Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13. Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11 . However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genomewide 3,12,13 , but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14 ; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13 .
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16 , but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17 . This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure  1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Video Link
The video component of this article can be found at http://www.jove.com/video/3912/

Probe Design
Design anti-sense DNA tiling probes for selective retrieval of RNA target by ChIRP.
1. Design anti-sense oligo probes using the online probe designer at singlemoleculefish.com 18 .
2. Use these parameters: number of probes = 1 probe /100 bp of RNA length; 2) Target GC% = 45; 3) Oligonucleotide length = 20; 4) Spacing length = 60-80. Break RNA into segments if too long for the designer. Omit regions of repeats or extensive homology. 3. Order anti-sense DNA probes with BiotinTEG at 3-prime end. 4. Label probes according to their positions along the RNA. Separate them into two pools so that the "even" pool contains all probes numbering 2, 4, 6, etc. and the "odd" pool contains probes numbering 1, 3, 5, etc. Dilute pool of probes to 100 μM concentration and store at -20 °C. 5. All experiments are to be performed using both pools, which serve as internal controls for each other. Real RNA-dependent signal would be present from both pools, while probe-specific noises would be unique to each pool. This applies for both ChIRP-qPCR and ChIRP-seq.

Harvest Cells
Collect cells that will be used for ChIRP experiment.

Cell Lysis
Lyse crosslinked cells to prepare cell lysate.

Sonication
Shear DNA by sonicating crosslinked cell lysates.
1. Sonicate cell lysate in Bioruptor in 15 ml Falcon tubes. Use <1.5 ml lysate in each tube, and for faster sonication, sonicate no more than two tubes at a time. 2. Sonicate in a 4 °C water bath at highest setting with 30 seconds ON, 45 seconds OFF pulse intervals. Check lysate every 30 min. Continue sonicating until the cell lysate is no longer turbid. This may take as little as 30 min and as many as 4 hrs. The number of tubes, the sample volume, the bath temperature, and the period of sonication time will affect how long the process takes. Tubes will likely sonicate at different rates, so pool them together every 30 min and redistribute into original tubes to ensure homogeneity. Note: glutaraldehyde-crosslinked cells take significantly longer to sonicate than formaldehyde equivalents. 3. When lysate turns clear, transfer 5 μL lysate to a fresh Eppendorf tube. Add 90 μL DNA Protease K (PK) Buffer (see buffer list) and 5 μL PK.
Vortex to mix and spin down briefly. Incubate for 45 min at 50 °C. 4. Extract DNA with Qiagen PCR purification kit. Elute DNA in 30 μL Qiagen Elution Buffer (EB) and check DNA size on 1% agarose gel. If bulk of the DNA smear is 100-500 bp, sonication is complete. If not, continue to sonicate. 5. Centrifuge sonicated samples at 16100RCF for 10 min at 4 °C. Combine supernatants, aliquot into 1 mL samples and flash-freeze in liquid nitrogen. Store at -80 °C.

ChIRP
Hybridize biotinylated DNA probes to RNA and isolate bound chromatin. Keep on ice till further use. 4. Transfer 1 mL chromatin to 15 mL Falcon tube. Add 2 mL Hybridization Buffer to each tube. For total volume <1.5 ml, use Eppendorf tubes. 5. Thaw probes at room temperature. Nanodrop probes to check amount if you haven't used it in a long time (100 μM probes should spec 500-600 ng/μl using single strand DNA setting). Add appropriate volume of probes to specific tubes (100 pmol probe per 1 mL chromatin, 1 μL of 100 pmol/μL probe per 1 mL chromatin). Mix well. Incubate at 37 °C for 4 hrs with shaking. 6. With 20 min remaining for hybridization, prepare the C-1 magnetic beads (stored at 4 °C). Use 100 μL per 100 pmol of probes. Wash with 1 mL unsupplemented Lysis Buffer three times, using the DynaMag-2 magnet strip to separate beads from buffer. 7. Resuspend beads in original volume of Lysis Buffer; supplement with fresh PMSF, P.I and Superase-in. After 4 hr hybridization reaction is complete, add 100 μL beads to each tube. Mix well. Incubate at 37 °C for 30 min with shaking. 8. Prepare Wash Buffer (5 mL per sample). Vortex to mix. Pre-warm to 37 °C. Add PMSF before use. Figure 1 depicts the ChIRP workflow. A successful ChIRP experiment typically enriches target RNA significantly over non-specific RNAs. Figure  2 shows enrichment of human telomerase RNA (TERC) from HeLa cells over GAPDH, an abundant cellular RNA that serves as a negative control. Majority of TERC RNAs (~88%) present in the cell were pulled down by performing ChIRP, whereas only 0.46% of GAPDH RNA was retrieved, demonstrating an enrichment factor of ~200 fold. Nonspecific probes such as probes targeting LacZ RNA, which is not expressed in mammalian cells (Figure 2), can be used as additional negative controls.

Representative Results
DNA regions expected to bind the target lncRNA are typically enriched over negative regions when measured by qPCR. Figure 3 shows qPCR validation of four HOTAIR-bound sites in primary human foreskin fibroblasts that we determined by performing ChIRP-seq in the same cell line, while TERC and GAPDH DNA sites serve as negative control regions. Both "even" and "odd" probe sets yielded comparable enrichment of expected HOTAIR-bound sites over negative regions, a hallmark of true lncRNA-binding sites.
High-throughput sequencing of ChIRP enriched DNA yields a global map of lncRNA-binding sites. The Drosophila lncRNA roX2 is known to interact with the X-chromosome in a manner that is required for dosage compensation. Figure 4 shows roX2 binding profile over a section of the X chromosome. Both "even" and "odd" samples have been sequenced and their unique noises have been eliminated to produce a track of overlapping signals. Each "peak" here indicates a strong site of roX2 binding. The complete track and list of roX2 target genes have been described in Chu et al. 2011

Discussion
Here we described ChIRP-seq, a method of mapping in vivo lncRNA binding sites genome-wide. The key parameters for success are the split pools of tiling oligonucleotide probes and glutaraldehyde crosslinking. The design of affinity-probes is straightforward given the RNA sequence and requires no prior knowledge of the RNA's structure or functional domains. Our success with roX2, TERC, and HOTAIR -three rather different RNAs in two species -suggests that ChIRP-seq is likely generalizable to many lncRNAs. As with all experiments, care and proper controls are required to interpret the results. Different lncRNA may require titration of conditions, and judicious change of conditions, such as selection of different affinity probes or crosslinkers, may highlight different aspects of RNA-chromatin interactions. Like ChIP-seq, not all binding events are necessarily functional, and additional studies are required to ascertain the biological consequences of RNA occupancy on chromatin. Nonetheless, we foresee many interesting applications of this technology for researchers of other chromatin-associated lncRNAs, which now number in the thousands 8,9 . Just as ChIP-seq has opened the door for genome-wide explorations of DNA-protein interactions, ChIRP-seq studies of the "RNA interactome" may reveal many new avenues of biology.