Introduction

The recent discovery of RNA interference and in particular the observation that siRNAs can modulate gene expression at the level of transcription, i.e. small-interfering RNA (siRNA) directed transcriptional gene silencing (TGS) in Human cells (Matzke and Birchler 2005; Morris 2005) has illustrated the fact that RNA may be far more intricately involved in epigenetics than was previously assumed. To determine more clearly how siRNAs are interacting with the homologous genomic regions in the nucleus in human cell cultures we designed several RNA-biotin based pulldown assays which can be used alone or in combination with other known assays such as ChIP and Flag-tagged pulldown assays. Three protocols are explained in detail here. The first protocol is essentially a dual-pulldown assay employing Flag-tagged DNMT3A or antibody of choice for an endogenous protein and 5' biotin linked antisense RNA (Figure 1a), while the second protocol is a triple pulldown assay which essentially expands upon the dual pulldown to incorporate a third pulldown which is an iteration of the ChIP and is a pulldown for H3K27me3+ (Figure 1b). The third assay described here is the biotin-RNA pulldown of a low-copy RNA that spans the siRNA targeted promoter region (Figure 2). Data generated from this assay is currently in submission.

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Procedure

Part I: Detection of Flag-tagged proteins binding 5´ biotin labeled siRNAs in vitro

1. A minimum of ~4.0×106 cells are required/sample and clearly the more cells to begin with the better the chances of a successful pulldown. A total of 4.0×106 293 HEK cells (~70% confluent) are plated and 24 hours later transfected with 15µg of a RNA Polymerase II expressing flag-tagged construct plasmid. We generally use Lipofectamine 2000™ (Invitrogen) at a 1:3 ratio (e.g. 1µl plasmid to 3µl lipofectamine). The plasmid and lipofectamine are mixed together, vortexed, pulse spun, and then 50µl of DMEM (without Fetal bovine serum, FBS) added. The entire mixture is again vortexed followed by a pulse spin, and then added drop wise (randomly around the plate) of the previously plated cell cultures (Figure 1a);

2. Forty-eight hours later the cell cultures are washed in cold 1xPBS, then scrapped from the plate and collected in 1ml of cold 1x PBS. The cultures are then pelleted at ~2,000 RPM for 4 minutes and the pellet re-suspended in 500µl of cold lysis buffer on ice for 10 minutes and then centrifuged 5,000 RPM for 5 minutes at 4°C. The pellet is considered the nuclear fraction while the solution is the cytoplasmic fraction. Save the cytoplasmic fraction on ice for step 3. To isolate the final nuclear fraction the pellet is re-suspended in 500µl of lysis buffer and incubated on ice for 10 minutes, centrifuged at 5,000 RPM for 5 minutes at 4°C and the solution saved as the nuclear fraction with the pellet discarded (comment 1 and comment 2);

3. The resultant lysates (cytoplasmic and nuclear fractions) are then mixed, 125µl of each of the cytoplasmic and nuclear fraction, and incubated for 3 hours at 4°C with 500nM 5'biotin end-labeled siRNA under gentle agitation, i.e. a rocking plate (Figure 1a). We utilized the EF1 alpha specific siRNA, EF52, shown previously to direct TGS in human cells (Morris et al. 2004). The EF52 siRNA was constructed with a 5' biotin tag on either the sense or antisense strand (City of Hope Protein and synthesis core facility) and various iterations were generated by first heating the RNAs (sense or antisense +/- biotin) to 70°C for 5 minutes and then mixed together and room temperature cooled, i.e. biotin tagged sense+antisense, sense+biotin tagged antisense, biotin tagged sense+biotin tagged antisense. The biotin tagged sense or antisense can also be used alone, as well as the sense and an antisense lacking the 5' biotin tag (as a controls). We have found that the antisense alone co-immunoprecpitates with the flag-tagged DNMT3A (Weinberg 2005). Biotin linked RNAs can easily be generated by numerous companies. We now generally use IDT technologies 5' biotin linked RNAs;

4. Following the 3 hour incubation Dynabeads™ M-280 Streptaviden magnetic beads (7x107 beads, ~100µl/sample) are added to the respective samples (note 1);

5. The resultant siRNA/Flag complexes are then eluted with magnetic bead binding, the solution can be discarded (or saved as input) and the beads (putatively bound with the siRNA/Flag complexes) washed 3 to 5 times with 2x wash buffer (note 2);

6. Following the final wash the bound siRNA/protein complexes are eluted from the avidin-biotin bound beads by incubation in 100µl of elution buffer at 65°C for 5 minutes;

7. he eluted complexes can then be electrophoresed in denaturing PAGE and subjected to western blot analysis with an anti-Flag antibody for detection of the particular flag-tagged protein of choice (Figure 1a).

Part II Triple immunoprecipitation: H3K27 ChIP/-flag-DNMT3A/biotin-RNA

1. A total of 4.0×106 293T cells are plated out and 24 hours later transfected plated (~70% confluent at the time of transfection) with the flag-tagged protein expressing plasmid of choice (15µg, Lipofectamine 2000™ Invitrogen). The following day the flag-tagged protein expressing plasmid transfected cultures are transfected again with 100nM EF52 biotin labeled siRNA (antisense or sense alone) using Lipofectamine 2000™ or stock 3.4µM MPG (3µl/ml of media described in (Morris et al. 2004)) (note 3).

2. Pulldown (1), Antibody specific

1. Forty-eight hours following the biotin linked sense or antisense siRNA transfection the cultures are collected and a chromatin immunoprecipitation (ChIP) assay is performed by first adding formaledehyde directly to tissue culture media to a final concentration of 1% for 10 minutes at room temperature under gentle rocking (Figure 1b);

2. Stop the crosslinking reaction by adding glycine to a final concentration of 0.125M and continue to rock or spin at room temp for 5 minutes;

3. Next, pour off media and rinse plates with cold 1X PBS+1/1000 PMSF, aspirate and then add 1.5ml of PBS+1/1000 PMSF and scrape the plates to remove the cells. Collect the cultures and centrifuge 4 minutes at 2000 RPM;

4. Remove the PBS+1/1000 PMSF by aspiration and re-suspend cell pellet in 1ml ChIP lysis buffer plus the protease inhibitors 1/1000 PMSF and 50 units of RNAse inhibitor. The final volume of cell lysis buffer should be sufficient so that there are no clumps of cells. Incubate on ice for 10 minutes;

5. Centrifuge at 5,000 rpm for 5 minutes at 4°C to pellet the nuclei;

6. Re-suspend nuclei in 600µl of ChIP lysis buffer+1/1000 PMSF+50 units of RNAse inhibitor and incubate on ice for 10 minutes;

7. Sonicate chromatin while keeping samples on ice (1 interval for 20 seconds at power setting of "3" on the Branson 50 cell machine). Immediately centrifuge at 14,000 RPM for 10 minutes at 4°C;

8. Carefully remove the supernatant and transfer to a new tube. Preclear chromatin by adding Protein A/Salmon Sperm (10µl). Incubate on a rotating platform at 4°C for 15 minutes, no longer. Centrifuge at 14,000 RPM for 5 minutes (comment 3);

9. Transfer supernatant to a clean tube and divide equally among your samples. Be sure to include a "no antibody" control sample. Adjust the final volume of each sample with lysis buffer if required. Sample volumes should be between 400-800µl/sample to allow ample mixing during the overnight incubation. Add 1µg of antibody to each sample and incubate on the rotating platform at 4°C overnight;

10. Add 20µl Protein A/Salmon sperm sephrose beads to each sample and incubate at room temperature for 15 minutes on a rotating platform (comment 3);

11. Centrifuge the samples containing the Protein A/Salmon spern sepharose beads (10,000 RPM for 1 minute at 4°C) and save the supernatants as unbound fraction (input for step 21 below);

12. Wash the pelleted Protein A/Salmon sperm sepharose beads bound to the putative protein of interest twice with: (note 4)

1. 1ml ChIP lysis buffer;

2. 1ml of ChIP high salt buffer;

3. 1ml of ChIP wash buffer;

4. Pulldown (2), Flag-tag specific

1. After the last wash elute the complex by adding 100µl of ChIP elution buffer. Incubate the samples at 65°C for 10 minutes and then centrifuge at 14,000 RPM for 3 minutes. Save 20µl of the elutes as (Flag-Input). Next, take the remaining ~80µl of elute to clean tubes containing 40µl of EZVIEW™ Red anti-Flag M2 affinity gel beads (Sigma™) which have been pre-treated with a wash in 250µl of TBS-mod buffer, vortexed and centrifuged for 30 seconds at 6,000 RPM (comment 4);

2. Gently mix (rotating plate) the cell lysate/ChIP pulldown with the antibody-M2 beads for 2-3 hours or overnight at 4°C;

3. Capture the Flag-antibody-M2 immunoprecipitated complexes by pulse centrifugation (15 seconds at 3,000 RPM). Wash the bead/complexes 3 times in 500µl ice-cold TBS-mod buffer (comment 4);

4. To elute the putative ChIP-Flag complexes (by competition with 3x Flag-peptide) add 100µl of TBS-mod buffer+3µl (5µg/µl 3X Flag Peptide) to each pelleted bead/complex, incubate 30-60 minutes at 4°C with gentle shaking;

5. Centrifuge the resin for 30 seconds at 10,000 RPM, transfer the supernatants (which should now contain the ChIP/Flag eluted complexes) to fresh tubes containing100µl (6-7×108 beads/ml) of Dynabeads™ M-280 Streptaviden (Figure 1b).

6. Pulldown (3), RNA-biotin specific

1. Incubate the elute/Dynabead™ slurry at 4°C for 15 minutes on an orbital shaker followed by capture with a magnetic bead separator. The captured beads are washed (carefully as to not agitate the beads) 3 times with 2x wash buffer;

2. After the last wash (2x wash buffer) the complexes are eluted in 100µl of 2x elute buffer at 65°C for 5 minutes (Figure 1b);

3. The elutes are then reverse cross-linked by adding 1µl Rnase A (10mg/ml) and 20µl of 5M NaCl. Incubate the samples in a 65° water bath or heat block for 4-5 hours to overnight (note 5);

4. After the reverse cross-linking the elutes are treated with 10µl of 0.5M EDTA, 20µl of 1M Tris-HCL, pH 6.5 and 2µl of 10mg/ml Proteinase K and incubated in 45°C water bath for 1 hour;

5. Recover DNA/RNA by Phenol/Chloroform extraction and ethanol precipitation (note 6);

6. Re-suspend the pelleted DNA in ~30µl of water and perform PCR or real-time PCR analysis for the siRNA targeted gene compared to the respective control(s).

7. Part III: Detection of pRNAs by 5'biotin EF52 pull-down assay

1. A minimum of ~4.0×106 cells are required/sample. Twenty-four hours following the plating (~70% confluent) the cultures are transfected with the 5' biotin linked sense (control) or antisense (treatment) RNAs (70-100nM final concentration (Ting et al. 2005; Kim, Villeneuve et al. 2006)) targeted to the particular promoter of choice with Lipofectamine 2000™ (Invitrogen) at a 1:3 ratio (e.g. 1µl biotin-tagged RNA (for final 70-100nM concentration) to 3µl lipofectamine). However, the RNAs are first heated to 70°C for 10 minutes to allow the relaxation of any untoward secondary structures. The RNAs and lipofectamine are then mixed together, vortexed, quickly spun, and then 50µl of DMEM (without fetal bovine serum, FBS) added. The entire mixture is again vortexed followed by a quick spin, and then added drop wise (randomly around the plate) to the previously plated cell cultures;

2. Twenty-four hours following transfection the treated cultures can be crosslinked by adding 1% formaledehyde directly to tissue culture media to a final concentration of 1% for 10 minutes at room temperature under gentle rocking and stopped by the addition of glycine to a final concentration of 0.125M and continue to rock or spin at room temp for 5 minutes. The cells are then washed (1xPBS), scrapped from the plate if adherent, and then genomic DNA extracted (Qiagen™ DNeasy kits). The extracted genomic DNA (200µl elute) is then used to detect promoter specific transcripts (pRNAs) with the biotin linked pull-down assay (Figure 2) (comment 5);
   From the 200µl elute 30µl should be saved as input and the remaining 170µl from the extracted genomic DNA (Qiagen™ DNeasy) is mixed with 170µl of modified lysis buffer. During this same time 100µl of Dynabeads™ M-280 Streptaviden should be washed with 300µl of modified lysis buffer (comment 6);

3. Next, mix the pre-washed Dynabeads™ M-280 Streptaviden beads with the 170µl of extracted genomic DNA elute/ modified lysis buffer solution (15 minutes at R/T under constant motion);

4. Following the 15 minute incubation the beads are captured using a magnetic bead separator (Invitrogen™) and washed 3 times with 2x wash buffer;

5. After the third wash the bound beads were eluted using elution buffer II, incubated at 65°C for 10 minutes and separated using the magnetic bead separator;

6. The final ~100µl elute is then assessed for promoter specific DNA and/or RNA by PCR and RT-PCR respectively (note 7).