This work was supported by Takeda Science Foundation (T.F.); the Asahi Glass Foundation; the Uehara Memorial Foundation (H.F.); the Kurata Memorial Hitachi Science and Technology Foundation (T.F. and H.F.); Grant-in-Aid for Young Scientists (B) (#25830131), Grant-in-Aid for Scientific Research (C) (#15K06895) (T.F.); and a Grant-in-Aid for Scientific Research on Innovative Areas &#39;Transcription Cycle&#39; (#25118512 &#38; #15H01354), Grant-in-Aid for Scientific Research (B) (#15H04329), Grant-in-Aid for Exploratory Research (#26650059) and &#39;Genome Support&#39; (#221S0002) (H.F.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

1. Design of Engineered DNA-binding Molecules Recognizing the Target Locus
<ol>
	<li>For enChIP using CRISPR complexes, use the CRISPRdirect web tool (http://crispr.dbcls.jp) to identify candidate gRNA target sequences in the genomic region of interest described as previously18. This web tool returns 23 bp genomic sites of the form 5&#39;-N20NGG-3&#39; within the target region.</li>
	<li>Synthesize a gBlock including the U6 promoter sequence and the sequence of 5&#39;-N20 in 5&#39;-N20NGG-3&#39; using commercial services (see Figure 2)19,20. For subcloning purposes, include appropriate restriction enzyme sites outside of the gBlock.</li>
	<li>Insert the gBlock into an appropriate vector as previously described16. Several retroviral vectors for gBlocks are available (see Materials).</li>
	<li>For enChIP using TAL proteins, design TAL proteins recognizing the target loci. Generate plasmids encoding TAL proteins using commercial services. Generate expression vectors containing the TAL proteins fused with one or more tags such as 3&#215;FLAG as previously described15.</li>
</ol>
2. Establishment of Cells for the enChIP Analysis
<ol>
	<li>Express 3&#215;FLAG-dCas9 and the gRNA (CRISPR-based procedure) or 3&#215;FLAG-TAL (TAL protein-based procedure) in the cells to be analyzed as described previously14-17.
	<ol>
		<li>Use transient transfection if the transfection efficiency is high. An expression vector containing 3&#215;FLAG-dCas9 and the CMV promoter is available (see Materials).</li>
		<li>Consider establishing stable transformants using conventional methods if the transient transfection efficiency is low. In addition to the aforementioned retroviral vectors for gBlock, retroviral vectors of 3&#215;FLAG-dCas9 are available (see Materials).</li>
	</ol>
	</li>
	<li>Confirm the expression of the 3xFLAG-dCas9 or 3xFLAG-TAL protein in the transfected cells by immunoblot analysis using an anti-FLAG antibody (Ab) as described previously14-17. 
	Note: Expression of these tagged proteins can also be confirmed by intracellular staining with anti-FLAG-FITC and a subsequent FACS analysis. A protocol for intracellular staining can be downloaded from our homepage (http://www.biken.osaka-u.ac.jp/lab/microimm/fujii/iChIP_protocols/english.html).</li>
	<li>Confirm expression of the gRNA by standard RT-PCR technique.</li>
	<li>For quantitative identification of proteins interacting with the genomic region of interest, consider use of a stable isotope labeling by amino acids in cell culture (SILAC) analysis combined with enChIP (enChIP-SILAC). 
	Note: Media for the SILAC analysis can be purchased from commercial vendors. SILAC is powerful in detecting specific interactions.
	<ol>
		<li>Culture cells in SILAC heavy medium. Prepare cells cultured in SILAC light medium as a negative control. Use at least 5 &#215; 107 cells for each SILAC medium (heavy or light). For efficient labeling, add both L-Lysine-2HCl, 13C6 and L-Arginine-HCl, 13C6, 15N4 in the SILAC Heavy media. Note: At least five cell divisions are necessary to label proteins.
		<ol>
			<li>For example, culture human fibrosarcoma HT1080 cells stably expressing 3xFLAG-dCas9 and a gRNA at 37 &#176;C and 5% CO2 in DMEM media for SILAC and dialyzed fetal bovine serum with Lysine-2HCl plus L-Arginine-HCl (Light medium) or 13C6 L-Lysine-2HCl plus 13C615N4 L-Arginine-HCl (Heavy medium) (see Materials)16. Add 50 mg of light or heavy L-Lysine-2HCl and L-Arginine-HCl in 500 ml of medium.</li>
			<li>Trypsinize and replate cells before they become confluent so that cells maintain exponential growth.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Crosslinking of Cells with Formaldehyde
<ol>
	<li>For cells in suspension culture, transfer 2 &#215; 107 cells expressing 3xFLAG-TAL proteins or 3xFLAG-dCas9 plus gRNA and suspend in 30 ml of regular culture medium in a 50 ml centrifuge tube. When more cells are used, increase the volumes and amounts of reagents proportionally. For SILAC experiments, mix the cells cultured in heavy medium or light medium (5 x 107 cells each) and divide the total of 1 x 108 cells into 5 tubes containing 2 x 107 cells.</li>
	<li>Add 810 &#181;l of 37% formaldehyde to 30 ml of the cell suspension (final concentration 1%) and incubate at 37 &#176;C for 5 min. For adherent cells, directly fix cells in culture dishes by adding 810 &#181;l of 37% formaldehyde to 30 ml of culture medium.</li>
	<li>Stop the crosslinking by adding 3.1 ml of 1.25 M glycine solution (final concentration 127 mM), and incubate at RT for 10 min.</li>
	<li>Collect cells by centrifugation (300 &#215; g for 5 min at 4 &#176;C). Carefully discard the supernatant including formaldehyde and store it in an appropriate waste bottle.
	<ol>
		<li>For adherent cells, detach cells with a cell scraper and harvest in a 50 ml tube, and collect cells as described in this step.</li>
		<li>For SILAC experiments, mix the detached cells cultured in heavy medium or light medium (5 x 107 cell each), divide the total of 1 x 108 cells into 5 tubes containing 2 x 107 cells, and collect cells as described in this step.</li>
	</ol>
	</li>
	<li>Wash the cell pellet with 30 ml of PBS twice per tube. Carefully discard the supernatant including formaldehyde and store it in an appropriate waste bottle.Handle the formaldehyde waste according to chemical safety guidelines. The fixed cells can be frozen and stored at -80 &#176;C.</li>
</ol>
4. Preparation of Chromatin (per 2 x 107 cells)
<ol>
	<li>Suspend the fixed cells in 10 ml of cell lysis buffer (10 mM Tris (pH 8.0), 1 mM EDTA, 0.5% IGEPAL CA-630, and 1&#215; protease inhibitors) and incubate on ice for 10 min.</li>
	<li>Centrifuge the sample (830 &#215; g at 4 &#176;C for 8 min) and discard the supernatant carefully.</li>
	<li>Suspend the pellet in 10 ml of nuclear lysis buffer (10 mM Tris (pH 8.0), 1 mM EDTA, 0.5 M NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.5% lauroylsarcosine, and 1x protease inhibitors). Incubate on ice for 10 min and vortex every 2-3 min.</li>
	<li>Centrifuge the sample (830 &#215; g at 4 &#176;C for 8 min) and discard the supernatant carefully.</li>
	<li>Wash the pellet in 10 ml of PBS. The pellet (chromatin fraction) can be stored at -80 &#176;C after immediate freezing in liquid nitrogen.</li>
</ol>
5. Sonication of Chromatin (per 2 x 107 cells)
<ol>
	<li>Suspend the chromatin fraction in 800 &#181;l of modified lysis buffer 3 (10 mM Tris (pH 8.0), 1 mM EDTA, 150 mM NaCl, 0.1% sodium deoxycholate, 0.1% SDS, and 1x protease inhibitors) and transfer into a 1.5 ml tube.</li>
	<li>Sonicate the chromatin using a sonicator (see Materials) and the following conditions: output: 3; duty: 100% (continuous); and time: free. Perform 10-18 cycles of sonication for 10 sec and cooling on ice for 20 sec. To avoid excessive heating, incubate the samples on ice for 2 min every 5-6 cycles. To avoid foaming, keep the position of the tip of the sonication probe 0.5 cm above the bottom of the tube.</li>
	<li>Centrifuge the sample at (16,000 &#215; g at 4 &#176;C for 10 min) and transfer the supernatant into a 1.5 ml tube. The sonicated chromatin can be stored at -80 &#176;C after immediate freezing in liquid nitrogen.</li>
</ol>
6. Evaluation of Chromatin Fragmentation
<ol>
	<li>Mix 10 &#181;l of the fragmented chromatin with 85 &#181;l of distilled water.</li>
	<li>Add 4 &#181;l of 5 M NaCl and incubate at 65 &#176;C O/N.</li>
	<li>Add 1 &#181;l of 10 mg/ml RNase A and incubate at 37 &#176;C for 45 min.</li>
	<li>Add 2 &#181;l of 0.5 M EDTA (pH 8.0), 4 &#181;l of 1 M Tris (pH 6.8), and 1 &#181;l of 20 mg/ml Proteinase K, and then incubate at 45 &#176;C for 1.5 hr.</li>
	<li>Separate 10 &#181;l of the sample by electrophoresis in a 1% agarose gel that does not contain staining dyes such as SYBR Green.</li>
	<li>Stain the gel to evaluate the distribution of the lengths of the fragmented chromatin. Conditions that generate an average length of 0.5-2 kbp (range of 0.2-4 kbp) are recommended.</li>
	<li>Purify DNA from the remaining samples by phenol-chloroform extraction or by using a DNA extraction kit (see Materials). The purified DNA can be used as input DNA to estimate the yields of enChIP analyses (see 8.11).</li>
</ol>
7. Preparation of Dynabeads Conjugated with Antibodies (per 2 x 107 cells)
<ol>
	<li>Prepare two 1.5 ml tubes, one for pre-clearing using normal mouse IgG and the other for incubation with the anti-FLAG Ab. Add 150 &#181;l of Protein G-conjugated magnetic beads (see Materials) to each tube.</li>
	<li>Place the tubes on a magnet stand and wait for 3 min. Discard the supernatant by pipetting.</li>
	<li>Suspend the beads in 1 ml of PBS containing 0.01% Tween-20 (PBS-T). Place the tubes on a magnetic stand and wait for 2 min. Discard the supernatant by pipetting, and then repeat the step.</li>
	<li>Suspend the beads in 1 ml of PBS-T containing 0.1% BSA.</li>
	<li>Add 15 &#181;g of normal mouse IgG or anti-FLAG Ab and rotate at 4 &#176;C O/N.</li>
	<li>Spin down briefly, then place the tubes on a magnet stand and wait for 3 min. Discard the supernatant by pipetting.</li>
	<li>Suspend the beads in 1 ml of PBS-T. Invert the sample several times and spin down briefly. Place the tubes on a magnet stand and wait for 3 min, then discard the supernatant by pipetting. Repeat the wash two more times with PBS-T (total of three wash steps).</li>
</ol>
8. Chromatin Immunoprecipitation (per 2 x 107 cells)
<ol>
	<li>Mix the fragmented chromatin prepared in 5.3) (approximately 800 &#181;l) with one-fifth of the volume (approximately 200 &#181;l) of modified lysis buffer 3 containing 5% Triton X-100 (final concentration of 1% Triton X-100 ).</li>
	<li>Add the chromatin solution to the tube in which Protein G-conjugated magnetic beads conjugated with normal mouse IgG were prepared. Rotate at 4 &#176;C for 1 hr.</li>
	<li>Place the tube on a magnet stand and wait for 3 min.</li>
	<li>Transfer the supernatant into the tube in which Protein G-conjugated magnetic beads conjugated with anti-FLAG Ab were prepared. Rotate at 4 &#176;C O/N.</li>
	<li>Place the tube on a magnet stand and wait for 3 min. Discard the supernatant by pipetting.</li>
	<li>Suspend the beads in 1 ml of low salt buffer (20 mM Tris (pH 8.0), 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, and 1x protease inhibitors) and rotate at 4 &#176;C for 5 min. Place the tube on a magnet stand and wait for 3 min. Discard the supernatant by pipetting and repeat the wash step.</li>
	<li>Wash the beads with high salt buffer (20 mM Tris (pH 8.0), 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% SDS, and 1x protease inhibitors) twice.</li>
	<li>Wash the beads with LiCl buffer (10 mM Tris (pH 8.0), 1 mM EDTA, 250 mM LiCl, 0.5% IGEPAL CA-630, 0.5% sodium deoxycholate, and 1x protease inhibitors) twice.</li>
	<li>Wash the beads with TBS-IGEPAL CA-630 (50 mM Tris (pH 7.5), 150 mM NaCl, 0.1% IGEPAL CA-630, and 1x protease inhibitors) once.</li>
	<li>Suspend the beads in 200 &#181;l of elution buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 0.1% IGEPAL CA-630, 1x protease inhibitors, and 500 &#181;g/ml 3&#215;FLAG peptide) and incubate at 37 &#176;C for 20 min. Place the tube on a magnet stand and wait for 3 min. Transfer the supernatant into a new 1.5 ml tube and repeat the elution step.</li>
	<li>Purify DNA from small portion (e.g.,&#160;5%) of the eluate by phenol-chloroform extraction or by using a DNA extraction kit (see Materials). The purified DNA can be used for PCR with specific primer sets to estimate the yields of enChIP analyses by comparing with input DNA prepared in Step 6.7 as described previously14,16.</li>
</ol>
9. SDS-PAGE, Staining, and Mass Spectrometric Analysis
<ol>
	<li>Mix the eluate (400 &#181;l) with 1 ml of 2-propanol, 50 &#181;l of 3M sodium acetate (pH 5.2), and 5 &#181;l of 20 mg/ml glycogen. Precipitate the chromatin at -20 &#176;C O/N.</li>
	<li>Centrifuge the sample (16,000 &#215; g at 4 &#176;C for 30 min) and discard the supernatant. Rinse the pellet with 1 ml of 70% ethanol and centrifuge again (16,000 &#215; g at 4 &#176;C for 10 min). Discard the supernatant completely by pipetting.</li>
	<li>Suspend the pellet in 40 &#181;l of 2x sample buffer (125 mM Tris (pH 6.8), 10% 2-mercaptoethanol, 4% SDS, 10% sucrose, and 0.004% bromophenol blue). Vortex for 5 min to dissolve the pellet completely, and then incubate at 100 &#176;C for 30 min to denature the proteins and reverse the crosslinking.</li>
	<li>For SDS-PAGE, run 40 &#181;l of the sample on the gel until the dye reaches 1 cm below the well. Note: We usually use 4-20% gradient gels, but other % gels can be used.</li>
	<li>Stain the gel with Coomassie Brilliant Blue or silver stain.</li>
	<li>Cut the gel into five pieces (2 mm height).</li>
	<li>Perform in gel digestion and mass spectrometric analysis as described previously13-16.</li>
	<li>For SILAC experiments with 5 x 107 cells each (total of 1 x 108 cells), suspend pellet from the initial five tubes in 40 &#181;l of 2x sample buffer (it is not necessary to scale up).</li>
</ol>
10. Purification of RNA and RNA-Seq Analysis
<ol>
	<li>To purify the RNA after enChIP, add 5 U/ml of RNase Inhibitor (see Materials) to all of the buffer solutions, except modified lysis buffer 3 and the elution buffer, to which add 40 U/ml of RNase Inhibitor.</li>
	<li>Mix the eluate (400 &#181;l) with 16 &#181;l of 5 M NaCl and incubate at 65 &#176;C for 2 hr.</li>
	<li>Add 1 ml of acid-guanidinium-phenol-based reagent (see Materials) to the sample. Vortex for 15 sec and then incubate at RT for 5-15 min. Centrifuge the sample&#160;for 15 min at 12,000 &#215; g and RT.</li>
	<li>Transfer the supernatant to a new 1.5 ml tube and add 5 &#181;l of p-bromoanisole. Vortex for 15 sec and then incubate at RT for 3-5 min. Centrifuge the sample for 10 min at 12,000 &#215; g and RT.</li>
	<li>Transfer the supernatant (approximately 1 ml) into a new 2 ml tube and add 1 ml of 2-propanol. Invert the tube and incubate at RT for 10 min. Load the mixture onto a column in an RNA purification kit (see Materials). Centrifuge the column for 1 min at 12,000 &#215; g and RT.</li>
	<li>Wash the column with 400 &#181;l of RNA Wash Buffer in an RNA purification kit (see Materials).</li>
	<li>Add 80 &#181;l of DNase I cocktail (mixture of 5 &#181;l of DNase I (1 U/&#181;l), 8 &#181;l of 10&#215; DNase I Reaction Buffer, 3 &#181;l of DNase/RNase-free water, and 64 &#181;l of RNA Wash Buffer in an RNA purification kit (see Materials)) to the column. Incubate the sample at 37 &#176;C for 15 min and then centrifuge &#160;for 30 sec at 12,000 &#215; g and RT.</li>
	<li>Wash the column with 400 &#181;l of RNA PreWash Buffer in an RNA purification kit (see Materials) twice.</li>
	<li>Elute the RNA with 50 &#181;l of DNase/RNase-free water. The eluted RNA can be used for RNA sequencing.</li>
</ol>

<ol>
	<li>Zhang, X. Y., Horz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+highly+purified+satellite+DNA+containing+chromatin+from+the+mouse.">Analysis of highly purified satellite DNA containing chromatin from the mouse.</a> Nucleic Acids Res. 10, 1481-1494 (1982).</li><li>Workman, J. L., Langmore, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleoprotein+hybridization:+a+method+for+isolating+specific+genes+as+high+molecular+weight+chromatin.">Nucleoprotein hybridization: a method for isolating specific genes as high molecular weight chromatin.</a> Biochemstry. 24, 7486-7497 (1985).</li><li>Boffa, L. C., Carpaneto, E. M., Allfrey, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+active+genes+containing+CAG+repeats+by+DNA+strand+invasion+by+a+peptide+nucleic+acid.">Isolation of active genes containing CAG repeats by DNA strand invasion by a peptide nucleic acid.</a> Proc. Natl. Acad. Sci. USA. 92, 1901-1905 (1995).</li><li>Jaskinskas, A., Hamkalo, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+initial+characterization+of+primate+satellite+chromatin.">Purification and initial characterization of primate satellite chromatin.</a> Chromosome Res. 7, 341-354 (1999).</li><li>Griesenbeck, J., Boeger, H., Strattan, J. S., Kornberg, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+purification+of+specific+chromatin+segments+from+chromosomal+loci+in+yeast.">Affinity purification of specific chromatin segments from chromosomal loci in yeast.</a> Mol. Cell Biol. 23, 9275-9282 (2003).</li><li>Ghirlando, R., Felsenfeld, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+studies+on+defined+heterochromatin+fragments+support+a+30-&#181;m+fiber+having+six+nucleosomes+per+turn.">Hydrodynamic studies on defined heterochromatin fragments support a 30-&#181;m fiber having six nucleosomes per turn.</a> J. Mol. Biol. 376, 1417-1425 (2008).</li><li>D&#233;jardin, J., Kingston, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+proteins+associated+with+specific+genomic+loci.">Purification of proteins associated with specific genomic loci.</a> Cell. 136, 175-186 (2009).</li><li>Hoshino, A., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertional+chromatin+immunoprecipitation:+a+method+for+isolating+specific+genomic+regions.">Insertional chromatin immunoprecipitation: a method for isolating specific genomic regions.</a> J. Biosci. Bioeng. 108, 446-449 (2009).</li><li>Fujita, T., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+idenification+of+insulator+components+by+insertional+chromatin+immunoprecipitation.">Direct idenification of insulator components by insertional chromatin immunoprecipitation.</a> PLoS One. 6, e26109 (2011).</li><li>Fujita, T., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+isolation+of+specific+genomic+regions+by+insertional+chromatin+immunoprecipitation+(iChIP)+with+a+second-generation+tagged+LexA+DNA-binding+domain.">Efficient isolation of specific genomic regions by insertional chromatin immunoprecipitation (iChIP) with a second-generation tagged LexA DNA-binding domain.</a> Adv. Biosci. Biotechnol. 3, 626-629 (2012).</li><li>Fujita, T., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locus-specific+biochemical+epigenetics+/+chromatin+biochemistry+by+insertional+chromatin+immunoprecipitation.">Locus-specific biochemical epigenetics / chromatin biochemistry by insertional chromatin immunoprecipitation.</a> ISRN Biochem. 2013, 913273 (2013).</li><li>Fujita, T., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+isolation+of+specific+genomic+regions+retaining+molecular+interactions+by+the+iChIP+system+using+recombinant+exogenous+DNA-binding+proteins.">Efficient isolation of specific genomic regions retaining molecular interactions by the iChIP system using recombinant exogenous DNA-binding proteins.</a> BMC Mol. Biol. 15, 26 (2014).</li><li>Fujita, T., Kitaura, F., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+of+the+Thy28-MYH9+axis+in+B+cell-specific+expression+of+the+Pax5+gene+in+chicken+B+cells.">A critical role of the Thy28-MYH9 axis in B cell-specific expression of the Pax5 gene in chicken B cells.</a> PLoS One. 10, (2015).</li><li>Fujita, T., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+isolation+of+specific+genomic+regions+and+identification+of+associated+proteins+by+engineered+DNA-binding+molecule-mediated+chromatin+immunoprecipitation+(enChIP)+using+CRISPR.">Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR.</a> Biochem. Biophys. Res. Commun. 439, 132-136 (2013).</li><li>Fujita, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+telomere-associated+molecules+by+engineered+DNA-binding+molecule-mediated+chromatin+immunoprecipitation+(enChIP).">Identification of telomere-associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP).</a> Sci. Rep. 3, 3171 (2013).</li><li>Fujita, T., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+proteins+associated+with+an+IFN&#947;-responsive+promoter+by+a+retroviral+expression+system+for+enChIP+using+CRISPR.">Identification of proteins associated with an IFN&#947;-responsive promoter by a retroviral expression system for enChIP using CRISPR.</a> PLoS One. 9, e103084 (2014).</li><li>Fujita, T., Yuno, M., Okuzaki, D., Ohki, R., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+non-coding+RNAs+associated+with+telomeres+using+a+combination+of+enChIP+and+RNA+sequencing.">Identification of non-coding RNAs associated with telomeres using a combination of enChIP and RNA sequencing.</a> PLoS One. 10, e0123387 (2015).</li><li>Naito, Y., Hino, K., Bono, H., Ui-Tei, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPRdirect:+software+for+designing+CRISPR/Cas+guide+RNA+with+reduced+off-target+sites.">CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites.</a> Bioinformatics. , (2014).</li><li>Mali, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-guided+human+genome+engineering+via+Cas9.">RNA-guided human genome engineering via Cas9.</a> Science. 339, 823-826 (2013).</li><li>Esvelt, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthogonal+Cas9+proteins+for+RNA-guided+gene+regulation+and+editing.">Orthogonal Cas9 proteins for RNA-guided gene regulation and editing.</a> Nat. Methods. 10, 1116-1121 (2013).</li><li>Qi, L. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repurposing+CRISPR+as+an+RNA-guided+platform+for+sequence-specific+control+of+gene+expression.">Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.</a> Cell. 152, 1173-1183 (2013).</li><li>Wu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+binding+of+the+CRISPR+endonuclease+Cas9+in+mammalian+cells.">Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells.</a> Nat. Biotechnol. 32, 670-676 (2014).</li><li>Kuscu, C., Arslan, S., Singh, R., Thorpe, J., Adli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+reveals+characteristics+of+off-target+sites+bound+by+the+Cas9+endonuclease.">Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease.</a> Nat Biotechnol. 32, 677-683 (2014).</li><li>Cencic, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protospacer+adjacent+motif+(PAM)-distal+sequences+engage+CRISPR+Cas9+DNA+target+cleavage.">Protospacer adjacent motif (PAM)-distal sequences engage CRISPR Cas9 DNA target cleavage.</a> PLoS One. 9, 109213 (2014).</li><li>O'Green, H., Henry, I. M., Bhakta, M. S., Meckler, J. F., Segal, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+analysis+of+Cas9+binding+specificity+using+ChIP-seq+and+targeted+sequence+capture.">A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture.</a> Nucleic Acids Res. 43, 3389-3404 (2015).</li><li>de Lange, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shelterin:+the+protein+complex+that+shapes+and+safeguards+human+telomeres.">Shelterin: the protein complex that shapes and safeguards human telomeres.</a> Genes Dev. 19, 2100-2110 (2005).</li></ol>

The authors have filed a patent on enChIP (Patent name: 'Method for isolating specific genomic regions using DNA-binding molecules recognizing endogenous DNA sequences'; Patent number: PCT/JP2013/74107). H.F. is a member of the Advisory Board of Addgene.

Here, we describe the purification of specific genomic regions using engineered DNA-binding molecules such as the CRISPR system and TAL proteins, and the identification of proteins and RNAs bound to these genomic regions. Binding of engineered DNA-binding molecules to the genome may affect chromatin structure, including nucleosome positioning, and may abrogate genomic functions, as described in CRISPR interference experiments21. To avoid these potential aberrant effects, we propose specific guidelines for choosing target genomic regions. First, to avoid potential inhibition of the recruitment of RNA polymerases and transcription factors, as well as disruption of nucleosome positioning around the transcription start site, the target regions for analyses of promoter regions should be several hundred base pairs upstream of (5' to) the transcription start site. By contrast, when analyzing genomic regions with distinct boundaries, such as enhancers and silencers, genomic regions that are directly juxtaposed to these regions can be targeted because it is less likely that the binding of engineered DNA-binding molecules will affect their functions. Furthermore, it is best to avoid using target regions that are conserved among different species, because important DNA-binding molecules often bind to evolutionarily conserved regions and inhibition of their binding might disrupt the functions of the target genomic regions. In this regard, it is always necessary to check that the function of the target genomic region is maintained in the established cells used for enChIP analyses. Because multiple gRNAs can be tested easily and it is tedious and expensive to generate multiple versions of TAL or zinc-finger proteins recognizing different target genomic regions, enChIP using CRISPR is more advantageous than enChIP using other proteins.
It has been shown that dCas9 binds to off-target sites although affinity to those sites might be weaker than that to the target sites22-25. There are several ways to manage contamination of molecules bound to those off-target sites. First, the use of several, at least two, different gRNAs would be recommended. Those molecules commonly observed in enChIP using distinct gRNAs would be true positives. Second, comparison of different conditions for enChIP would be effective in cancelling contamination of non-specific molecules and molecules bound to off-target sites. Examples of those comparison sets would be (i) stimulation (-) and (+), or (ii) different cell types such as T cells vs. B cells. Finally, quantitative analysis of binding of candidate molecules should be performed to confirm their specific binding to the target sites. It is preferable to prepare cells expressing only dCas9 but not gRNA as a negative control.
Using enChIP analyses, we were able to successfully identify a number of known and novel molecules interacting with specific genomic regions (Tables 1-3)14-17. However, this technique failed to detect some other known proteins interacting with these regions. For example, STAT1 reportedly associates with the IRF-1 promoter upon IFN&#947; stimulation8, but our enChIP-SILAC analysis did not detect STAT1 as a protein induced to interact with this genomic region16. In addition, in the enChIP-MS analysis of telomeres, we did not detect shelterin proteins consisting of TRF-1 and TRF-215, which have been shown to interact with telomeres26. There are a few potential reasons for these discrepancies. First, the stoichiometry of binding of Stat1 to the IRF-1 promoter might be very low. It is reasonable that enChIP-MS, including enChIP-SILAC, detects proteins that are more abundantly associated with target genomic regions; hence, the analysis of more cells might be necessary to detect these proteins. Increases in the sensitivities of MS instruments would also contribute to the efficient detection of proteins with low stoichiometric binding. Second, some proteins, possibly including Stat1 and shelterins, might be difficult targets for MS analyses. Third, in our analysis of telomere-binding proteins15, the 3&#215;FLAG-TAL proteins recognizing telomeres (3&#215;FN-Tel-TAL) might have blocked the binding of shelterins to telomeres in a competitive fashion.
In contrast to the relative difficulty of detecting transcription factors binding to specific genomic regions using enChIP, we successfully identified epigenetic regulators such as histone modification enzymes using enChIP analyses. The success of this technique may be due to the fact that epigenetic regulators bind to a broad range of genomic regions; hence, more proteins per genomic region are available for MS. Because epigenetic regulators are increasingly recognized as important targets for drugs against intractable diseases such as cancer, enChIP would be a useful tool for the identification of epigenetic drug targets.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of genomic functions such as transcription and epigenetic regulation. Although several techniques have been developed for biochemical analysis of specific genomic regions1-7, they are not used widely at this stage because of their intrinsic problems such as limited application (e.g., only for high-copy number loci or loci with repeats) and too much time and efforts required.
In order to perform biochemical analysis of specific genomic regions easily, we have developed two locus-specific chromatin immunoprecipitation (ChIP) technologies, namely insertional ChIP (iChIP)8-13 and engineered DNA-binding molecule-mediated ChIP (enChIP)14-17. In iChIP, a locus of interest is tagged by inserting recognition sequences of an exogenous DNA-binding protein such as LexA. The locus is then isolated by affinity purification using the tagged DNA-binding protein. In enChIP, engineered DNA-binding molecules, such as zinc-finger proteins, transcription activator-like (TAL) proteins, and clustered regularly interspaced short palindromic repeats (CRISPR) complexes, are used to tag a locus of interest (Figure 1). Subsequently, the genomic region is isolated by affinity purification of the tagged DNA-binding molecules.
One of the advantages of enChIP over iChIP is that insertion of recognition sequences of an exogenous DNA-binding protein is not necessary. Targeting of loci using CRISPR complexes consisting of a catalytically inactive form of Cas9 (dCas9) and a guide RNA (gRNA) is much easier than targeting of these regions by iChIP or enChIP using TAL and zinc-finger proteins. Here, we describe a step-by-step protocol for enChIP combined with mass spectrometry and RNA sequencing (RNA-Seq) to identify locus-associated proteins and RNAs, respectively.

<table><tbody><tr><td>gBlock synthesis service</td><td>Life Technologies</td><td>Gene Synthesis by GeneArt</td><td></td></tr><tr><td>gBlock synthesis service</td><td>IDT (Integrated DNA Technologies)</td><td>gBlocks Gene Fragments</td><td></td></tr><tr><td>pSIR-neo</td><td>Addgene</td><td>51128</td><td></td></tr><tr><td>pSIR-GFP</td><td>Addgene</td><td>51134</td><td></td></tr><tr><td>pSIR-DsRed-Express2</td><td>Addgene</td><td>51135</td><td></td></tr><tr><td>pSIR-hCD2</td><td>Addgene</td><td>51143</td><td></td></tr><tr><td>TAL synthesis service</td><td>Life Technologies</td><td>GeneArt Precision TALs</td><td></td></tr><tr><td>3xFLAG-dCas9/pCMV-7.1</td><td>Addgene</td><td>47948</td><td></td></tr><tr><td>3&amp;times;FLAG-dCas9/pMXs-puro</td><td>Addgene</td><td>51240</td><td></td></tr><tr><td>3&amp;times;FLAG-dCas9/pMXs-IG</td><td>Addgene</td><td>51258</td><td></td></tr><tr><td>3&amp;times;FLAG-dCas9/pMXs-I2</td><td>Addgene</td><td>51259</td><td></td></tr><tr><td>3&amp;times;FLAG-dCas9/pMXs-neo</td><td>Addgene</td><td>51260</td><td></td></tr><tr><td>anti-FLAG M2 Ab</td><td>Sigma-Aldrich</td><td>F1804</td><td></td></tr><tr><td>FITC-conjugated anti-FLAG M2</td><td>Sigma-Aldrich</td><td>F4049</td><td></td></tr><tr><td>DMEM medium for SILAC</td><td>Life Technologies</td><td>89985</td><td>Other medium can be purchased from Life Technologies</td></tr><tr><td>Dialyzed FBS for SILAC</td><td>Life Technologies</td><td>89986</td><td></td></tr><tr><td>L-Lysine-2HCl for SILAC</td><td>Life Technologies</td><td>89987</td><td>For Light medium</td></tr><tr><td>L-Arginine-HCl for SILAC</td><td>Life Technologies</td><td>89989</td><td>For Light medium</td></tr><tr><td>L-Lysine-2HCl, 13C6 for SILAC</td><td>Life Technologies</td><td>89988</td><td>For Heavy medium</td></tr><tr><td>L-Arginine-HCl, 13C6, 15N4 for SILAC</td><td>Life Technologies</td><td>89990</td><td>For Heavy medium</td></tr><tr><td>Complete, mini, EDTA-free</td><td>Roche Diagnostics</td><td>4693159</td><td></td></tr><tr><td>Ultrasonic Disruptor UD-201</td><td>Tomy Seiko</td><td></td><td></td></tr><tr><td>ChIP DNA Clean &amp;amp; Concentrator</td><td>Zymo Research</td><td>D5205</td><td></td></tr><tr><td>Dynabeads-Protein G</td><td>Life Technologies</td><td>DB10004</td><td></td></tr><tr><td>RNasin Plus RNase Inhibitor</td><td>Promega</td><td>N2611</td><td></td></tr><tr><td>Isogen II</td><td>Nippon Gene</td><td>311-07361</td><td></td></tr><tr><td>Direct-zol RNA Miniprep kit</td><td>Zymo Research</td><td>R2050</td><td></td></tr><tr><td>LTQ Orbitrap Velos</td><td>Thermo Fisher Scientific</td><td></td><td>A component of a nanoLC-MS/MS system for MS analysis</td></tr><tr><td>nanoLC</td><td>Advance, Michrom Bioresources</td><td></td><td>A component of a nanoLC-MS/MS system for MS analysis</td></tr><tr><td>HTC-PAL autosampler</td><td>CTC Analytics</td><td></td><td>A component of a nanoLC-MS/MS system for MS analysis</td></tr></tbody></table>

isolation of specific genomic regions and identification of associated molecules by enchip

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

Overall, 1-30% of target genomic regions can be purified using enChIP. Figure 3 includes representative data showing the yields of enChIP analyses targeting telomeres and the promoter of the interferon (IFN) regulatory factor-1 (IRF-1) gene. As examples of typical results, Table 1 lists the proteins associated with the IRF-1 promoter in an IFN&#947;-specific manner identified by enChIP-SILAC, Table 2 lists the telomere-binding proteins identified by enChIP combined with mass spectrometry (enChIP-MS), and Table 3 lists the RNAs associated with telomeres identified by enChIP-RNA-Seq.
<img alt="Figure 1" src="/files/ftp_upload/53478/53478fig1.jpg" /> 
Figure 1. Overview of enChIP. enChIP using CRISPR (A, B) and TAL (C, D, E) is shown. A locus of interest is tagged with engineered DNA-binding molecules such as a TAL protein or the CRISPR system consisting of a catalytically inactive form of Cas9 (dCas) and guide RNA (gRNA). The molecular interactions are fixed with formaldehyde or other crosslinkers, if necessary. Subsequently, fixed chromatin is fragmented by sonication or enzymatic digestion. The tagged genomic regions are purified by affinity purification. Finally, crosslinking is reversed, and interacting molecules (genomic regions, RNAs, and proteins) are identified using next generation sequencing and mass spectrometry. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53478/53478fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53478/53478fig2.jpg" /> 
Figure 2. Sequence of gBlock. The U6 promoter (in black), the G nucleotide before the guide sequence (in blue), the guide sequence (gRNA spacer) (in red), the scaffold sequence (in green), and the terminator sequence (in orange) are shown.
<img alt="Figure 3" src="/files/ftp_upload/53478/53478fig3.jpg" /> 
Figure 3. Yields of representative enChIP analyses. (A) Percentage inputs for the target IRF-1 locus and the non-target Sox2 locus. (B) Percentage inputs for the target telomeres and non-target &#947;-satellites. The figures have been adapted and modified from previous publications15,16.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Categories</td>
			<td>Proteins</td>
		</tr>
		<tr>
			<td>Transcription</td>
			<td>DDX1, PARP1, CKAP4, Pescadillo homolog, PUR&#946;, 
			activated RNA polymerase II transcriptional activator p15, 
			BTF3, Myb-binding protein 1A</td>
		</tr>
		<tr>
			<td>Histone&#160; deacetylation, 
			corepressor components</td>
			<td>RBBP4, PA2G4, TBL3</td>
		</tr>
		<tr>
			<td>Acetyltransferase</td>
			<td>Protein arginine N-methyltransferase 1</td>
		</tr>
		<tr>
			<td>DNA topoisomerase</td>
			<td>DNA topoisomerase 2&#945;</td>
		</tr>
		<tr>
			<td>Histones</td>
			<td>Histone H2A.Z, histone H3.2</td>
		</tr>
	</tbody>
</table>
Table 1: Examples of proteins associated with the human IRF-1 promoter region in an IFN&#947;-specific manner identified by enChIP-SILAC. The table has been adapted from a previous publication16.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Categories</td>
			<td>Proteins</td>
		</tr>
		<tr>
			<td>Mammalian telomere-binding proteins</td>
			<td>PML, RPA, CDK1, PARP1, PCBP1</td>
		</tr>
		<tr>
			<td>Telomere-binding proteins in yeast 
			or other organisms</td>
			<td>IMP4</td>
		</tr>
		<tr>
			<td>Proteins interacting with telomere-binding 
			proteins [associated telomere-binding protein]</td>
			<td>DNA polymerase &#945; (POLA1) [Cdc13p], 
			ARMC6 [TRF2], CTBP1 [FoxP2-POT1], 
			exportin-5 [TERT], GNL3L [TRF1], 
			exportin-1 [TERT], 14-3-3 [TERT]</td>
		</tr>
		<tr>
			<td>Proteins localizing to heterochromatin</td>
			<td>BEND3</td>
		</tr>
		<tr>
			<td>Proteins regulating epigenetic marks</td>
			<td>KDM5C</td>
		</tr>
		<tr>
			<td>Proteins whose mutations affect telomere function</td>
			<td>DNA polymerase &#945; (POLA1), HAT1, Nup133, 
			CDK7, DPOE1, PRDX1, TYSY, 
			glutamate-cysteine ligase, glutaredoxin, SMRC1</td>
		</tr>
	</tbody>
</table>
Table 2: Examples of proteins associated with mouse telomeres identified by enChIP-MS. The table has been adapted from a previous publication15.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Categories</td>
			<td>RNAs</td>
		</tr>
		<tr>
			<td>Telomerase components</td>
			<td>Terc, Rmrp</td>
		</tr>
		<tr>
			<td>Telomeric RNAs</td>
			<td>TERRAs</td>
		</tr>
		<tr>
			<td>scaRNAs</td>
			<td>Scarna6, Scarna10, Scarna13, Scarna2</td>
		</tr>
		<tr>
			<td>H/ACA snoRNAs</td>
			<td>Snora23, Snora74a, Snora73b, Snora73a</td>
		</tr>
		<tr>
			<td>C/D snoRNAs</td>
			<td>Snord17, Snord15a, Snord118</td>
		</tr>
		<tr>
			<td>lncRNA</td>
			<td>Neat1</td>
		</tr>
	</tbody>
</table>
Table 3: Examples of RNAs associated with mouse telomeres identified by enChIP-RNA-Seq. The table has been adapted from a previous publication17.

Watch this Scientific Journal Video about Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP at JoVE.com

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

isolation-specific-genomic-regions-identification-associated

Nanyang Technological University

Research

JoVE Journal

Biology

10.4K Views.  Osaka University. The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

Video: Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

Genetics

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo)

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) is widely used to identify genome-wide binding locations of DNA-binding proteins. However, the ChIP-seq method is limited by its heterogeneity in length of sonicated DNA fragments and non-specific background DNA, resulting in low mapping resolution and uncertainty in DNA-binding sites. To overcome these limitations, the combination of ChIP with exonuclease digestion (ChIP-exo) utilizes 5&#8217; to 3&#8217; exonuclease digestion to trim the heterogeneously sized immunoprecipitated DNA to the protein-DNA crosslinking site. Exonuclease treatment also eliminates non-specific background DNA. The library-prepared and exonuclease-digested DNA can be sent for high-throughput sequencing. The ChIP-exo method allows for near base-pair mapping resolution with greater detection sensitivity and reduced background signal. An optimized ChIP-exo protocol for mammalian cells and next-generation sequencing is described below.

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease ChIP-Exo

Precise determination of protein-binding locations across the genome is important for understanding gene regulation. Here we describe a&#160;genomic mapping method that treats chromatin-immunoprecipitated DNA with exonuclease digestion (ChIP-exo) followed by high-throughput sequencing. This method detects protein-DNA interactions with near base-pair mapping resolution and high signal-to-noise ratio in mammalian neurons.

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled ...

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

Advancing iChip Method for Culturing Novel Microorganisms

Isolation of Soil Microorganisms Using iChip Technology

The iChip isolation technique uses an in-situ isolation device that increases the cultivability of previously unculturable microorganisms. Microorganisms are an important source of novel chemistries and potentially bioactive molecules. However, only 1% of environmental microorganisms can be cultured using conventional laboratory methods. With the rise in antimicrobial resistance, finding new drugs to combat infections and diseases is of foremost importance, and a critical method to finding new molecules is the discovery of new microorganisms. By incubating colonies of soil microorganisms in the wells of a 96-well plate, sealed with a semipermeable membrane and incubated on top of soil, the microbes are in contact with water and growth factors from the soil, allowing for the isolation of novel microbes in a laboratory setting. After a period of domestication in an iChip, microorganisms can potentially be subcultured onto conventional media and used for further study. This device is valuable to bioactive molecule discovery and soil microbiome research and has been used previously in both applications.

The iChip technique makes use of an inexpensive and simple in-situ isolation device that increases the rates of novel microorganism discovery from the soil. Novel microorganisms can be used for further study pertaining to the soil microbiome or natural product discovery, amongst other applications.

The iChip isolation technique uses an in-situ isolation device that increases the cultivability of previously unculturable microorganisms. Microorganisms ...

Biochemistry

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. 
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. 
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.

Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks.
ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs&#39; position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome.
Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final&#160;evaluation in the therapeutically relevant setting of interest.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among ...

Bioengineering

DNA-affinity-purified Chip (DAP-chip) Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of limited use in exploring bacterial two component regulatory systems with uncharacterized activation conditions. Such systems regulate transcription only when activated in the presence of unique signals. Since these signals are often unknown, the in vitro microarray based method described in this video article can be used to determine gene targets and binding sites for response regulators. This DNA-affinity-purified-chip method may be used for any purified regulator in any organism with a sequenced genome. The protocol involves allowing the purified tagged protein to bind to sheared genomic DNA and then affinity purifying the protein-bound DNA, followed by fluorescent labeling of the DNA and hybridization to a custom tiling array. Preceding steps that may be used to optimize the assay for specific regulators are also described. The peaks generated by the array data analysis are used to predict binding site motifs, which are then experimentally validated. The motif predictions can be further used to determine gene targets of orthologous response regulators in closely related species. We demonstrate the applicability of this method by determining the gene targets and binding site motifs and thus predicting the function for a sigma54-dependent response regulator DVU3023 in the environmental bacterium Desulfovibrio vulgaris Hildenborough.

DNA-affinity-purified Chip DAP-chip Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

This video article describes an in vitro microarray based method to determine the gene targets and binding sites for two component system response regulators.

In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of ...

Single-Cell Factor Localization on Chromatin using Ultra-Low Input Cleavage Under Targets and Release using Nuclease

Determining the binding locations of a protein on chromatin is essential for understanding its function and potential regulatory targets. Chromatin Immunoprecipitation (ChIP) has been the gold standard for determining protein localization for over 30 years and is defined by the use of an antibody to pull out the protein of interest from sonicated or enzymatically digested chromatin. More recently, antibody tethering techniques have become popular for assessing protein localization on chromatin due to their increased sensitivity. Cleavage Under Targets &amp; Release Under Nuclease (CUT&amp;RUN) is the genome-wide derivative of Chromatin Immunocleavage (ChIC) and utilizes recombinant Protein A tethered to micrococcal nuclease (pA-MNase) to identify the IgG constant region of the antibody targeting a protein of interest, therefore enabling site-specific cleavage of the DNA flanking the protein of interest. CUT&amp;RUN can be used to profile histone modifications, transcription factors, and other chromatin-binding proteins such as nucleosome remodeling factors. Importantly, CUT&amp;RUN can be used to assess the localization of either euchromatic- or heterochromatic-associated proteins and histone modifications. For these reasons, CUT&amp;RUN is a powerful method for determining the binding profiles of a wide range of proteins. Recently, CUT&amp;RUN has been optimized for transcription factor profiling in low populations of cells and single cells and the optimized protocol has been termed ultra-low input CUT&amp;RUN (uliCUT&amp;RUN). Here, a detailed protocol is presented for single-cell factor profiling using uliCUT&amp;RUN in a manual 96-well format.

CUT&#38;RUN and its variants can be used to determine protein occupancy on chromatin. This protocol describes how to determine protein localization on chromatin using single-cell uliCUT&#38;RUN.

Determining the binding locations of a protein on chromatin is essential for understanding its function and potential regulatory targets. Chromatin ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

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