We thank Drs. L. Vales, D. Ozata and H. Mou for revision of the manuscript. The D.R. Lab is supported by the Howard Hughes Medical Institute and the National Institutes of Health (R01CA199652 and R01NS100897).

Generation of inducible EED rescue mESCs
1. Cell culture
<ol>
	<li>Use feeder-free C57BL/6 mouse embryonic stem cells (mESCs) possessing a stably integrated CreERT2 transgene, which can translocate to the nucleus upon administration of 4-Hydroxytamoxifen (4-OHT)13.</li>
	<li>Grow mESCs in conventional ESC medium14,15, supplemented with 1000 U/mL LIF, 1 &#956;M ERK inhibitor PD0325901 and 3 &#956;M GSK3 inhibitor CHIR99021. For conventional ESC medium, use knockout DMEM containing 15% fetal bovine serum (FBS), 2 mM&#160;L-glutamine, 1X penicillin/streptomycin and 0.1 mM 2-mercaptoethanol.</li>
	<li>Use plates coated with 0.1% gelatin solution for culturing mESCs.</li>
</ol>
2. Generation of clonal EED knockout (KO) mESCs
<ol>
	<li>Design guide RNAs (gRNAs) to delete Exon 10 and Exon 11 of endogenous copy of EED in mESCs using the CRISPR design tool in Benchling16. EED-KO-gRNA-1 targets the intron immediately upstream of exon 10 and EED-KO-gRNA-2 targets the intron immediately downstream of Exon 11. The simultaneous application of these gRNAs deletes both Exon 10 and Exon 11 by non-homologous end joining (NHEJ).</li>
	<li>Clone gRNAs into pSpCas9(BB)-2A-GFP (PX458) using instructions in Ran et al., 201317.</li>
	<li>In a 6-well plate format, transfect 2 x 105 mESCs with 1 &#956;g of each EED-KO-gRNA-1 and EED-KO-gRNA-2 (see Table of Materials) using transfection reagent by following the manufacturer&#39;s instructions. Change the media 24 h after transfection.</li>
	<li>Two days after the transfection, isolate GFP positive cells using Fluorescence-activated cell sorting (FACS). Expect the transfection efficiency to vary around 10-30 %. Sort around 5 x 105 cells to capture sufficient GFP positive cells for plating.</li>
	<li>Plate the isolated GFP positive mESCs into 15 cm plates pre-coated with 0.1% gelatin (10-20 x103 cells per plate) for colony picking.</li>
	<li>Grow the cells in ESC medium for about one week until single colonies are visible. Change the media every 2 days.</li>
	<li>Pick a minimum of 48 colonies using 20 &#956;L micropipette tip. Scrape over the colony while aspirating into the micropipette tip. Do not break up the colony into single cells. Transfer the colony into an accutase-containing (20 &#956;L) 96 well plate.</li>
	<li>Incubate the colonies for 10 min at 37 &#176;C until all cells are dissociated.</li>
	<li>Add 200 &#956;L of ESC media into each well using multichannel pipette.</li>
	<li>Mix well and plate the cells into two separate 96 well plates using multichannel pipette.</li>
	<li>Use one of the 96 well plates for genotyping and keep the other growing until genotyping is concluded. Use DNA extraction solution to extract DNA from the 96 well plate by following the manufacturer&#39;s instructions.</li>
	<li>Use genotyping primers Gnt_EED-KO-up and Gnt_EED-KO_down, which span the deleted site and perform genotyping PCR with Taq DNA polymerase following the manufacturer&#8217;s instructions. 
	NOTE: Other types of DNA polymerases can also be used for genotyping, however, Taq DNA polymerase offers convenience as the PCR reaction can directly be loaded on a gel when its colored reaction buffer is used.
	<ol>
		<li>Observe a DNA product of lower molecular weight in cells with a homozygous deletion relative to the wild-type (WT) case. 
		NOTE: To generate PRC2 null cells, homozygous deletion of exons 10 and 11 is necessary to destabilize and degrade EED, an essential subunit of core PRC213. The CRISPR targeting efficiency is around 10 %.</li>
	</ol>
	</li>
	<li>Validate the deletion of EED exon 10 and 11 and the loss of EED protein by Sanger sequencing and Western blotting, respectively.</li>
	<li>Confirm the depletion of EED and H3K27me2/me3 from chromatin in EED KO cells by ChIP-seq using antibodies against H3K27me2/me3 and EED. Use the protocol given in Oksuz et al., 201715 for ChIP-seq experiments.</li>
</ol>
3. Engineering the EED knockout mESCs to harbor Cre-ERT2 based inducible EED expression
<ol>
	<li>Design gRNA (EED-gRNA-inducible) to introduce a cut within the intron following exon 9 of EED using the CRISPR design tool in Benchling16 (see Table of Materials).</li>
	<li>Clone gRNAs into pSpCas9(BB)-2A-GFP (PX458) using instructions in Ran et al., 201317.</li>
	<li>Design a donor template DNA that comprises EED cDNA sequence after exon 9 and a C-terminal Flag-HA tag upstream of a T2A-GFP sequence, all in reverse orientation with respect to the endogenous gene sequence.
	<ol>
		<li>Flank the cassette with a splice-acceptor and a polyadenylation sequence nested between heterologous inverted loxP sites (lox66 and lox71)18.</li>
		<li>Include at least 500 bp of homology arms from each end. Split the donor template into 2 segments of gBlocks gene fragments (see gBlock-1, gBlock-2-WT &#34;https://benchling.com/s/seq-l2LLlWNEnLrfGXcbdCxI&#34;&#160;and gBlock-2-cage-mutant &#34;https://benchling.com/s/seq-n8eiZCB2XAkOuzzpv6qM&#34;) and assemble them into PCR Blunt vector using Gibson cloning following the manufacturer&#39;s instructions. 
		NOTE: gblock-2 contains an aromatic residue (Y365) within the cage of EED that is important for interaction with H3K27me312. gBlock-2-Wt contains wild type residue, whereas gblock-2-cage-mutant contains the cage-mutant of EED (Y365A), incapable of binding to H3K27me3. Inducible WT EED rescue is denoted as i-WT-r and inducible cage-mutant EED rescue is denoted as i-MT-r for convenience.</li>
	</ol>
	</li>
	<li>In a 6-well plate format, transfect 2 x 105 mESCs with 1 &#956;g of the gRNA (EED-gRNA-inducible) and 1 &#956;g of the Donor templates (i-WT-r or i-MT-r) using transfection reagent and isolate GFP positive cells using FACS.</li>
	<li>Follow steps 2.3-2.11 to isolate individual colonies ready for genotyping for successful targeting.</li>
	<li>Use genotyping primers Inducible_Genotype-FW-1 and Inducible_Genotype-REV-1, which span the inserted cassette and perform genotyping PCR using Taq DNA polymerase. 
	NOTE: The targeting efficiency for CRISPR is around 10 %.
	<ol>
		<li>Confirm correct integration of the cassette by PCR using Inducible_Genotype-FW-2 and Inducible_Genotype-REV-2 primers, which are outside of the homology arms. 
		NOTE: Homozygous integration of the cassette is necessary to accomplish efficient rescue of EED. Note that cells with homozygous integration will produce a single large DNA product as compared to WT EED, which will produce a short product.</li>
	</ol>
	</li>
	<li>Confirm the integration of the cassette by Sanger sequencing.</li>
	<li>Confirm flipping of the cassette and the expression of EED and other PRC2 core components (e.g., EZH2 and SUZ12) upon 4-OHT administration by Western blotting.</li>
	<li>Confirm the expression of T2A-GFP and percentage of flip by flow cytometry. Note that expression of GFP is indicative of flipping of the cassette and the expression of EED.</li>
</ol>
4. Following nucleation and spreading of PRC2 activity on chromatin
<ol>
	<li>Confirm that i-WT-r and i-MT-r mESCs do not have leaky expression of GFP by flow cytometry. In the case of leaky expression of GFP, isolate GFP-negative cells by FACS before starting the experiment.</li>
	<li>Expand the i-WT-r and i-MT-r mESCs into five 15 cm plates (5x 106 cells per plate).</li>
	<li>Induce expression of WT or cage-mutant EED by administration of 0.5 &#956;M 4-OHT for 0 h, 12 h, 24 h, 36 h and 8 days (one 15 cm plate per condition). Change the media after 12 h for treatments longer than 12 h. Isolate the successfully recombined cells by FACS using GFP. Adjust the time of the treatments such that all of the conditions are collected at once.</li>
	<li>Perform ChIP-seq for H3K27me2, H3K27me3 to investigate their temporal deposition to chromatin in response to re-expression of WT or cage-mutant EED. Use ChIP-seq protocol including library preparation detailed in Oksuz et al., 201715.</li>
	<li>Use spike-in control in each sample to allow for quantitative comparison among different time points19.
	<ol>
		<li>For spike-in control, use chromatin from Drosophila melanogaster (in a 1:50 ratio to the mESC-derived chromatin) as well as Drosophila specific H2Av antibody (1 &#956;L of H2Av antibody per 4 &#956;g of Drosophila chromatin according to manufacturer&#8217;s instructions) in each sample.</li>
		<li>Prepare the chromatin from Drosophila in a manner similar to that from mESCs using the protocol detailed in Oksuz et al., 201715.</li>
	</ol>
	</li>
	<li>Map the sequence reads for ChIP-seq to mm10 genome with Bowtie 2 using default parameters20.</li>
	<li>Normalize the mouse ChIP-seq reads to spike-in Drosophila read counts21. Calculate the spike-in normalization factor using the following formula: 1 x 106/unique Drosophila read counts. Expect to get around 1 x 106 Drosophila read counts and 20 x 106 mouse read counts per experiment.
	<ol>
		<li>Do not include the Drosophila chromatin when sequencing the input samples.</li>
	</ol>
	</li>
	<li>Use genomecov tool from bedtools to convert bam file into bedgraph22. Next, convert the bedgraph into bigwig file using bedGraphToBigWig tool from USCS23,24. See the following script: 
	gen=&#34;path to mm10 genome&#34; 
	chr=&#34;path to mm10 chromosome sizes&#34; 
	inp_bam=&#34;path to input bam file &#34; 
	multiply=&#34;calculate the spike-in normalization factor&#34; 
	bedtools genomecov -bg -scale $multiply -ibam $inp_bam -g $gen &#62; output.bedGraph 
	sort -k1,1 -k2,2n output.bedGraph &#62; output_sorted.bedGraph 
	bedGraphToBigWig output_sorted.bedGraph $chr output.bw</li>
	<li>Visualize the ChIP-seq read densities by uploading the bigwig files on the USCS genome browser24.</li>
</ol>
5. Monitoring emergence and growth of the H3K27me3 foci in the mESCs nuclei
<ol>
	<li>Plate 1x 104 mESCs into 8-well chamber slides pre-coated with 0.1% gelatin.</li>
	<li>Next day, start performing consecutive 4-OHT (0.5 &#956;M) inductions to collect the cells expressing EED for indicated time points (0 h, 12 h, 24 h and 36 h). Change the media after 12 h for treatments longer than 12 h.</li>
	<li>Fix the mESCs with 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature (RT) for 10 min.</li>
	<li>Permeabilize the cells with PBS/0.25% Triton X-100 at RT for 30 min.</li>
	<li>Block the cells with PBS/5% donkey serum/0.1% Triton X-100 (blocking buffer) at RT for 30 min.</li>
	<li>Dilute H3K27me3 primary antibody 1:500 in the blocking buffer and add onto the cells.</li>
	<li>Incubate the cells with primary antibody overnight at 4 &#176;C.</li>
	<li>Next day, perform 3 washes with PBS/0.1% Triton X-100.</li>
	<li>Dilute secondary antibody (Alexa fluor 595) 1:1000 in the blocking buffer and add onto cells.
	<ol>
		<li>Perform all incubations in dark in the following steps as secondary antibodies conjugated with Alexa Fluor are light sensitive.</li>
	</ol>
	</li>
	<li>Incubate the secondary antibody for 2 h at RT.</li>
	<li>Perform 3 washes with PBS/0.1% Triton X-100.</li>
	<li>Dilute DAPI (1 mg/mL) 1:4000 with PBS/0.1% Triton X-100 and add onto cells.</li>
	<li>Incubate the cells with DAPI for 10 min at RT.</li>
	<li>Mount the cells with Aqua mount.</li>
	<li>Image the cells with confocal microscopy at 63X magnification.</li>
	<li>Process and pseudo-color the images using a distribution of ImageJ, Fiji25.&#160;</li>
</ol>

<ol>
	<li>Bonev, B., Cavalli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+function+of+the+3D+genome.">Organization and function of the 3D genome.</a> Nature Reviews Genetics. 17 (11), 661-678 (2016).</li><li>Dixon, J. 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=Topological+domains+in+mammalian+genomes+identified+by+analysis+of+chromatin+interactions.">Topological domains in mammalian genomes identified by analysis of chromatin interactions.</a> Nature. 485 (7398), 376-380 (2012).</li><li>Pope, B. D., 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=Topologically+associating+domains+are+stable+units+of+replication-timing+regulation.">Topologically associating domains are stable units of replication-timing regulation.</a> Nature. 515 (7527), 402-405 (2014).</li><li>Carelli, F. N., Sharma, G., Broad Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+Domains:+An+Important+Facet+of+Genome+Regulation.">Chromatin Domains: An Important Facet of Genome Regulation.</a> Bioessays. 39 (12), (2017).</li><li>Margueron, R., Reinberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Polycomb+complex+PRC2+and+its+mark+in+life.">The Polycomb complex PRC2 and its mark in life.</a> Nature. 469 (7330), 343-349 (2011).</li><li>Holoch, D., Margueron, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+Regulating+PRC2+Recruitment+and+Enzymatic+Activity.">Mechanisms Regulating PRC2 Recruitment and Enzymatic Activity.</a> Trends in Biochemical Sciences. 42 (7), 531-542 (2017).</li><li>Cao, 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=Role+of+histone+H3+lysine+27+methylation+in+polycomb-group+silencing.">Role of histone H3 lysine 27 methylation in polycomb-group silencing.</a> Science. 298 (5595), 1039-1043 (2002).</li><li>Kuzmichev, A., Nishioka, K., Erdjument-Bromage, H., Tempst, P., Reinberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+methyltransferase+activity+associated+with+a+human+multiprotein+complex+containing+the+enhancer+of+zeste+protein.">Histone methyltransferase activity associated with a human multiprotein complex containing the enhancer of zeste protein.</a> Genes and Development. 16 (22), 2893-2905 (2002).</li><li>Czermin, B., Melfi, R., McCabe, D., Seitz, V., Imhof, A., Pirrotta, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+enhancer+of+Zeste/ESC+complexes+have+a+histone+H3+methyltransferase+activity+that+marks+chromosomal+Polycomb+sites.">Drosophila enhancer of Zeste/ESC complexes have a histone H3 methyltransferase activity that marks chromosomal Polycomb sites.</a> Cell. 111 (2), 185-196 (2002).</li><li>M&#252;ller, J., 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=Histone+methyltransferase+activity+of+a+Drosophila+Polycomb+group+repressor+complex.">Histone methyltransferase activity of a Drosophila Polycomb group repressor complex.</a> Cell. 111 (2), 197-208 (2002).</li><li>Ferrari, K. J., 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=Polycomb-Dependent+H3K27me1+and+H3K27me2+Regulate+Active+Transcription+and+Enhancer+Fidelity.">Polycomb-Dependent H3K27me1 and H3K27me2 Regulate Active Transcription and Enhancer Fidelity.</a> Molecular Cell. 53 (1), 49-62 (2014).</li><li>Margueron, 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=Role+of+the+polycomb+protein+EED+in+the+propagation+of+repressive+histone+marks.">Role of the polycomb protein EED in the propagation of repressive histone marks.</a> Nature. 461 (7265), 762-767 (2009).</li><li>Oksuz, O., 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=Capturing+the+Onset+of+PRC2-Mediated+Repressive+Domain+Formation.">Capturing the Onset of PRC2-Mediated Repressive Domain Formation.</a> Molecular cell. 70 (6), 1149-1162 (2018).</li><li>Tee, W. W., Shen, S. S., Oksuz, O., Narendra, V., Reinberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erk1/2+activity+promotes+chromatin+features+and+RNAPII+phosphorylation+at+developmental+promoters+in+mouse+ESCs.">Erk1/2 activity promotes chromatin features and RNAPII phosphorylation at developmental promoters in mouse ESCs.</a> Cell. 156 (4), 678-690 (2014).</li><li>Oksuz, O., Tee, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+chromatin+modifications+in+response+to+ERK+signaling.">Probing chromatin modifications in response to ERK signaling.</a> Methods in Molecular Biology. 1487, 289-301 (2017).</li><li>Benchling for Academics. Benchling Available from: <a target="_blank" href="https://benchling.com">https://benchling.com</a> (2018)</li><li>Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+using+the+CRISPR-Cas9+system.">Genome engineering using the CRISPR-Cas9 system.</a> Nature Protocols. 8 (11), 2281-2308 (2013).</li><li>Zhang, Z., Lutz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cre+recombinase-mediated+inversion+using+lox66+and+lox71:+method+to+introduce+conditional+point+mutations+into+the+CREB-binding+protein.">Cre recombinase-mediated inversion using lox66 and lox71: method to introduce conditional point mutations into the CREB-binding protein.</a> Nucleic acids research. 30 (17), 90 (2002).</li><li>Orlando, D. A., 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=Quantitative+ChIP-Seq+normalization+reveals+global+modulation+of+the+epigenome.">Quantitative ChIP-Seq normalization reveals global modulation of the epigenome.</a> Cell reports. 9 (3), 1163-1170 (2014).</li><li>Langmead, B., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+gapped-read+alignment+with+Bowtie+2.">Fast gapped-read alignment with Bowtie 2.</a> Nature Methods. 9 (4), 357-359 (2012).</li><li>Descostes, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIPSeqSpike:+ChIP-Seq+data+scaling+according+to+spike-in+control.">ChIPSeqSpike: ChIP-Seq data scaling according to spike-in control.</a> R package version 1.2.1. , (2019).</li><li>Quinlan, A. R., Hall, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BEDTools:+a+flexible+suite+of+utilities+for+comparing+genomic+features.">BEDTools: a flexible suite of utilities for comparing genomic features.</a> Bioinformatics. 26 (6), 841-842 (2010).</li><li>Kent, W. J., Zweig, A. S., Barber, G., Hinrichs, A. S., Karolchik, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BigWig+and+BigBed:+enabling+browsing+of+large+distributed+datasets.">BigWig and BigBed: enabling browsing of large distributed datasets.</a> Bioinformatics. 26 (17), 2204-2207 (2010).</li><li>. UCSC Genome Browser Home Available from: <a target="_blank" href="https://genome.ucsc.edu">https://genome.ucsc.edu</a> (2019)</li><li>Schindelin, J., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Nishimura, K., Fukagawa, T., Takisawa, H., Kakimoto, T., Kanemaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+auxin-based+degron+system+for+the+rapid+depletion+of+proteins+in+nonplant+cells.">An auxin-based degron system for the rapid depletion of proteins in nonplant cells.</a> Nature Methods. 6 (12), 917-922 (2009).</li><li>Banaszynski, L. A., Chen, L., Maynard-Smith, L. A., Ooi, A. G. L., Wandless, T. 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+Rapid,+Reversible,+and+Tunable+Method+to+Regulate+Protein+Function+in+Living+Cells+Using+Synthetic+Small+Molecules.">A Rapid, Reversible, and Tunable Method to Regulate Protein Function in Living Cells Using Synthetic Small Molecules.</a> Cell. 126 (5), 995-1004 (2006).</li><li>H&#248;jfeldt, J. W., 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=Accurate+H3K27+methylation+can+be+established+de+novo+by+SUZ12-directed+PRC2.">Accurate H3K27 methylation can be established de novo by SUZ12-directed PRC2.</a> Nature Structural &amp; Molecular Biology. 25 (3), 225-232 (2018).</li></ol>

D.R. is a co-founder of Constellation Pharmaceuticals and Fulcrum Therapeutics. Authors declare that they have no competing interests.

A powerful approach towards understanding the mechanistic details during the formation of a given chromatin domain, is to first disrupt the domain and then track its reconstruction in progress within cells. The process can be paused at any time during the reconstruction to analyze in detail the events in progress. Previous studies on chromatin domains were unable to resolve such events as they were performed under steady-state conditions (e.g., comparing wild-type and gene knockout). Here, we outline a system to assess t...

Eukaryotic genomes are highly organized and changes in the chromatin accessibility directly controls gene transcription1. The genome contains distinct types of chromatin domains, which correlate with transcriptional activity and replication timing2,3. These chromatin domains range in size from a few kilobases (kb) to more than 100 kb and are characterized by an enrichment in distinct histone modifications4. The central questions are: how are these domains formed and how are they propagated?
One of the most well-characterized chromatin domains is fostered through the activity of the Polycomb repressive complex 2 (PRC2). PRC2 is a multi-subunit complex composed of a subset of the Polycomb Group (PcG) of proteins5,6, and catalyzes the mono-, di- and trimethylation of lysine 27 of histone H3 (H3K27me1/me2/me3)7,8,9,10. H3K27me2/me3 are associated with a repressive chromatin state, but the function of H3K27me1 is unclear6,11. One of the core components of PRC2, embryonic ectoderm development (EED), binds to the end product of PRC2 catalysis, H3K27me3, through its aromatic cage and this feature results in the allosteric stimulation of PRC212,13. The PRC2 enzymatic activity is crucial for preserving cellular identity during development as the inappropriate expression of certain developmental genes that are contraindicated for a specific lineage, would be detrimental5,6. Hence, unraveling the mechanisms by which PRC2 fosters the formation of repressive chromatin domains in mammals is of fundamental importance to understanding cellular identity.
All of the past experimental systems designed to investigate chromatin domain formation including PRC2-mediated chromatin domains, were performed under steady-state conditions, which are unable to track the unfolding events of chromatin domain formation in cells. Here, we present a detailed protocol to generate an inducible cellular system which monitors the initial recruitment and propagation of chromatin domains. Specifically, we focus on tracking the formation of PRC2-mediated repressive chromatin domains that comprise H3K27me2/3. This system that can capture the mechanistic details of chromatin domain formation, could be adapted to incorporate other chromatin domains, such as the widely studied domains comprising either H2AK119ub or H3K9me. In combination with genomics and imaging technologies, this approach has the potential to successfully address various, key questions in chromatin biology.

<table border="0" cellpadding="0" cellspacing="0" style="width:1045px;" width="1045">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">(Z)-4-Hydroxytamoxifen (5 mg)</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:101px;">H7904-5MG</td>
			<td style="width:368px;">For induction of EED expression</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">16% Paraformaldehyde aqueous solution (10x10 ml)</td>
			<td style="width:317px;">Electron Microscopy Sciences</td>
			<td style="width:101px;">15710</td>
			<td>For immunofluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">2-mercaptoethanol</td>
			<td style="width:317px;">LifeTechnologies</td>
			<td style="width:101px;">21985-023</td>
			<td style="width:368px;">For mESCs culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">2% Gelatin Solution</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:101px;">G1393-100ml</td>
			<td style="width:368px;">For mESCs culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Accutase 500 ML</td>
			<td style="width:317px;">Innovative Cell Tech/FISHER</td>
			<td style="width:101px;">AT 104-500</td>
			<td>For mESCs culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">Alexa Fluor 594 AffiniPure Donkey Anti-Rabbit IgG (H+L)</td>
			<td style="width:317px;">Jackson immunoresaerch</td>
			<td style="width:101px;">711-585-152</td>
			<td>For immunofluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Aqua-Mount Mounting Medium</td>
			<td style="width:317px;">FISHER/VWR</td>
			<td style="width:101px;">41799-008</td>
			<td>For immunofluorescence</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">CHAMBER SLD TC PRMA 8-CHM 16 PK</td>
			<td style="width:317px;">Fisher Sci</td>
			<td style="width:101px;">177445PK</td>
			<td>For immunofluorescence</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride) - 10 mg</td>
			<td style="width:317px;">Life Tech</td>
			<td style="width:101px;">D1306</td>
			<td>For immunofluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">ERK inhibitor, PD0325901</td>
			<td style="width:317px;">Stemgent</td>
			<td style="width:101px;">04-0006</td>
			<td style="width:368px;">For mESCs culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ESGRO Recombinant Mouse LIF Protein</td>
			<td>Millipore/Fisher</td>
			<td>ESG1107</td>
			<td>For mESCs culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">FBS Stem Cell Qualified</td>
			<td style="width:317px;">Atlanta</td>
			<td style="width:101px;">S10250</td>
			<td style="width:368px;">For mESCs culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Gibson Assembly Master Mix</td>
			<td style="width:317px;">NEB</td>
			<td style="width:101px;">E2611L</td>
			<td>For Donor template cloning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">GSK3 inhibitor, CHIR99021</td>
			<td style="width:317px;">Stemgent</td>
			<td style="width:101px;">04-0004</td>
			<td style="width:368px;">For mESCs culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">H3K27me2 (D18C8) rabbit mAB</td>
			<td style="width:317px;">Cell Signaling</td>
			<td style="width:101px;">9728S</td>
			<td>Antibody for ChIP-seq</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">H3K27me3</td>
			<td style="width:317px;">Cell Signaling</td>
			<td style="width:101px;">9733S</td>
			<td>Antibody for ChIP-seq</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Histone H2Av antibody (pAb)</td>
			<td style="width:317px;">Active motif</td>
			<td style="width:101px;">39715</td>
			<td>Spike-in control for ChIP-seq</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Knockout DMEM</td>
			<td style="width:317px;">Invitrogen</td>
			<td style="width:101px;">10829-018</td>
			<td style="width:368px;">For mESCs culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">L-glutamine</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:101px;">G7513</td>
			<td style="width:368px;">For mESCs culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Lipofectamine 2000</td>
			<td style="width:317px;">LifeTech</td>
			<td style="width:101px;">11668019</td>
			<td>For transfection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">MangoTaq DNA Polymerase</td>
			<td style="width:317px;">Bioline</td>
			<td style="width:101px;">BIO-21079</td>
			<td>For Genotyping PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Normal donkey serum (10 mL)</td>
			<td style="width:317px;">Jackson ImmunoResearch</td>
			<td style="width:101px;">017-000-121</td>
			<td>For immunofluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Penicillin-Streptomycin</td>
			<td style="width:317px;">Sigma/Roche</td>
			<td style="width:101px;">P0781</td>
			<td style="width:368px;">For mESCs culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">pSpCas9(BB)-2A-GFP (PX458)</td>
			<td style="width:317px;">Addgene</td>
			<td style="width:101px;">48138</td>
			<td style="width:368px;">For gRNA cloning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">QuickExtract DNA Extraction Solution</td>
			<td style="width:317px;">Lucigen</td>
			<td style="width:101px;">QE0905T</td>
			<td style="width:368px;">For Genotyping PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Triton X-100</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:101px;">T8787-250ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zero Blunt PCR Cloning Kit</td>
			<td style="width:317px;">Thermo Fisher</td>
			<td>K270020</td>
			<td>For Donor template cloning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primers/gBlocks</td>
			<td style="width:317px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EED-KO-gRNA-1</td>
			<td style="width:317px;"></td>
			<td></td>
			<td>Sequence: ctctggctactgtcaactag. gRNAs pairs to knockout EED in C57BL/6 ESCs for i-WT-r and i-MT-r systems.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EED-KO-gRNA-2</td>
			<td style="width:317px;"></td>
			<td></td>
			<td>Sequence: TAGGCTATGACGCAGCTCAG. gRNAs pairs to knockout EED in C57BL/6 ESCs for i-WT-r and i-MT-r systems.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EED-gRNA-inducible</td>
			<td style="width:317px;"></td>
			<td></td>
			<td>Sequence: atggcaccccgaaattagaa. gRNA and Donor to generate i-WT-r system in the EED-KO background.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">i-WT-r Donor</td>
			<td style="width:317px;"></td>
			<td></td>
			<td>https://benchling.com/s/seq-l2LLlWNEnLrfGXcbdCxI. gRNA and Donor to generate i-WT-r system in the EED-KO background.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EED-gRNA-inducible</td>
			<td style="width:317px;"></td>
			<td></td>
			<td>Sequence: atggcaccccgaaattagaa. gRNA and Donor to generate i-WT-r system in the EED-KO background.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:259px;">i-MT-r Donor</td>
			<td style="width:317px;"></td>
			<td style="width:101px;"></td>
			<td style="width:368px;">https://benchling.com/s/seq-n8eiZCB2XAkOuzzpv6qM. gRNA and Donor to generate i-MT-r system in the EED-KO background.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Genotyping Primers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gnt-EED-KO-FW-1</td>
			<td></td>
			<td></td>
			<td>Sequence: ctgtaggctgccatctgtga. Wild type allele will produce a product of 1.9 kb. Knockout allele will produce a product of 200 bp.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gnt-EED-KO-REV-1</td>
			<td></td>
			<td></td>
			<td>Sequence: agccagggctacacagagaa. Wild type allele will produce a product of 1.9 kb. Knockout allele will produce a product of 200 bp.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inducible_Genotype-FW-1</td>
			<td></td>
			<td></td>
			<td>Sequence: tgcagtgaaacaaatttggaa. When the casette is inserted, the primers will produce 1863 bp. The wild type allele will produce a product of ~200 bp.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inducible_Genotype-REV-1</td>
			<td></td>
			<td></td>
			<td>Sequence: gagaggggtggcactgtaaa. When the casette is inserted, the primers will produce 1863 bp. The wild type allele will produce a product of ~200 bp.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inducible_Genotype-FW-2</td>
			<td></td>
			<td></td>
			<td>Sequence: ccccctctttctccttttct. When the casette is inserted, the primers will produce 3200 bp. The wild type allele will produce a product of 1560 bp.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Inducible_Genotype-REV-2</td>
			<td style="width:317px;"></td>
			<td style="width:101px;"></td>
			<td>Sequence: atgcctgggtgaatgaaaaa. When the casette is inserted, the primers will produce 3200 bp. The wild type allele will produce a product of 1560 bp.</td>
		</tr>
	</tbody>
</table>

a method to study de novo formation of chromatin domains

The organization and structure of chromatin domains are unique to individual cell lineages. Their misregulation might lead to a loss in cellular identity and/or disease. Despite tremendous efforts, our understanding of the formation and propagation of chromatin domains is still limited. Chromatin domains have been studied under steady-state conditions, which are not conducive to following the initial events during their establishment. Here, we present a method to inducibly reconstruct chromatin domains and follow their re-formation as a function of time. Although, first applied to the case of PRC2-mediated repressive chromatin domain formation, it could be easily adapted to other chromatin domains. The modification of and/or the combination of this method with genomics and imaging technologies will provide invaluable tools to study the establishment of chromatin domains in great detail. We believe that this method will revolutionize our understanding of how chromatin domains form and interact with each other.

A general scheme of the conditional rescue system 
Figure 1 shows the targeting scheme to conditionally rescue EED KO cells with either WT or cage-mutant (Y365A) EED that is expressed from the endogenous EED locus. After knocking out EED, a core subunit of PRC2 that is essential for its stability and enzymatic activity, a cassette within the intron following exon 9 of EED is introduced (Figure 1). The cassette consists of the remaining 3&#3...

Watch this Scientific Journal Video about A Method to Study de novo Formation of Chromatin Domains at JoVE.com

A Method to Study de novo Formation of Chromatin Domains

This method is designed to follow formation of PRC2-mediated chromatin domains in cell lines, and the method can be adapted to many other systems.

A Method to Study de novo Formation of Chromatin Domains

The organization and structure of chromatin domains are unique to individual cell lineages. Their misregulation might lead to a loss in cellular identity ...

a-method-to-study-de-novo-formation-of-chromatin-domains

Research

JoVE Journal

Genetics

5.3K Views.  New York University School of Medicine. This method is designed to follow formation of PRC2-mediated chromatin domains in cell lines, and the method can be adapted to many other systems.

Video: A Method to Study de novo Formation of Chromatin Domains

Leveraging CyVerse Resources for De Novo Comparative Transcriptomics of Underserved (Non-model) Organisms

This workflow allows novice researchers to leverage advanced computational resources such as cloud computing to carry out pairwise comparative transcriptomics. It also serves as a primer for biologists to develop data scientist computational skills, e.g. executing bash commands, visualization and management of large data sets. All command line code and further explanations of each command or step can be found on the wiki (<a href="https://wiki.cyverse.org/wiki/x/dgGtAQ">https://wiki.cyverse.org/wiki/x/dgGtAQ</a>). The Discovery Environment and Atmosphere platforms are connected together through the CyVerse Data Store. As such, once the initial raw sequencing data has been uploaded there is no more need to transfer large data files over an Internet connection, minimizing the amount of time needed to conduct analyses. This protocol is designed to analyze only two experimental treatments or conditions. Differential gene expression analysis is conducted through pairwise comparisons, and will not be suitable to test multiple factors. This workflow is also designed to be manual rather than automated. Each step must be executed and investigated by the user, yielding a better understanding of data and analytical outputs, and therefore better results for the user. Once complete, this protocol will yield de novo assembled transcriptome(s) for underserved (non-model) organisms without the need to map to previously assembled reference genomes (which are usually not available in underserved organism). These de novo transcriptomes are further used in pairwise differential gene expression analysis to investigate genes differing between two experimental conditions. Differentially expressed genes are then functionally annotated to understand the genetic response organisms have to experimental conditions. In total, the data derived from this protocol is used to test hypotheses about biological responses of underserved organisms.

Leveraging CyVerse Resources for De Novo Comparative Transcriptomics of Underserved Non-model Organisms

This protocol outlines a comparative de novo transcriptome assembly and annotation workflow for novice bioinformaticians. The workflow is available for free entirely through CyVerse and connected by the Data Store. Command line and graphical user interfaces are used, but all code needed is available to copy and paste.

This workflow allows novice researchers to leverage advanced computational resources such as cloud computing to carry out pairwise comparative ...

Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous nature of most mammalian chromatin-modifying complexes. In order to overcome these hurdles, we have adapted a sequential salt extraction (SSE) assay for evaluating the relative binding affinities of chromatin-bound complexes. This easy and straightforward assay can be used by non-experts to evaluate the relative difference in binding affinity of two related complexes, the changes in affinity of a complex when a subunit is lost or an individual domain is inactivated, and the change in binding affinity after alterations to the chromatin landscape. By sequentially re-suspending bulk chromatin in increasing amounts of salt, we are able to profile the elution of a particular protein from chromatin. Using these profiles, we are able to determine how alterations in a chromatin-modifying complex or alterations to the chromatin environment affect binding interactions. Coupling SSE with other in vitro and in vivo assays, we can determine the roles of individual domains and proteins on the functionality of a complex in a variety of chromatin environments.

Sequential salt extraction of chromatin bound proteins is a useful tool for determining the binding properties of large protein complexes. This method can be employed to evaluate the role of individual subunits or domains in the overall affinity of a protein complex to bulk chromatin.

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous ...

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV integration sites can be used as functional markers of active regulatory elements. Here, we present a retroviral scanning tool, which allows the genome-wide identification of cell-specific enhancers and promoters. Briefly, the target cell population is transduced with an mLV-derived vector and genomic DNA is digested with a frequently cutting restriction enzyme. After ligation of genomic fragments with a compatible DNA linker, linker-mediated polymerase chain reaction (LM-PCR) allows the amplification of the virus-host genome junctions. Massive sequencing of the amplicons is used to define the mLV integration profile genome-wide. Finally, clusters of recurrent integrations are defined to identify cell-specific regulatory regions, responsible for the activation of cell-type specific transcriptional programs.
The retroviral scanning tool allows the genome-wide identification of cell-specific promoters and enhancers in prospectively isolated target cell populations. Notably, retroviral scanning represents an instrumental technique for the retrospective identification of rare populations (e.g. somatic stem cells) that lack robust markers for prospective isolation.

Here, we describe a protocol for genome-wide mapping of the integration sites of Moloney murine leukemia virus-based retroviral vectors in human cells.

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein interactions in yeast. It allows analysis of a target region of interest without the need for prior knowledge about likely proteins bound to the target region. This, in theory, allows HyCCAPP to be used to analyze any genomic region of interest, and it provides sufficient flexibility to work in different cell systems. This method is not meant to study binding sites of known transcription factors, a task better suited for Chromatin Immunoprecipitation (ChIP) and ChIP-like methods. The strength of HyCCAPP lies in its ability to explore DNA regions for which there is limited or no knowledge about the proteins bound to it. It can also be a convenient method to avoid biases (present in ChIP-like methods) introduced by protein-based chromatin enrichment using antibodies. Potentially, HyCCAPP can be a powerful tool to uncover truly novel DNA-protein interactions. To date, the technology has been predominantly applied to yeast cells or to high copy repeat sequences in mammalian cells. In order to become the powerful tool we envision, HyCCAPP approaches need to be optimized to efficiently capture single-copy loci in mammalian cells. Here, we present our adaptation of the initial yeast HyCCAPP capture protocol to human cell lines, and show that single-copy chromatin regions can be efficiently isolated with this modified protocol.

This is a method to identify novel DNA-interacting proteins at specific target loci, relying on sequence-specific capture of crosslinked chromatin for subsequent proteomic analyses. No prior knowledge about potential binding proteins, nor cell modifications are required. Initially developed for yeast, the technology has now been adapted for mammalian cells.

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein ...

A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in atherogenesis progression, ischemia, and myocardial infarction. Here we present a methodology of patient genotyping to investigate the post-transcriptional role of the C924T polymorphism (rs4523) situated at the 3&#39; region of the TBXA2 receptor gene. This method relies on DNA extraction from whole blood, polymerase chain reaction (PCR) amplification of the TBXA2 gene portion containing the C924T mutation, and identification of wild type and/or mutant genotypes using a restriction digest analysis, specifically a restriction fragment length polymorphism (RFLP) on agarose gel. In addition, the results were confirmed by sequencing the TBXA2R gene. This method features several potential advantages, such as high efficiency and the rapid identification of the C924T polymorphism by PCR and restriction enzyme analysis. This approach allows a predictive study for plaque formation and atherosclerosis progression by analyzing patient genotypes for the TBXA2R C924T polymorphism. Application of this method has the potential to identify subjects who are more susceptible to atherothrombotic processes, in particular subjects in a high-risk, aspirin-treated group.

In the present study, we describe a methodology to analyze the C924T genotype. The protocol consists of three phases: DNA extraction, amplification by polymerase chain reaction (PCR), and analysis of the restriction fragment length polymorphism (RFLP) on agarose gel.

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in ...

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a routine procedure is widely utilized throughout the world and its main limitation resides in the poor efficacy of transgene integration, resulting in a low yield of founder animals. Only few percent of animals born after implantation of injected fertilized oocytes have integrated the transgene. In contrast, lentiviral vectors are powerful tools for integrative gene transfer and their use to transduce fertilized oocytes allows highly efficient production of founder transgenic mice with an average yield above 70%. Furthermore, any mouse strain can be used to produce transgenic animal and the penetrance of transgene expression is extremely high, above 80% with lentiviral mediated transgenesis compared to DNA microinjection. The size of the DNA fragment that can be cargo by the lentiviral vector is restricted to 10 kb and represents the major limitation of this method. Using a simple and easy to perform injection procedure beneath the zona pellucida of fertilized oocytes, more than 50 founder animals can be produced in a single session of microinjection. Such a method is highly adapted to perform, directly in founder animals, rapid gain and loss of function studies or to screen genomic DNA regions for their ability to control and regulate gene expression in vivo.

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a complementary DNA (cDNA) intermediate. However, such approaches are rarely applied as a de novo RNA sequencing method, but used mainly as a tool that can assist in quality assurance for confirming known sequences of purified single-stranded RNA samples. Recently, we developed&#160;a direct RNA sequencing method by integrating a 2-dimensional mass-retention time hydrophobic end-labeling strategy into MS-based sequencing (2D-HELS MS Seq). This method is capable of accurately sequencing single RNA sequences as well as mixtures containing up to 12 distinct RNA sequences. In addition to the four canonical ribonucleotides (A, C, G, and U), the method has the capacity to sequence RNA oligonucleotides containing modified nucleotides. This is possible because the modified nucleobase either has an intrinsically unique mass that can help in its identification and its location in the RNA sequence, or can be converted into a product with a unique mass. In this study, we have used RNA, incorporating two representative modified nucleotides&#160;(pseudouridine (&#936;) and 5-methylcytosine (m5C)), to illustrate the application of the method for the de novo sequencing of a single RNA oligonucleotide&#160;as well as a mixture of RNA oligonucleotides, each with a different sequence and/or modified nucleotides. The procedures and protocols described here to sequence these model RNAs will be applicable to other short RNA samples (&#60;35 nt) when using a standard high-resolution LC-MS system, and can also be used for&#160;sequence verification of modified therapeutic RNA oligonucleotides.&#160;In the future, with the development of more robust algorithms and with better instruments, this method could allow sequencing of more complex biological samples.&#160;

Here, we describe a detailed protocol for an LC-MS-based sequencing method that can be used as a direct method to sequence short RNA (&#60;35 nt per run) without a cDNA intermediate, and as a general method to sequence different nucleotide modifications in a single study at single-base precision.