We thank Ross Dickins of the University of Melbourne for graciously providing the shRNA library used in the screen. This work was funded by the Rett Syndrome Research Trust (A.B.).

All steps involving animals were carried out using protocols approved by the Fred Hutchinson Cancer Research Center Institutional Animal Care and Use Committee (IACUC). No reagents used here are known to pose significant human health risks except for 5-azacytidine (5-AZA).
1. Generating a reporter cell line with MeCP2-LUC-HR transgene on the Xi
<ol>
	<li>Preparing mouse tendon fibroblasts from a female XMeCP2-LUC-HR /XMeCP2 mouse
	<ol>
		<li>Euthanize a 10-12 day-old female XMeCP2-LUC-HR/XMeCP2 mouse.</li>
		<li>Using straight dissection scissors, make a circumferential incision through the skin of the proximal tail portion.</li>
		<li>Holding the carcass near the base of the tail, pull the skin away from the mouse to expose the fibrocartilaginous portion of the tail. Remove and cut the terminal 10 mm of the exposed tissue into 1 mm long fragments using a surgical scalpel.</li>
		<li>Transfer the fragmented tissue to a 15 mL conical tube containing 5 mL of Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 1 U/mL penicillin, 1 &#181;g/mL streptomycin, and 1.5 mg/mL collagenase, pre-filtered through 0.45 &#181;m and then 0.2 &#181;m mesh filters. Incubate for 3-4 h at 37 &#176;C with continuous rotation.</li>
		<li>Centrifuge the sample at room temperature at 500 x g for 10 min. Resuspend the pellet in 10 mL of DMEM supplemented with 10% fetal bovine serum, 1 U/mL penicillin, and 1 &#181;g/mL streptomycin (DMEM-10) and plate on a 6-well tissue culture plate. Passage the cells once and expand to a 10 cm plate before proceeding to the next step.</li>
		<li>Immortalize the cells by performing a transduction with an expression vector carrying HPV-16 viral oncogenes (E6/E7), as described previously7.</li>
	</ol>
	</li>
	<li>Selecting single cell clones and characterizing MeCP2 reporter expression
	<ol>
		<li>Harvest the cells from a 10 cm plate. First, aspirate the media, add 1 mL of 0.05% trypsin-EDTA and incubate for 2-5 min at 37 &#176;C. Quench with 9 mL of DMEM-10 and collect the resuspended cells in a 15 mL conical tube.</li>
		<li>Dilute the cells with DMEM-10 to a final concentration of 1 cell/100 &#181;L. Transfer to 96-well plates by pipetting 100 &#181;L of the suspension into each well.</li>
		<li>Incubate at 37 &#176;C until the wells are confluent, which can take up to 2-3 weeks. Replace the media with fresh DMEM-10 every 3-5 days. When confluent, mobilize the cells with 20 &#181;L of 0.05% trypsin-EDTA per well and split them into two duplicate sets of 96-well plates.</li>
		<li>Use one set of plates to assay for luciferase activity. Use a homogeneous firefly luciferase assay kit which does not require media removal. Follow the protocol included with the kit.</li>
		<li>Use the results from step 1.2.4 to select clones with no detectable luciferase activity from the remaining set of plates. Expand these clones to 24-well and then 6-well plates.</li>
		<li>Perform the same for a clone with the highest luciferase activity; this will be used as a positive control for validation purposes.</li>
		<li>Harvest an aliquot of each well in the 6-well plate for a Western blot. Mobilize the cells with 200 &#181;L of 0.05% trypsin-EDTA. Incubate for 2-5 min at 37 &#176;C. Quench with 2 mL of DMEM-10, and transfer half of the cells to 1.5ml centrifuge tube. Wash the cells once with PBS before lysing for Western blot. Alternatively, transfer half of the cells to another 6-well plate. When the cells attach to the bottom of the plate, rinse with PBS and lyse for Western blot.&#160;</li>
		<li>Perform a Western blot with anti-MeCP2 antibody to confirm that luciferase-negative cells express,&#160;while the luciferase-positive cells do not express wild-type MeCP2 from the active X chromosome; follow the previously described protocol8. Use brain tissue lysate from any wild type mouse as a positive control.</li>
		<li>Plate several negative clones at 2 x 105 cells/well in a 6-well plate. Treat once with 10 &#181;M 5-AZA, incubate for 72 h without changing the media, and assay for luciferase activity as described in step 1.2.3.</li>
		<li>Choose one negative clone (Xi clone) that gained luciferase activity with 5-AZA and begin expanding it to multiple 10 cm plates. Freeze the remaining negative clones in liquid nitrogen as a backup. The positive clone (Xa clone) can be maintained in the tissue culture or frozen until further use.</li>
	</ol>
	</li>
</ol>
2. Screening the library for reactivation using hygromycin B selection
<ol>
	<li>Infecting the test clone, applying selection, and harvesting DNA for sequencing
	<ol>
		<li>Obtain viral concentrates of retroviral constructs expressing hairpins from an shRNA library, or produce concentrated viral supernatant using the protocol included with the desired library.</li>
		<li>Before proceeding with the screen, titrate the library (using a GFP marker, if available). Aim for a multiplicity of infection between 0.3 and 0.5. 
		NOTE: Perform at least four independent screens to minimize false positive rates. Use 20 plates per screen to obtain 100-fold coverage of the library to maintain adequate representation of hairpins and avoid random dropouts9. However, given positive selection screen and a large library size, it is likely that a lower coverage would suffice.</li>
		<li>In the morning, plate 1 x 106 Xi cells onto 10 cm tissue culture plates.</li>
		<li>In the afternoon, infect the plates by replacing the media with fresh DMEM-10 containing viral supernatant and 5-10 &#181;g/mL of polybrene. Use the amount of viral supernatant determined in step 2.1.2.</li>
		<li>After 18-24 h, aspirate the media and add 10 mL of fresh DMEM-10 to each plate. Culture for another 48 h.</li>
		<li>In the morning, mobilize the cells using 1 mL of 0.05% trypsin-EDTA per plate and quench with 9 mL of DMEM-10 per plate. Run the suspension through a 100 &#181;m mesh filter. Pool the filtered suspensions together, and seed two sets of 10 cm plates at 1 x 106 cells/plate.</li>
		<li>In the afternoon, replace the media in one set of plates with DMEM-10 containing 15 &#181;g/mL of hygromycin B (day 0). In the other set, replace the media with fresh DMEM-10 only.</li>
		<li>After 48 hours, mobilize the cells from untreated set of plates using 1 ml of 0.05% trypsin-EDTA. Incubate for 2-5 min at 37 &#176;C. Quench with 9 mL of DMEM-10. Transfer the cells to 15 ml conical tubes and centrifuge at 500 x g for 5 minutes at room temperature. Aspirate the supernatants and proceed immediately with extracting DNA from the pellets. Alternatively, pool the cells into larger tubes before the centrifugation step.</li>
		<li>Extract DNA using a mixture of phenol and chroroform and precipitate DNA with sodium acetate and cold ethanol as previously described10. Pool the individual pre-selection DNA samples and store at -20 &#176;C until further use.</li>
		<li>Continue culturing hygromycin B selection set for 4 more days. Replenish the selection media with fresh DMEM-10 containing hygromycin B after 2 days.</li>
		<li>On day 7, replace the selection media with fresh DMEM-10 only. Allow 2 to 4 days for recovery, and then harvest the cells as described in step 2.1.8. Proceed with DNA extraction as described in step 2.1.9.</li>
	</ol>
	</li>
	<li>Identifying hairpins enriched in the post-selection samples
	<ol>
		<li>Use 5&#39; context (5&#39;-ATCGTTGCCTGCACATCTTGG-3&#39;) and 3&#39; reverse loop primers (5&#39;-ACATCTGTGGCTTCACTA-3&#39;) to amplify half-hairpins from pre- and post-selection DNA samples. Use all extracted DNA in separate PCR reactions with 100-200 ng of DNA per reaction. 
		NOTE: These 5&#39; context primer and 3&#39; reverse loop primers correspond to the miR-30 context sequence upstream of the shRNA insert and the sequence at the shRNA loop, respectively. Different libraries will require different oligos. Adjust purification steps to isolate different product sizes as necessary.</li>
		<li>Use size-selection beads to remove primers and genomic DNA, and recover PCR products between 100 and 200 bp. Follow the protocol included with the beads.</li>
		<li>Prepare a sequencing library to sequence the half-hairpins. Generate at least 50 million reads per screen. Follow the protocol appropriate for the sequencing kit and equipment. 
		NOTE: 50 million reads per screen provides 1000-fold representation of each shRNA in the library and approximately 10-fold coverage of each independent infection (carried out at 100-fold representation of each shRNA), which ensures adequate representation of each infection event.</li>
		<li>Enumerate each shRNA using a Perl script that counts sequences with perfect matches. Chose shRNAs that had at least 10 reads in the preselection pool for further analysis. Calculate the enrichments as ratios of post- to preselection counts and determine false discovery rate (FDR) on the basis of 1000 permutations. Chose genes targeted by shRNAs with &#60;5% FDR as &#34;hits&#34; for further testing.</li>
	</ol>
	</li>
</ol>
3. Validating the identified hairpins (&#34;hits&#34;)
<ol>
	<li>Packaging lentiviruses containing shRNAs identified in the screen
	<ol>
		<li>Seed 8 x 106 293FT cells per plate on 10 cm plates.</li>
		<li>The following day, replace the media with 9 mL of antibiotic-free DMEM-10 per plate; do this 30 minutes before transfection.</li>
		<li>During the 30-minute interval, prepare two mixes for transfection:
		<ol>
			<li>Prepare Mix 1 (per plate/shRNA; scale up according to the total number of shRNAs): 10 &#181;L of 2 mg/mL polyethylenimine (PEI) in 0.5 mL of optimal minimum essential media. Mix by pipetting and incubate at room temperature for 5 min.</li>
			<li>Prepare Mix 2 (per shRNA): 4 &#181;g of the shRNA vector to be packaged, 1.5 &#181;g of the VSV-G envelope vector (pMD2.G), and 2.5 &#181;g of the packaging vector (psPAX2), mixed in 0.5 mL of optimal minimum essential media. 
			NOTE: Include an empty lentiviral vector in the packaging to use in later infections as a negative control.</li>
		</ol>
		</li>
		<li>Add 0.5 mL of mix 1 to each aliquot of mix 2, and mix gently by pipetting. Incubate in the dark at room temperature for 20 min.</li>
		<li>Apply the mixture onto each plate from step 3.1.2 in a dropwise fashion; swirl the plate gently to mix.</li>
		<li>The following day, replace the media with 5 mL of fresh DMEM-10.</li>
		<li>48 hours after initial transfection, collect the supernatant and run it through a 0.22 &#181;m filter. Pre-wet the filter with DMEM-10 to maximize recovery.</li>
		<li>Aliquot the filtered supernatant into amounts ideal for infecting a 6-well plate well.</li>
		<li>Freeze the aliquots at -80 &#176;C for later use, or proceed immediately with transduction.</li>
	</ol>
	</li>
	<li>Transduction and testing of individual hairpins for luciferase reporter reactivation and reactivation of wild-type MeCP2 gene on the Xi.
	<ol>
		<li>In the morning, plate 1 x 105 Xi cells/well onto 6 well plates.</li>
		<li>In the afternoon, infect the plates by replacing the media with 2 mL of fresh DMEM-10 containing lentiviral supernatant and 5-10 &#181;g/mL of polybrene. Infect a control well with viral supernatant produced from an empty lentiviral vector.</li>
		<li>The following day, replace the media with 2 mL of DMEM-10 containing 1 mg/mL puromycin. Culture for four days.</li>
		<li>Replace the media with 2 mL of fresh DMEM-10 and allow 48-72 h for recovery.</li>
		<li>Mobilize the cells with 150 &#181;L of 0.05% trypsin-EDTA. Incubate for 2-5 min at 37 &#176;C. Quench with 1.5 mL of DMEM-10. Transfer the cells to 15 ml conical tubes and centrifuge at 500 x g for 10 minutes at room temperature.</li>
		<li>Remove the media and resuspend the cells in 100 &#181;L lysis buffer provided in the luciferase assay kit. Incubate at room temperature for 5 minutes. Transfer the lysates to a white 96-well plate and assay for luciferase activity using the protocol included with the kit. Use Xa cells as a positive control and Xi cells infected with an empty lentiviral vector as a negative control. 
		NOTE: Avoid exposing the white plates to fluorescent light prior to the assay; this will reduce the background and increase assay sensitivity. The protocol provided in the assay kit was found to be most effective if performed in the dark whenever possible.</li>
		<li>To determine whether the hairpin had an effect on the expression level of MeCP2 on the active X-chromosome, isolate RNA from tranduced Xi cells and measure MeCP2 mRNA level using quantitative PCR with primers designed to amplify only wild type MeCP2 (F: 5&#39;-TTCCATGCCAAGGCCAAACAG-3&#39;, R: 5&#39;-CCCATAAGGAGAAGAGACAACAGC-3&#39;).</li>
		<li>Optional: Increase the sensitivity of the assay by adding 5-AZA to a final concentration of 0.2 &#181;M after the cells have recovered from puromycin selection. Culture the cells for 72 h without replacing the media, and then assay for luciferase activity using the commercial luciferase assay kit.</li>
		<li>Measure reactivation of the silenced wild-type MeCP2. Infect Xa cells, which harbor the wild type MeCP2 on the Xi, with the vector containing individual hairpins, following the protocol steps 3.2.1. - 3.2.4. Isolate RNA from cells and measure MeCP2 mRNA level using quantitative PCR with primers designed to amplify only wild type MeCP2 (F: 5&#39;-TTCCATGCCAAGGCCAAACAG-3&#39;, R: 5&#39;-CCCATAAGGAGAAGAGACAACAGC-3&#39;).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Amir, R. E., 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=Rett+syndrome+is+caused+by+mutations+in+X-linked+MECP2,+encoding+methyl-CpG-binding+protein+2.">Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2.</a> Nat Genet. 23, 185-188 (1999).</li><li>Guy, J., Gan, J., Selfridge, J., Cobb, S., Bird, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversal+of+neurological+defects+in+a+mouse+model+of+Rett+syndrome.">Reversal of neurological defects in a mouse model of Rett syndrome.</a> Science. 315, 1143-1147 (2007).</li><li>Maduro, C., de Hoon, B., Gribnau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fitting+the+Puzzle+Pieces:+the+Bigger+Picture+of+XCI.">Fitting the Puzzle Pieces: the Bigger Picture of XCI.</a> Trends Biochem Sci. 41, 138-147 (2016).</li><li>Disteche, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dosage+compensation+of+the+sex+chromosomes+and+autosomes.">Dosage compensation of the sex chromosomes and autosomes.</a> Semin Cell Dev Biol. 56, 9-18 (2016).</li><li>Wang, 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=Wild-type+microglia+do+not+reverse+pathology+in+mouse+models+of+Rett+syndrome.">Wild-type microglia do not reverse pathology in mouse models of Rett syndrome.</a> Nature. 521, E1-E4 (2015).</li><li>Sripathy, 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=Screen+for+reactivation+of+MeCP2+on+the+inactive+X+chromosome+identifies+the+BMP/TGF-beta+superfamily+as+a+regulator+of+XIST+expression.">Screen for reactivation of MeCP2 on the inactive X chromosome identifies the BMP/TGF-beta superfamily as a regulator of XIST expression.</a> Proc Natl Acad Sci U S A. 114, 1619-1624 (2017).</li><li>Foster, S. A., Galloway, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+papillomavirus+type+16+E7+alleviates+a+proliferation+block+in+early+passage+human+mammary+epithelial+cells.">Human papillomavirus type 16 E7 alleviates a proliferation block in early passage human mammary epithelial cells.</a> Oncogene. 12, 1773-1779 (1996).</li><li>Hnasko, T. S., Hnasko, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Western+Blot.">The Western Blot.</a> Methods Mol Biol. 1318, 87-96 (2015).</li><li>Strezoska, Z., 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=Optimized+PCR+conditions+and+increased+shRNA+fold+representation+improve+reproducibility+of+pooled+shRNA+screens.">Optimized PCR conditions and increased shRNA fold representation improve reproducibility of pooled shRNA screens.</a> PLoS One. 7, e42341 (2012).</li><li>Green, M. R., Sambrook, J. <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+High-Molecular-Weight+DNA+from+Mammalian+Tissues+Using+Proteinase+K+and+Phenol.">Isolation of High-Molecular-Weight DNA from Mammalian Tissues Using Proteinase K and Phenol.</a> Cold Spring Harb Protoc. 2017, (2017).</li><li>Goto, Y., Takagi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetraploid+embryos+rescue+embryonic+lethality+caused+by+an+additional+maternally+inherited+X+chromosome+in+the+mouse.">Tetraploid embryos rescue embryonic lethality caused by an additional maternally inherited X chromosome in the mouse.</a> Development. 125, 3353-3363 (1998).</li><li>Csankovszki, G., Nagy, A., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergism+of+Xist+RNA,+DNA+methylation,+and+histone+hypoacetylation+in+maintaining+X+chromosome+inactivation.">Synergism of Xist RNA, DNA methylation, and histone hypoacetylation in maintaining X chromosome inactivation.</a> J Cell Biol. 153, 773-784 (2001).</li><li>Minajigi, 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=Chromosomes.+A+comprehensive+Xist+interactome+reveals+cohesin+repulsion+and+an+RNA-directed+chromosome+conformation.">Chromosomes. A comprehensive Xist interactome reveals cohesin repulsion and an RNA-directed chromosome conformation.</a> Science. 349, (2015).</li><li>McHugh, C. 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=The+Xist+lncRNA+interacts+directly+with+SHARP+to+silence+transcription+through+HDAC3.">The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3.</a> Nature. 521, 232-236 (2015).</li><li>Bhatnagar, 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=Genetic+and+pharmacological+reactivation+of+the+mammalian+inactive+X+chromosome.">Genetic and pharmacological reactivation of the mammalian inactive X chromosome.</a> Proc Natl Acad Sci U S A. 111, 12591-12598 (2014).</li><li>Minkovsky, 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=The+Mbd1-Atf7ip-Setdb1+pathway+contributes+to+the+maintenance+of+X+chromosome+inactivation.">The Mbd1-Atf7ip-Setdb1 pathway contributes to the maintenance of X chromosome inactivation.</a> Epigenetics Chromatin. 7, 12 (2014).</li><li>Minkovsky, 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=A+high-throughput+screen+of+inactive+X+chromosome+reactivation+identifies+the+enhancement+of+DNA+demethylation+by+5-aza-2'-dC+upon+inhibition+of+ribonucleotide+reductase.">A high-throughput screen of inactive X chromosome reactivation identifies the enhancement of DNA demethylation by 5-aza-2'-dC upon inhibition of ribonucleotide reductase.</a> Epigenetics Chromatin. 8, 42 (2015).</li></ol>

Dr. Leko contributed to this article as an employee of the Fred Hutchinson Cancer Research Center.&#160; The views expressed are his own and do not necessarily represent the views of the National Institutes of Health or the United States Government

In our recent study6, we generated a murine cell line with luciferase and hygromycin resistance genes fused to MeCP2 on the Xi, and transduced it with a library of &#62;60,000 shRNAs targeting &#62;25,000 genes. We found 30 genes whose knock-down conferred survival advantage under hygromycin B selection, suggesting their role in control of MeCP2 and X-chromosome silencing. These results were validated by transducing the reported cell line with individual hairpins and assessing re...

One the most common forms of hereditary mental impairment in females, Rett syndrome, is caused by heterozygous mutations in MeCP2, an X-chromosome gene that encodes a protein essential for normal neuronal function1. A potential approach to treating this disorder would be reactivation of the wild-type MeCP2 allele on the Xi, as restoration of MeCP2 expression was shown to reverse neurological deficits in a mouse model of this disease1,2,4. However, epigenetic silencing of one of the two X chromosomes in female cells is tightly maintained throughout the lifespan of an organism3,4, and robust reactivation of an Xi gene would likely require a major disruption in multiple epigenetic regulatory pathways.
To identify factors necessary for maintenance of MeCP2 silencing, we first developed a transgenic mouse model carrying a MeCP2-luciferase- hygromycin resistance gene fusion (MeCP2-LUC-HR) on one of the two X chromosomes (XMeCP2-LUC-HR/XMeCP2)5. Although the MeCP2 expressed from the fusion construct proved to be unstable and resulted in a loss of function, phenotypically mimicking MeCP2 deletion in hemizygous males (XMeCP2-LUC-HR/Y), the expression of the reporter genes was readily detectable in a pattern consistent with the expression of endogenous MeCP25. We then generated immortalized fibroblast clones with the construct on either inactive or active X-chromosome, and confirmed that former expressed wild type MeCP2 and not the reporter construct; the inverse was true for the latter. When exposed to a DNA demethylating agent known to abrogate gene silencing, 5-azacytidine (5-AZA), cells with the reporter on the Xi (&#34;reporter cells&#34;) gained activity in a bioluminescence assay, indicating that our construct could be reactivated and therefore used for genetic screening.
We next developed a high-throughput genetic screen for regulators of MeCP2 silencing. The reporter cell line was first infected with a retroviral library containing &#62;60,000 different shRNAs targeting &#62;25,000 genes throughout the mouse genome5,6, and then subjected to hygromycin B selection. Hairpin frequency was compared in pre- and post-selection samples using next generation sequencing, as reporter reactivation conferred growth advantage under hygromycin B selection and resulted in enrichment of responsible hairpins. Using this approach, we identified 30 genes implicated in MeCP2 silencing, and subsequently confirmed the findings by transducing the reporter cells with individual hairpins and measuring their luciferase&#160; activity.

<table><tbody><tr><td>293FT Cell Line</td><td>Invitrogen</td><td>R700-07</td><td></td></tr><tr><td>5-Azacytidine</td><td>Sigma</td><td>A2385-100MG</td><td></td></tr><tr><td>Agencourt AMPure XP</td><td>Beckman-Coulter</td><td>A63881</td><td></td></tr><tr><td>Anti-MECP2 antibody</td><td>Millipore</td><td>07-013</td><td></td></tr><tr><td>Collagenase</td><td>Sigma</td><td>C2674-1G</td><td></td></tr><tr><td>Corning 96-Well Solid White Polystyrene Microplates</td><td>Fisher Scientific</td><td>07-200-336</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Gibco</td><td>11965-092</td><td></td></tr><tr><td>Expression Arrest microRNA-adapted Retroviral Vector (pMSCV)</td><td>Open Biosystems</td><td>EAV4679</td><td></td></tr><tr><td>Expression Arrest pSM2 Retroviral shRNAmir library</td><td>Open Biosystems</td><td>RMM3796</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Fisherbrand</td><td>03-600-511</td><td></td></tr><tr><td>HiSeq 2500</td><td>Illumina</td><td>SY&amp;ndash;401&amp;ndash;2501</td><td>Or equivalent</td></tr><tr><td>Hygromycin B</td><td>Calbiochem</td><td>400051-1MU</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen</td><td>11668-019</td><td></td></tr><tr><td>Bright-Glo Luciferase Assay System</td><td>Promega</td><td>E2610</td><td>Homogenous Assay for Screening Colonies</td></tr><tr><td>Luciferase Assay System</td><td>Promega</td><td>E4530</td><td>Non-Homogenous Assay for Testing Individual Hairpins</td></tr><tr><td>Luminometer TopCount NXT</td><td>Perkin Elmer</td><td>N/A</td><td>Or similar luminometer</td></tr><tr><td>MiSeq Reagent Kit v2</td><td>Illumina</td><td>MS-102-2001</td><td>Use kit compatible with your equipment</td></tr><tr><td>Opti-MEM</td><td>Gibco</td><td>31985-070</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>pMD2.G</td><td>Addgene</td><td>#12259</td><td>VSV-G envelope vector</td></tr><tr><td>Polybrene</td><td>Sigma</td><td>TR-1003-G</td><td></td></tr><tr><td>Polyethylenimine</td><td>Polysciences</td><td>23966-1</td><td></td></tr><tr><td>psPAX2</td><td>Addgene</td><td>#12260</td><td>Packaging vector</td></tr><tr><td>Puromycin</td><td>Gibco</td><td>A11138-02</td><td></td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>Gibco</td><td>25300-054</td><td></td></tr></tbody></table>

pooled shrna screen for reactivation of mecp2 on the inactive x chromosome

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control in model organisms. Technologies including short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats (CRISPR) have enabled such screens in diploid mammalian cells. Here we describe a large-scale shRNA screen for regulators of X-chromosome inactivation (XCI), using a murine cell line with firefly luciferase and hygromycin resistance genes knocked in at the C-terminus of the methyl CpG binding protein 2 (MeCP2) gene on the inactive X-chromosome (Xi). Reactivation of the construct in the reporter cell line conferred survival advantage under hygromycin B selection, enabling us to screen a large shRNA library and identify hairpins that reactivated the reporter by measuring their post-selection enrichment using next-generation sequencing. The enriched hairpins were then individually validated by testing their ability to activate the luciferase reporter on Xi.

Mouse tendon fibroblasts were harvested from a previously described XMeCP2-LUC-HR/XMeCP2 female mouse, immortalized by retroviral transduction of E6 and E7 oncogenes from HPV-16 virus7, cloned using limiting dilution, and tested for luciferase activity and expression of wild-type MeCP2 protein (Figure 1A and 1B). Luciferase activity was robust if the reporter was on Xa, and undetectable if on Xi; the native MeCP2 expression exhibited a reciprocal patter...

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Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome

We report a small hairpin RNA (shRNA) and next generation sequencing-based protocol for identifying regulators of X-chromosome inactivation in a murine cell line with firefly luciferase and hygromycin resistance genes fused to the methyl CpG binding protein 2 (MeCP2) gene on the inactive X chromosome.

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control ...

pooled-shrna-screen-for-reactivation-mecp2-on-inactive-x

Research

JoVE Journal

Genetics

7.2K Views.  Fred Hutchinson Cancer Research Center. Wij rapporteren een kleine haarspeld RNA (shRNA) en de volgende generatie sequencing-gebaseerd protocol voor het identificeren van de regelgevers van de inactivering van het x-chromosoom in een lymfkliertest cellijn met firefly luciferase en hygromycin resistentiegenen gesmolten aan de methyl CpG bindende eiwit 2) MeCP2) gen op het X-chromosoom inactief.

Video: Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome

A Non-random Mouse Model for Pharmacological Reactivation of Mecp2 on the Inactive X Chromosome

X chromosome inactivation (XCI) is the random silencing of one X chromosome in females to achieve gene dosage balance between the sexes. As a result, all females are heterozygous for X-linked gene expression. One of the key regulators of XCI is Xist, which is essential for the initiation and maintenance of XCI. Previous studies have identified 13 trans acting X chromosome inactivation factors (XCIFs) using a large-scale, loss-of-function genetic screen. Inhibition of XCIFs, such as ACVR1 and PDPK1, using short-hairpin RNA or small molecule inhibitors, reactivates X chromosome-linked genes in cultured cells. But the feasibility and tolerability of reactivating the inactive X chromosome in vivo remains to be determined. Towards this goal, a Xist&#916;:Mecp2/Xist:Mecp2-Gfp mouse model has been generated with non-random XCI due to deletion of Xist on one X chromosome. Using this model, the extent of inactive X reactivation was quantitated in the mouse brain following treatment with XCIF inhibitors. Recently published results show, for the first time, that pharmacological inhibition of XCIFs reactivates Mecp2 from the inactive X chromosome in cortical neurons of the living mouse brain.

Here, we describe a protocol to generate a viable female murine model with non-random X chromosome inactivation, i.e., the maternally-inherited X chromosome is inactive in 100% of the cells. We also describe a protocol to test feasibility, tolerability, and safety of pharmacological reactivation of the inactive X chromosome in vivo.

X chromosome inactivation (XCI) is the random silencing of one X chromosome in females to achieve gene dosage balance between the sexes. As a result, all ...

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian cells, this technology represents a novel means to perform genome-wide genetic screens for functional genomics studies. Libraries of guide RNAs (sgRNA) targeting all open reading frames permit the facile generation of thousands of genetic perturbations in a single pool of cells that can be screened for specific phenotypes to implicate gene function and cellular processes in an unbiased and systematic way. CRISPR-Cas screens provide researchers with a simple, efficient, and inexpensive method to uncover the genetic blueprints for cellular phenotypes. Furthermore, differential analysis of screens performed in various cell lines and from different cancer types can identify genes that are contextually essential in tumor cells, revealing potential targets for specific anticancer therapies. Performing genome-wide screens in human cells can be daunting, as this involves the handling of tens of millions of cells and requires analysis of large sets of data. The details of these screens, such as cell line characterization, CRISPR library considerations, and understanding the limitations and capabilities of CRISPR technology during analysis, are often overlooked. Provided here is a detailed protocol for the successful performance of pooled genome-wide CRISPR-Cas9 based screens.

CRISPR-Cas9 technology provides an efficient method to precisely edit the mammalian genome in any cell type and represents a novel means to perform genome-wide genetic screens. A detailed protocol discussing the steps required for the successful performance of pooled genome-wide CRISPR-Cas9 screens is provided here.

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian ...

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

Cancer Research

An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants

The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, this assay was used to analyze intrinsic levels of MeCP2 in mouse brain tissue and the uptake of TAT-MeCP2 in human dermal fibroblasts. The MeCP2-ECLIA produces highly accurate and reproducible measurements with low intra- and inter-assay error. In summary, we developed a quantitative method for the evaluation of MeCP2 protein variants that can be utilized in high-throughput screens.

The electrochemiluminescence immunoassay (ECLIA) is a novel approach for quantitative detection of endogenous and exogenously applied MeCP2 protein variants, which produces highly quantitative, accurate and reproducible measurements with low intra- and inter-assay error over a wide working range. Here, the protocol for the MeCP2-ECLIA in a 96-well format is described.

The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, ...

Biochemistry

Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve survival in patients with acute myeloid leukemia (AML). A small fraction of leukemic cells that survive chemotherapy have a poised epigenetic state to tolerate chemotherapeutic insult. Further exposure to chemotherapy allows these drug persister cells to attain a fixed epigenetic state, which leads to altered gene expression, resulting in the proliferation of these drug-resistant populations and eventually relapse or refractory disease. Therefore, identifying epigenetic modulations that necessitate the survival of drug-resistant leukemic cells is critical. We detail a protocol to identify epigenetic modulators that mediate resistance to the nucleoside analog cytarabine (AraC) using pooled shRNA library screening in an acquired cytarabine-resistant AML cell line. The library consists of 5,485 shRNA constructs targeting 407 human epigenetic factors, which allows high-throughput epigenetic factor screening.

High-throughput RNA interference (RNAi) screening using a pool of lentiviral shRNAs can be a tool to detect therapeutically relevant synthetic lethal targets in malignancies. We provide a pooled shRNA screening approach to investigate the epigenetic effectors in acute myeloid leukemia (AML).

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve ...

Genome-Scale Screening to Identify and Compare Therapeutic Targets in Tumors

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome neurofibromatosis type 1 (NF1), MPNSTs are the most common malignancy and the leading cause of death. These rare and aggressive soft-tissue sarcomas offer a stark future, with 5-year disease-free survival rates of 34-60%. Treatment options for individuals with MPNSTs are disappointingly limited, with disfiguring surgery being the foremost treatment option. Many once-promising therapies such as tipifarnib, an inhibitor of Ras signaling, have failed clinically. Likewise, phase II clinical trials with erlotinib, which targets the epidermal growth factor (EFGR), and sorafenib, which targets the vascular endothelial growth factor receptor (VEGF), platelet-derived growth factor receptor (PDGF), and Raf, in combination with standard chemotherapy, have also failed to produce a response in patients. 
In recent years, functional genomic screening methods combined with genetic profiling of cancer cell lines have proven useful for identifying essential cytoplasmic signaling pathways and the development of target-specific therapies. In the case of rare tumor types, a variation of this approach known as cross-species comparative oncogenomics is increasingly being used to identify novel therapeutic targets. In cross-species comparative oncogenomics, genetic profiling and functional genomics are performed in genetically engineered mouse (GEM) models and the results are then validated in the rare human specimens and cell lines that are available. 
This paper describes how to identify candidate driver gene mutations in human and mouse MPNST cells using whole exome sequencing (WES). We then describe how to perform genome-scale shRNA screens to identify and compare critical signaling pathways in mouse and human MPNST cells and identify druggable targets in these pathways. These methodologies provide an effective approach to identifying new therapeutic targets in a variety of human cancer types.

Author Spotlight: Finding New Therapeutic Targets for Malignant Peripheral Nerve Sheath Tumor Through Genome-Scale shRNA Screens 

We have developed a cross-species comparative oncogenomics approach utilizing genomic analyses and functional genomic screens to identify and compare therapeutic targets in tumors arising in genetically engineered mouse models and the corresponding human tumor type.

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome ...

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies.
There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids 1. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library.
The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes 2. Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 108 TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification.
Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line 3, 4. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.

Here we use a human LentiPlex pooled library and traditional sequencing methods to identify gene targets promoting cell survival. We demonstrate how to set up and deconvolute a LentiPlex screen and validate the results.

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to ...

Biology

Screening Peptides that Activate MRGPRX2 using Engineered HEK Cells

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new therapeutics. Mas related G-protein receptor-X2 (MRGPRX2) is an important receptor that regulates mast cell activation and, thus, directs the general immune response. Numerous ligands for MRGPRX2 have been identified and include endogenous peptides like PAMPs, defensins, LL-37 and other protein fragments (i.e., degraded albumin). Further identification of MRGPRX2 specific ligands requires the screening of a large number of peptides (i.e., peptide library); however, mast cells are difficult and expensive to maintain in vitro and, therefore, not economical to use for screening large numbers of molecules. The present paper demonstrates a method to design, develop, and screen a library of small peptide molecules using MRGPRX2 expressing HEK cells. This cell line is relatively easy and inexpensive to maintain and can be used for in vitro high-throughput analysis. A calcium sensitive Fura-2 fluorescent dye to mark intracellular calcium flux upon activation was used to monitor the activation. The ratio of fluorescence intensity of Fura-2 at 510 nm against excitation wavelengths of 340 and 380 nm was used to calculate calcium concentration. The peptide library used to verify this system was based on the endogenous proadrenomedullin N-terminal 12 (PAMP-12) secretagogue, which is known to bind MRGPRX2 with high specificity and affinity. Subsequent peptides were generated through amino acid truncation and alanine scanning techniques applied to PAMP-12. The method described here is simple and inexpensive yet robust for screening a large library of compounds to identify binding domains and other important parameters that play an important role in receptor activation.

Techniques for generating a library of short peptides that can activate mast cells via the MRGPRX2 receptor are described. Associated techniques are easy, inexpensive, and can be extended to other cell receptors.

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new ...

Immunology and Infection

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome

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