Formaldehyde Assisted Isolation of Regulatory Elements

In eukaryotic cells, regulation of the chromatin structure is an important component in the control of transcription. Formaldehyde-Assisted Isolation of Regulatory- Elements (FAIRE) is one of the new method used for the study of chromatin structure.  This method is used to identify and isolate specific genomic DNA sequences that are not readily trapped by formaldehyde crosslinking of chromatin. These DNA regions contain information about the organizational principle of chromatin.

Principle of FAIRE

When formaldehyde is present, all the regions of chromosomal DNA do not bond well with the chromosomal protein. FAIRE is based on this property. Phenol-chloroform extraction tetains DNA segments which are trapped by crosslinked DNA binding proteins. The DNA segments remain in the aqueous phase and are not protein associated.

The method involves the following steps:

  1. Crosslinking the cells of interest using formaldehyde.
  2. DNA fragments are sonicated to a length in hundreds of nucleotide sequence.
  3. Extraction of crosslinked material after sonication using phenol-chloroform extraction.
  4. DNA (enriched) in the aqueous phase pis precipitated.
  5. Identification of the DNA sequences by either microarray analysis or direct sequencing.

To avoid the loss of DNA to the phenol-chloroform interphase overnight, reversal of cross-links is done.  The qualitative property that is interlinked to the DNA is not understood during the initial use of FAIRE. The discovery of FAIRE was to contribute a ChIP (chromatin immunoprecipitation) experiment to map the distribution of mono, di, and trimethylated histone in various mutants of the Saccharomyces cerevisiae set 1 methyltransferase complex. DNA extracted from untreated cells are used as a control. Initially, this experiment with untreated control suggested that there is an increase in methylated nucleosome in coding regions of the genome. However, the same results were obtained from nonmethylated yeast strains. Upon investigation, it was concluded that this false interpretation happens due to the loss of coding regions in the interphase of phenol-chloroform extraction and it lead to the discovery of FAIRE.

Detection by FAIRE

Finding the non-coding regions of the genome is enriched by phenol-chloroform extraction. The chromatin from these regions is sonicated and analyzed. The analysis results showed that the coding regions is significantly lower in these chromatins.  Formaldehyde penetrates organic material and forms and a stable- reversible methylene bridges with the protein molecules. For a DNA to react with formaldehyde it should be partially denatured to expose N-1 position of guanine, or the exocyclic group’s adenine, guanine or cytosine. Due to the high amount of lysine and crosslinking capacity of nucleosomes, uneven nucleosome distribution along the chromatin is detected by FAIRE. Regulatory regions always have fewer nucleosomes than the coding regions. Enrichment of these non-coding regions does not correlate with the level of transcribed. They concluded that the relationship between the chromatin remodeling and level of transcription is gene specific. Remodelling does not always result in transcription. Nucleosome dynamics in the promoter region is fundamentally different from coding regions. The mechanism of the difference in the dynamics and crosslinkability is due to the property that some proteins do not expose the exocyclic amino acid for formaldehyde bonding.

FUNCTIONAL CHARACTERISTICS OF FAIRE-ENRICHED DNA SEQUENCES

FAIRE has been applied to both human and yeast chromatin. As discussed earlier, there is an inherent property of difference and crosslinkability between coding and noncoding regions of yeast chromatin. In many genomic regions, the FAIRE fragments overlapped with DNase I hypersensitive sites, RNase II transcription starts sites and histone modification associated with active transcription.  There is a strong correlation between the localization of FAIRE peaks with DNase I hypersensitivity regions. It represents active chromatin regions.  FAIRE peaks are significantly reduced in regions of inactive chromatin, this is defined by lack of RNase II polymerase.

PRACTICAL USES OF FAIRE

FAIRE is a simple reproducible method that provides the genomic information of the cells at the time of fixation. Other methods take some preparatory steps which may be lead to some transcriptional changes during the analysis process. These changes may be unintended protein DNA modification and degradation. Pilot studies also are required to determine the activity of an enzyme. FAIRE has no limitation of availability of antibodies generated against a specific epitope of interest. It is neither limited to obtaining intact active target protein. These lead to a limitation in another aspect functional significance of the enrichment is difficult to explain unless provided with other studies.  FAIRE enables direct isolation and identification of genomic regions which is otherwise identified by their absence. This property enables as to identify and isolate regulatory regions of the organism for which there is no information is available.

Insulator elements were not significantly enriched by FAIRE. When  FAIRE is used to isolate regulatory regions of human genome it produced a genome which is 20 times higher than the nonenriched DNA domain content. Another successful application of FAIRE was to demonstrate nucleosome deposition onto Cytomegalovirus (CMV) genomes following entry of the viral DNA into the nucleus.  Nucleosome-depleted chromatin was enriched by FAIRE at specific times after infection. The ration of glyceraldehyde-3- phosphate dehydrogenase and viral DNA from fixed and nonfixed cells are analyzed. This is done by q-PCR with primer pairs corresponding to the various functionally relevant regions of the viral genome. The results showed the viral replication origin region remain free of nucleosome though rest of the virus became extensively chromantinized.

FAIRE analysis

  1. First, samples are treated with formaldehyde to cross-link DNA and proteins. 1% formaldehyde is used for fixation, at a concentration commonly employed in FAIRE and ChIP studies. In general, cross-linking occurs more efficiently between histones and DNA than between transcription factors and DNA, for two reasons. First, crosslinking requires the direct interaction between DNA and protein which occurs 10–15 times between histone proteins and DNA strands (as the DNA strand two times surrounds the histone proteins) whereas a typical regulatory protein interacts with DNA only via a small number of bases (5–15 bp) which potentially allows for one or two direct interactions. Second, histones have a high percentage of basic amino acids which favors crosslinking to DNA.
  2. The second major step in FAIRE is chromatin isolation which in contrast to ChIP does not require the use of antibodies. Note that prior to the extraction of chromatin, nuclei are isolated (enriched) from both samples (FAIRE and UN-FAIRE; steps 9–20).
  • In this step, chromatin is sheared to DNA fragments of 0.2– 0.8 kb length (note, that fragment length determines the resolution of the FAIRE peaks); the exact experimental conditions need to be optimized for each experiment and tissue. Checking fragment sizes can be done by analyzing aliquots of the unsheared (from step 20) and sheared chromatin samples (from step 22) on standard 1% agarose gels after decross‐inking and PCI extraction, This releases the DNA molecules from bound proteins.
  1. In this step, which distinguishes FAIRE from ChIP, the sheared chromatin is directly subjected to PCI extraction to separate NDRs (i.e. the open chromatin regions partitioning into the aqueous phase) from chromatin containing bound nucleosomes (which will accumulate in the interphase). In the UN-FAIRE control sample, all DNA regions will be extracted into the aqueous phase.
  2. FAIRE and control samples are compared to identify FAIRE peaks. These peaks indicate the potential regulatory elements.

MATERIALS AND METHODS

Plants

Arabidopsis thaliana (L.) Heynh. seeds were obtained. In brief, seeds were surface sterilized using sodium hypochlorite solution (50%), sown on agar medium plates (1 x Murashige Skoog (MS), 1% sucrose, pH 5.8), and plates were stored for 2 d under vernalization condition and then transferred to long-day growth condition (16 h light, 22 °C and 8 h dark, 18 °C). After 2 weeks, seedlings were transferred to liquid MS medium (1% sucrose) and kept overnight under the same environmental condition, and then treated for 3 h with 3-amino-1,2,4-triazole (3-AT) to block catalase activity. Seedlings were fixed with formaldehyde and processed further as explained in the protocol.

Reagents

The following reagents and consumables were used to set up the FAIRE protocol for A. thaliana: 1.5 and 2mL safe-lock tubes (Eppendorf tubes); b-ME (b-mercaptoethanol; double-distilled autoclaved water (cold and room temperature (RT)); ethylenediaminetetraacetic acid (EDTA; 1 M, adjusted to pH 8.0 with NaOH), autoclaved; ethanol; Falcon tubes (15 and 50mL); formaldehyde; GelRed; glycine (2.5M stock solution; glycogen; HyperLadder 1 kb; liquid nitrogen; MgCl2 ; Miracloth or nylon mesh (50–75mm); NaCl (5M stock solution; PCI, 25:24:1 ; phenylmethylsulfonyl fluoride (PMSF); Complete Protease Inhibitor Tablets ; Proteinase K; RNase A; sodium dodecylsulfate (SDS); sucrose; SYBR Green PCR Master Mix; TE buffer (10mM Tris-HCl, 1 mM EDTA, pH 8.0); Tris-HCl (1M, adjusted to pH 8.0 with HCl); Triton X-100.

Solutions

All solutions should be prepared freshly and kept on ice or in a fridge until use (unless stated otherwise).

Buffer 1 (crosslinking buffer) for 100 mL: 400 mM sucrose (20 mL of 2Mstock), 10mMTris-HCl, pH 8.0 (1 mL of 1Mstock), 5 mM b-ME (35 mL of 14.3 Mstock), 0.1 mM PMSF (50 mL of 0.2 M stock). Add 1 tablet of Complete Protease Inhibitor Cocktail to 50 mL Buffer 1 immediately before use.

Buffer 2 for 10 mL: 250 m M sucrose (1.25 mL of 2 M stock), 10 mM Tris-HCl, pH 8.0 (100 mL of 1Mstock), 10 mM MgCl2 (100 mL of 1 Mstock), 1% Triton X-100 (0.5 mL of 20% stock), 5 mM b-ME (3.5 mL of 14.3Mstock), 0.1 mM PMSF (m mL of 0.2 M stock). Immediately before use dissolve half a Complete Protease Inhibitor Tablet in Buffer 2.

Buffer 3 for 10 mL: 1.7 M Sucrose (8.2 mL of 2 M stock), 10 mM Tris-HCl, pH 8.0 (100 mL of 1 M stock), 0.15% Triton X-100 (75 mL of 20% stock), 2 mM MgCl2 (20 mL of 1Mstock), 5 mM b-ME (3.5 mL of 14.3 M stock), 0.1 mM PMSF (5 mL of 0.2 M stock). Immediately before use dissolve half a Complete Protease Inhibitor Tablet in Buffer 3.

Nuclei Lysis Buffer (NLB) for 5 mL: 50 mM Tris-HCl, pH 8.0 (0.25 mL of 1 M), 10 mM EDTA (24 mL of 0.5 M), 1% SDS (0.25 mL of 20%), 0.1 mM PMSF (5 mL of 0.2 M stock). Immediately before use dissolve one fourth of a Complete Protease Inhibitor Tablet in NLB. TE buffer for 10 mL: 10 mM Tris-HCl, pH 8.0 (100 mL of 1 M stock), 1 mM EDTA (10 mL of 1 M stock). TE buffer can be kept at RT.

Step-by-step protocol for NDR isolation by FAIRE Sampling of plant material

  1. Sow Arabidopsis seeds on soil or on semi-solid MS plates.
  2. Harvest 1–2 g fresh tissue of preferably 2–3-week old seedlings and transfer to a 50mL Falcon tube. To minimize environmental impact on the FAIRE patterns, sampling should be done as quickly as possible. Generally, all types of tissues suitable for ChIP studies can also be used for FAIRE experiments. However, younger tissues tend to have less lignin and woody structures than older ones which facilitates formaldehyde penetration, cross-linking, and chromatin isolation.
  3. Wash seedlings three times with RT, double-distilled autoclaved water and every time gently invert the tube two to four times and discard the water. This step cleans the tissues and wet. This tissues is now ready for formaldehyde penetration. In case a treatment was applied to the plants (e.g., hormone or stress treatment), a fraction of the seedlings can be harvested for RNA extraction and gene expression analysis using microarrays, by RNA-seq, or by qRT-PCR. By checking transcript levels, potential regulatory elements identified through FAIRE may be linked to differentially expressed genes.

Formaldehyde cross-linking (fixation)

  1. Thoroughly remove the water and fix tissues in 40 mL of RT cross-linking buffer (Buffer 1) supplemented with 1% formaldehyde (1,010 mL of 37% stock); vacuum-infiltrate for 8–10 min. After 5 min of vacuum infiltration, stop vacuum pump and shake Falcons to remove air bubbles. Note, that fixation time is dependent on the tissue and age of the samples, but often the fixation time for FAIRE experiments is shorter than for ChIP experiments on similar samples. Make sure that seedlings or tissues are kept in fixation solution while the vacuum pump is working; it may be necessary to softly push back the tissue into the buffer; this can be done with the help of a blue pipette tip or a little sponge.
  2. Stop the cross-linking process by adding glycine to a final concentration of 0.125M (2 mL of 2.5 M glycine in 40 mL of Buffer 1) and applying vacuum for 5 min to allow the glycine to penetrate the tissue. At this stage, leaves of seedlings should turn translucent and develop a slightly darker green appearance (due to penetration of liquid into the tissue).
  3. Discard Buffer 1 from the Falcon tube.
  4. Quickly rinse seedlings twice with cold double-distilled, autoclaved water (to clean the tissue from formaldehyde and glycine).
  5. Remove the water and quickly dry tissues between paper towels before transferring them to a new Falcon tube. Cross-linked samples can either be snap-frozen in liquid nitrogen and stored at –80 °C until use, or be directly processed for chromatin isolation. For the preparation of control (i.e., non-cross-linked) samples (“UN-FAIRE”), seedlings are processed in the same way with the exception that formaldehyde and glycine are omitted from Buffer 1.

Isolation and sonication of chromatin

  1. Grind tissues to a fine powder using a pre-cooled mortar and pestle. This step is very important for efficient chromatin isolation. Grinding samples for 10–15 min (without thawing) should produce a fine powder suitable for chromatin isolation.
  2. Resuspend the powder in 30 mL Buffer 1 (4 °C) in a new 50 mL Falcon tube. Incubate for 10–15 min on ice to obtain a completely homogenized suspension (if needed, gently shake the Falcon tube).
  3. Filter the homogenized suspension through four layers of Miracloth or nylon mesh (50–75mm) into a new pre-cooled 50 mL Falcon tube kept on ice.
  4. Centrifuge for 20 min at 2,880 g and 4 °C. A white pellet (with a green layer) should form at the bottom of the Falcon tube.
  5. Carefully remove the supernatant and resuspend the pellet in 1mL of Buffer 2.
  6. Transfer the suspension to a 1.5 mL Eppendorf tube.
  7. Centrifuge at 12,000 g for 10 min at 4 °C.
  8. Repeat steps 13–15 for two more times to wash the pellet. The green overlay should disappear (or diminish) after three times of washing.
  9. Discard the supernatant and resuspend pellet in 300 mL of ice-cold Buffer 3.
  10. Overlay the resuspended pellet onto 300 mL of ice-cold Buffer 3 in a fresh pre-cooled 1.5 mL Eppendorf tube.
  11. Spin at 16,000 g for 70 min at 4 °C.
  12. Carefully discard the supernatant and resuspend the chromatin pellet in 300mL of ice-cold nuclei lysis buffer (NLB) by vortexing or pipetting up and down on the ice. Keep an aliquot (5–10 mL) of the suspension to check for sonication efficiency by gel electrophoresis (see step 22). This will represent unsheared chromatin.
  13. Once resuspended, sonicate the chromatin for 10 cycles on the ice, using a sonicator (each cycle 15 s, on 70% power, setting 7 (x 10), with 100 s breaks between cycles, on ice), to shear DNA to fragments of approximately 0.2–0.8 kb length. Sonication conditions may vary depending on the equipment used; therefore, test experiments should be run to determine instrumental settings and treatment times appropriate for the generation of DNA fragments of 0.2–0.8 kb length. During sonication, the temperature of the chromatin suspension should not rise and the formation of foam and bubbles should be prevented. The sonicated chromatin can be stored at 80 °C until use or directly processed further in the next experimental steps.
  14. Spin the sonicated chromatin suspension for 10 min at 4 °C (16,000 g) to pellet debris. Transfer the supernatant to a new tube and store at 20 °C for later use or directly proceed to the isolation of nucleosome-depleted regions using PCI extraction (step 23 and following). Use an aliquot (5–10 mL) from this step (sheared chromatin) and step 20 (unsheared chromatin) to check sonication efficiency. To this end, both samples are de-cross-linked by overnight incubation at 65 °C and a 1–2 h treatment with 1 mL Proteinase K at 37 °C. Check de-cross-linked DNA by electrophoresis on a standard 1% agarose gel. Although samples can also be checked without de-crosslinking, the separation of the DNA fragments will work better upon removal of the bound histones prior to electrophoresis. Note, that in some studies only a limited amount of tissue is available for FAIRE analysis. In such cases, a third or half (i.e., 100–150 mL) of the secured supernatant may be retained and subjected to de-cross-linking. To this end, incubate at 65 °C overnight, add 10 mL Proteinase K and incubate for another 2 h at 37 °C. The de-cross-linked DNA is then subjected to PCI extraction (essentially as described in step 23 and following) and may serve as an internal control for the identification of enriched genomic regions (NDRs) in the non-de-cross-linked fraction of the same sample.

Isolation of NDRs

  1. Add an equal volume of PCI to the sample (300 mL).
  2. Vortex for 2 min, to establish a milky suspension.
  3. Spin at 12,000 g for 10 min at RT. Three different layers should be visible; the upper aqueous phase contains the NDRs, the milky inter-phase contains proteins and cross-linked genomic DNA (nucleosome-rich regions), and the lower yellowish phase is the organic phase. This step should be repeated if three layers are not well separated.
  4. Transfer the upper, aqueous phase to a new Eppendorf tube.
  5. Repeat steps 23–26 two more times to gain highly pure DNA.
  6. Add 0.1 volume of 3M sodium acetate (to a final concentration of 0.3 M).
  7. Add 2.5 volume of absolute pure ethanol and mix well. To increase the yield of DNA, 1 mL glycogen suspension may be added.
  8. Keep the solution at 20 °C overnight or at 80 °C for 1 h.
  9. Spin at 16,000 g (maximum speed) for 45 min at 4 °C.
  10. Discard the supernatant.
  11. Wash the pellet with 1mL of 70% ethanol (RT).
  12. Spin at 11,000 g for 5–10 min at RT.
  13. Discard the supernatant.
  14. Repeat steps 33–35 for two more times to increase the purity of the DNA pellet.
  15. Dry DNA pellet at RT (or at 56 °C for 5–10 min).
  16. Dissolve DNA pellet in TE buffer (pH 8.0) or DNase-free water.
  17. Check DNA concentration using a Nanodrop.
  18. Check fragment sizes by electrophoresis on a standard 1% agarose gel (fragment lengths should range from 0.2–0.8 kb, with a peak concentration of ~0.5 kb).
  19. The isolated DNA can be used directly for the identification of nucleosome-depleted regions by qPCR (for a small number of loci), or by microarray hybridization or next-generation sequencing (for genome-wide assessment).