phenol-chloroform extraction of the crosslinked sonicated material. 4) Precipitation ofDNA enriched in the aqueous phase. 5) Identification of the DNA by microarray analysis ordirect sequencing. The observation, that DNA fragments that crosslink poorly to proteinsaccumulate in the aqueous phase while the majority of DNA trapped by crosslinked proteincomponents of chromatin forms a thick interphase, is hardly surprising [10]. To avoid lossof immunoprecipitated DNA to the phenol-chloroform interphase, ChIP protocols normallyinclude overnight reversal of crosslinks before the immunoprecipitated material is phenol-chloroform extracted [2]. However, at the time of the discovery of FAIRE, it was not widelyappreciated that DNA extracted from crosslinked chromatin would be qualitatively differentfrom that obtained from non-crosslinked samples [7]. The original discovery of FAIRE wasfortuitous and came during a ChIP-Chip (chromatin immunoprecipitation coupled withanalyses of the enriched DNA fragments using genomic microarray) experiment to map thedistribution of mono- di- and trimethylated histone tails in various mutants of the S.
cerevisiae Set1 methyltransferase complex. Instead of using DNA extracted from untreatedcells as a control, total DNA extracted from crosslinked cells was used as a reference for theChIP’ed material. The result was a striking apparent enrichment for coding over non-codingregions in the immunoprecipitated material. Initially, this observation suggested that
methylated nucleosomes were enriched in coding regions of the genome, however, similarresults were obtained from mutant yeast strains that lacked H3K4 methylation. To obtain anexplanation for this methylation independent enrichment of coding regions, material fromevery step was meticulously tested. It was concluded that the reference DNA isolated fromcrosslinked lysates was enriched for noncoding regions due to loss of coding regions trappedby crosslinked protein to the interphase during the phenol-chloroform extraction. Thus uponcomparison of this reference with the ChIP-ed material, the latter appeared to be enrichedfor the coding regions. Later work showed that FAIRE was also able to detect variations inthe crosslinkability of the significantly more complex human chromatin [8].
What does FAIRE detect?
The finding that noncoding regions of the yeast genome could be enriched by phenol/chloroform extraction of crosslinked and sonicated chromatin indicated that the
crosslinkable protein concentration in these regions was lower than on the coding portion ofRNA polymerase II transcribed genes. Formaldehyde penetrates organic materials quicklyand forms stable but reversible methylene bridges, mainly between proteins, via the ε-nitrogen atom of lysine and an adjacent amide nitrogen of a peptide linkage. For DNA toreact with formaldehyde, it must be partially denatured to expose the –CO-NH grouping atthe N-1 position of guanine, or the exocyclic amino groups of adenine, guanine, or cytosine
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[11]. Because the bulk of chromatin is accounted for by the high lysine content and thushighly crosslinkable histone subunits of nucleosomes, it was proposed that the phenomenonthat FAIRE detected uneven nucleosome distribution along chromatin [7]. Several otherpublications have since demonstrated that consistent with this hypothesis, regulatory regionsin general have fewer nucleosomes than coding regions [12-15]. Enrichment for non-codingregions was more pronounced for highly transcribed genes, but was also obtained for silentgenes [7]. Because the enrichment did not correlate with the level of transcription, the
possibility of a role of specific DNA sequences or general base composition was examined[7,16-18]. However, no such correlation was found. A recent finding by Shivaswamy et al.provides an alternative explanation. These authors showed that specific nucleosomalrearrangements in promoters changed even in the absence of transcription [19]. Theyconcluded that the relationship between chromatin remodeling and transcription is genespecific and that remodeling does not necessarily result in transcription. There are other datafrom the Lieb laboratory indicating that nucleosome dynamics in promoter regions isfundamentally different from that seen in coding regions [15]. In synchronized yeast cellpopulations progressing through the cell cycle, variations in crosslinkability are limited topromoters of cell cycle regulated genes.
Transcription factor occupancy also affects nucleosome density in regulatory regions.
Destabilization of nucleosomes in the promoter regions of actively transcribed genes is welldocumented [5,6]. The question arises: why would it make a difference whether DNA isbound to histones or transcription factors? Shouldn’t they be able to crosslink equally well?The answer to this question lies in the crosslinking characteristics of formaldehyde. Proteinsthat do not expose the exocyclic amino-groups of DNA do not crosslink to DNA directly,but depending on their steric relationship with DNA might simply trap it following intra-protein crosslinking [11]. In the original paper in which Solomon and Varshavsky proposedformaldehyde mediated DNA-protein crosslinking as a probe for in vivo analysis of
chromatin structure, they show that purified lac repressor does not crosslink to lac operator-containing DNA [9]. They also obtained similar results with (A + T) DNA-binding protein(alpha-protein) to its cognate DNA.
It is also important to note that nucleosome density might not be the only determinant ofchromatin crosslinkability. Methylation of the lysine residue K36 in the N-terminal tail ofhistone H3 (H3K36) has been shown to correlate extremely well with regulatory versus non-regulatory yeast chromatin [20]. This modification is most common in the body oftranscribed genes and is responsible for recruitment of a histone deacetylase complexnecessary for reestablishment of chromatin structure following passage of the polymerase[21]. Whether there is a causative relationship between FAIRE enrichment and the absenceof the H3K36 dimethylation requires further study. Although methylation might affect thecrosslinkability of specific lysine residues, it might have a more significant indirect effectthrough alteration of nucleosome compaction. FAIRE might be detecting the three-dimensional proximity of one nucleosome to others, irrespective of the linear distance
between them. However, the degree of compaction of yeast chromatin is still unclear [18]. Inhigher eukaryotes the existence of histone variants that incorporate into chromatin in areplication independent manner could also potentially alter the ability of formaldehydetreated chromatin to trap associated DNA segments [22] .
NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFunctional characteristics of FAIRE enriched DNA sequences
FAIRE has been applied to analysis of both yeast and human chromatin. As discussed
above, the original FAIRE studies in yeast demonstrated that there is an inherent differencebetween the crosslinkability of chromatin in coding regions versus noncoding regions.Giresi et al. performed a FAIRE study in human fibroblasts across the ENCODE regions
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[23] which cover about 1% of the human genome [8]. In many genomic regions FAIREfragments overlapped with DNase I hypersensitive sites, RNA polymerase II transcriptionstart sites, and histone modifications associated with active transcription [4,24-27].
Figure 1 shows a 268 kb region of human chromosome 19q13.42 displaying the resultsobtained with different techniques designed to probe chromatin structure in a variety of
different cell types. This region contains 18 annotated genes and a large number of repetitiveelements. ChIP-chip results are shown from the Ludwig Institute which map the locations ofRNA polymerase II, histone modifications associated with active chromatin (H3 acetylationand H3K4 dimethylation) as well as repressive chromatin marks (SUZ12 and H3K27
trimethylation) [24]. Data from Duke/NHGRI that maps sites of DNase I hypersensitivity [4]and FAIRE data from the Lieb laboratory [8] are also displayed. As described earlier [8],there is strong correlation between the localization of the FAIRE peaks with regions ofDNase I hypersensitivity, as well as indicators of active chromatin (Fig. 1, between59,295,000 and 59.410,000). On the contrary, FAIRE peaks are significantly reduced inregions of inactive chromatin defined by the lack RNA polymerase II and the presence ofrepressive chromatin marks (Fig. 1, <59,295,000 and >59.410,000). Interestingly, DNase Ihypersensitivity correlates with FAIRE in both active and inactive chromatin (indicated bystars). The region of transition between active and inactive chromatin regions (59,380,000 to59,430,000) was expanded and is shown in the lower panel of Figure 1. At this resolution itis clear that FAIRE correlates with transcriptional start sites, as was found earlier [8]. This isseen in the region containing three promoters in close proximity to each other (one for
MBOAT7 and two for TSEN34) and on the promoter of the highly active ribosomal proteingene RPS9. In addition, FAIRE signals are relatively high throughout the coding region aswell as downstream of the 3′ end of the RPS9 gene. These regions are exactly where RNApolymerase II maps, suggesting that some FAIRE signals are due to displacement ofnucleosomes by RNA polymerase II. However, FAIRE signals, accompanied by DNase Ihypersensitivity sites, are also found in inactive chromatin (indicated by stars)
demonstrating that not all FAIRE sites are caused by RNA polymerase II transcription.Some FAIRE sites are considered “orphan” sites because they do not coincide with othermarks of chromatin structure. Giresi et al. noted that the 40% of FAIRE peaks that fell intothat category and attributed this finding to “ the difference in cell types used among the
experiments being compared and the sparse state of current human genome annotations” [8].The functional importance of FAIRE elements is supported by the approximately 2-folddecrease in the frequency of insertion-deletion mutations in such regions compared to non-conserved, non-coding genomic regions [28]. To find a function for “orphan “FAIREelements would be potentially helped by studies examining the localization of prominentFAIRE enriched regions in the nucleus. It would also be of interest to examine how FAIREis affected by sudden changes in the nuclear compaction, for example during the activationof quiescent lymphocytes by phytohemagglutinins. Similarly, it would be of interest toexamine how FAIRE is affected by transformation of cervical epithelial cells by humanpapilloma virus induced changes in nuclear morphology.
NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptPractical uses of FAIRE
FAIRE is a simple, reproducible method that provides information about genomicorganization as it is present in the cells at the time of fixation. Other methods, such asDNase I hypersensitivity studies require more extensive preparatory steps that allow
compensatory transcriptional changes to occur and also increase the chance of unintendedprotein and DNA modifications and potentially degradation. There is also a need to performpilot experiments to determine the activity of individual batches of the enzyme. FAIRE isnot limited by availability of antibodies generated against a specific epitope of interest, or
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the inability to obtain a functionally intact tagged version of the target protein. This strengthof FAIRE is also its major weakness; the functional significance of the enrichment observedmay be difficult to explain, unless there is data from other ChIP or hypersensitivity studies.FAIRE allows for physical isolation and identification by direct sequencing of genomicregions that are otherwise identified by their absence, such as DNase I hypersensitive sites.This in theory would make it possible to identify regulatory regions in organisms for whichno sequence information is available. A caveat is that one study found that Negative
Regulatory Elements (NRE), such as insulator elements, were not significantly enriched byFAIRE [29]. Yaragatti et al. [30] were successful using a method reminiscent of FAIRE toisolate regulatory regions of the human genome. They formaldehyde crosslinked F9embryonal carcinoma cells, permeabilized their nuclei, and then used HaeIII digestion tofragment the genomic DNA trapped in the nucleus. Nucleosome free regions that wereaccessible to digestion produced small HaeIII fragments able to diffuse out of the nucleus,while DNA in tightly packaged, transcriptionally inactive regions was not released. Theynoted that there was some background release of DNA fragments that were associated withpromoters inactive in F9 cells, which they removed using FAIRE. They used the DNA fromthe aqueous phase to generate a library that had about 20 times higher regulatory domaincontent than non-enriched DNA. In transcriptional reporter assays, about 20% of thefragments obtained showed significant promoter activity compared to the 1% in non-selected DNA. They were successful in identifying both promoter and enhancer regionsusing this methodology [30]. Another successful application of FAIRE was to demonstratenucleosome deposition onto Cytomegalovirus (CMV) genomes following entry of the viralDNA into the nucleus [31]. Nucleosome-depleted chromatin was enriched by FAIRE atspecific times following infection. The ratio of GAPDH and viral DNA recovered fromfixed and nonfixed cells was compared using quantitative PCR studies using seven primerpairs corresponding to various functionally relevant regions of the viral genome. The resultsshowed that the region corresponding to the viral replication origin (oriLyt) remained free ofnucleosomes even when the rest of the virus became extensively chromatinized. There isreason to expect that similar studies will be conducted in the characterization of otherviruses in the future.
NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptConclusion
FAIRE provides a snapshot view of the tremendously complex organization of chromatin.Most FAIRE enriched elements represent regulatory regions of the genome but some do nothave easily attributable functional roles as of today. This might change as our understandingof chromatin structure grows, and as more people use the method to characterize various celltypes and transcriptional states. The appearance of deep sequencing methodologies willallow better quantification of the enrichment accomplished by FAIRE and thus will allow asemiquantitative measurement of the crosslinkability of genomic DNA packaged intochromatin. The improved sensitivity and higher resolution promised by these technologieswill allow for the use of FAIRE in characterization of the alterations in chromatin structurethat is a hallmark of a number of genetic disorders such as laminopathies as well as variousmalignancies [32,33].
Acknowledgments
We thank Arkady Khodursky for critical reading of the manuscript. P.L.N. is supported by grant from the AmericanHeart Association (0655618Z) and the NIH (NS064253). D.H.P is supported by NIH (GM35500).
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Figure 1. Comparison of FAIRE with other techniques probing chromatin structure
A region of human chromosome 19 was analyzed using the UCSC genome browser
(http://genome.ucsc.edu/) as described in the text. Numbers at the top of both panels indicatechromosomal coordinates. In both panels the positions of genes in the regions covered, aswell as the locations of repeated sequences are indicated. Stars denote position of FAIREsignals that overlap with sites of DNase I hypersensitivity in inactive chromatin.
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