Name: pooled shrna screen for reactivation of mecp2 on the inactive x chromosome
Uploaded: 2018-03-02T13:00:00
Description: Watch this Scientific Journal Video about Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome at JoVE.com

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>Eu...

<ol>
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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. 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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. 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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>

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&#39;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&#8211;401&#8211;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...

Watch this Scientific Journal Video about Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome at JoVE.com

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 ...

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