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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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 ("reporter cells") 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 >60,000 different shRNAs targeting >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  activity.

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Protocol

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

  1. Preparing mouse tendon fibroblasts from a female XMeCP2-LUC-HR /XMeCP2 mouse
    1. Euthanize a 10-12 day-old female XMeCP2-LUC-HR/XMeCP2 mouse.
    2. Using straight dissection scissors, make a circumferential incision through the skin of the proximal tail portion.
    3. 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.
    4. Transfer the fragmented tissue to a 15 mL conical tube containing 5 mL of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 1 U/mL penicillin, 1 µg/mL streptomycin, and 1.5 mg/mL collagenase, pre-filtered through 0.45 µm and then 0.2 µm mesh filters. Incubate for 3-4 h at 37 °C with continuous rotation.
    5. 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 µg/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.
    6. Immortalize the cells by performing a transduction with an expression vector carrying HPV-16 viral oncogenes (E6/E7), as described previously7.
  2. Selecting single cell clones and characterizing MeCP2 reporter expression
    1. 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 °C. Quench with 9 mL of DMEM-10 and collect the resuspended cells in a 15 mL conical tube.
    2. Dilute the cells with DMEM-10 to a final concentration of 1 cell/100 µL. Transfer to 96-well plates by pipetting 100 µL of the suspension into each well.
    3. Incubate at 37 °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 µL of 0.05% trypsin-EDTA per well and split them into two duplicate sets of 96-well plates.
    4. 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.
    5. 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.
    6. Perform the same for a clone with the highest luciferase activity; this will be used as a positive control for validation purposes.
    7. Harvest an aliquot of each well in the 6-well plate for a Western blot. Mobilize the cells with 200 µL of 0.05% trypsin-EDTA. Incubate for 2-5 min at 37 °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. 
    8. Perform a Western blot with anti-MeCP2 antibody to confirm that luciferase-negative cells express, 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.
    9. Plate several negative clones at 2 x 105 cells/well in a 6-well plate. Treat once with 10 µM 5-AZA, incubate for 72 h without changing the media, and assay for luciferase activity as described in step 1.2.3.
    10. 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.

2. Screening the library for reactivation using hygromycin B selection

  1. Infecting the test clone, applying selection, and harvesting DNA for sequencing
    1. 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.
    2. 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.
    3. In the morning, plate 1 x 106 Xi cells onto 10 cm tissue culture plates.
    4. In the afternoon, infect the plates by replacing the media with fresh DMEM-10 containing viral supernatant and 5-10 µg/mL of polybrene. Use the amount of viral supernatant determined in step 2.1.2.
    5. After 18-24 h, aspirate the media and add 10 mL of fresh DMEM-10 to each plate. Culture for another 48 h.
    6. 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 µm mesh filter. Pool the filtered suspensions together, and seed two sets of 10 cm plates at 1 x 106 cells/plate.
    7. In the afternoon, replace the media in one set of plates with DMEM-10 containing 15 µg/mL of hygromycin B (day 0). In the other set, replace the media with fresh DMEM-10 only.
    8. 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 °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.
    9. 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 °C until further use.
    10. Continue culturing hygromycin B selection set for 4 more days. Replenish the selection media with fresh DMEM-10 containing hygromycin B after 2 days.
    11. 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.
  2. Identifying hairpins enriched in the post-selection samples
    1. Use 5' context (5'-ATCGTTGCCTGCACATCTTGG-3') and 3' reverse loop primers (5'-ACATCTGTGGCTTCACTA-3') 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' context primer and 3' 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.
    2. 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.
    3. 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.
    4. 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 <5% FDR as "hits" for further testing.

3. Validating the identified hairpins ("hits")

  1. Packaging lentiviruses containing shRNAs identified in the screen
    1. Seed 8 x 106 293FT cells per plate on 10 cm plates.
    2. The following day, replace the media with 9 mL of antibiotic-free DMEM-10 per plate; do this 30 minutes before transfection.
    3. During the 30-minute interval, prepare two mixes for transfection:
      1. Prepare Mix 1 (per plate/shRNA; scale up according to the total number of shRNAs): 10 µL 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.
      2. Prepare Mix 2 (per shRNA): 4 µg of the shRNA vector to be packaged, 1.5 µg of the VSV-G envelope vector (pMD2.G), and 2.5 µg 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.
    4. 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.
    5. Apply the mixture onto each plate from step 3.1.2 in a dropwise fashion; swirl the plate gently to mix.
    6. The following day, replace the media with 5 mL of fresh DMEM-10.
    7. 48 hours after initial transfection, collect the supernatant and run it through a 0.22 µm filter. Pre-wet the filter with DMEM-10 to maximize recovery.
    8. Aliquot the filtered supernatant into amounts ideal for infecting a 6-well plate well.
    9. Freeze the aliquots at -80 °C for later use, or proceed immediately with transduction.
  2. Transduction and testing of individual hairpins for luciferase reporter reactivation and reactivation of wild-type MeCP2 gene on the Xi.
    1. In the morning, plate 1 x 105 Xi cells/well onto 6 well plates.
    2. In the afternoon, infect the plates by replacing the media with 2 mL of fresh DMEM-10 containing lentiviral supernatant and 5-10 µg/mL of polybrene. Infect a control well with viral supernatant produced from an empty lentiviral vector.
    3. The following day, replace the media with 2 mL of DMEM-10 containing 1 mg/mL puromycin. Culture for four days.
    4. Replace the media with 2 mL of fresh DMEM-10 and allow 48-72 h for recovery.
    5. Mobilize the cells with 150 µL of 0.05% trypsin-EDTA. Incubate for 2-5 min at 37 °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.
    6. Remove the media and resuspend the cells in 100 µL 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.
    7. 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'-TTCCATGCCAAGGCCAAACAG-3', R: 5'-CCCATAAGGAGAAGAGACAACAGC-3').
    8. Optional: Increase the sensitivity of the assay by adding 5-AZA to a final concentration of 0.2 µM 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.
    9. 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'-TTCCATGCCAAGGCCAAACAG-3', R: 5'-CCCATAAGGAGAAGAGACAACAGC-3').

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Results

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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Discussion

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 >60,000 shRNAs targeting >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...

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Disclosures

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

Acknowledgements

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.).

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Materials

NameCompanyCatalog NumberComments
293FT Cell LineInvitrogenR700-07
5-AzacytidineSigmaA2385-100MG
Agencourt AMPure XPBeckman-CoulterA63881
Anti-MECP2 antibodyMillipore07-013
CollagenaseSigmaC2674-1G
Corning 96-Well Solid White Polystyrene MicroplatesFisher Scientific07-200-336
Dulbecco's Modified Eagle MediumGibco11965-092
Expression Arrest microRNA-adapted Retroviral Vector (pMSCV)Open BiosystemsEAV4679
Expression Arrest pSM2 Retroviral shRNAmir libraryOpen BiosystemsRMM3796
Fetal Bovine SerumFisherbrand03-600-511
HiSeq 2500IlluminaSY–401–2501Or equivalent
Hygromycin BCalbiochem400051-1MU
Lipofectamine 2000Invitrogen11668-019
Bright-Glo Luciferase Assay SystemPromegaE2610Homogenous Assay for Screening Colonies
Luciferase Assay SystemPromegaE4530Non-Homogenous Assay for Testing Individual Hairpins
Luminometer TopCount NXTPerkin ElmerN/AOr similar luminometer
MiSeq Reagent Kit v2IlluminaMS-102-2001Use kit compatible with your equipment
Opti-MEMGibco31985-070
Penicillin-StreptomycinGibco15140-122
pMD2.GAddgene#12259VSV-G envelope vector
PolybreneSigmaTR-1003-G
PolyethyleniminePolysciences23966-1
psPAX2Addgene#12260Packaging vector
PuromycinGibcoA11138-02
Trypsin-EDTA (0.05%), phenol redGibco25300-054

References

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  2. Guy, J., Gan, J., Selfridge, J., Cobb, S., Bird, A. Reversal of neurological defects in a mouse model of Rett syndrome. Science. 315, 1143-1147 (2007).
  3. Maduro, C., de Hoon, B., Gribnau, J. Fitting the Puzzle Pieces: the Bigger Picture of XCI. Trends Biochem Sci. 41, 138-147 (2016).
  4. Disteche, C. M. Dosage compensation of the sex chromosomes and autosomes. Semin Cell Dev Biol. 56, 9-18 (2016).
  5. Wang, J., et al. Wild-type microglia do not reverse pathology in mouse models of Rett syndrome. Nature. 521, E1-E4 (2015).
  6. Sripathy, S., et al. Screen for reactivation of MeCP2 on the inactive X chromosome identifies the BMP/TGF-beta superfamily as a regulator of XIST expression. Proc Natl Acad Sci U S A. 114, 1619-1624 (2017).
  7. Foster, S. A., Galloway, D. A. Human papillomavirus type 16 E7 alleviates a proliferation block in early passage human mammary epithelial cells. Oncogene. 12, 1773-1779 (1996).
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  9. Strezoska, Z., et al. Optimized PCR conditions and increased shRNA fold representation improve reproducibility of pooled shRNA screens. PLoS One. 7, e42341(2012).
  10. Green, M. R., Sambrook, J. Isolation of High-Molecular-Weight DNA from Mammalian Tissues Using Proteinase K and Phenol. Cold Spring Harb Protoc. 2017, (2017).
  11. Goto, Y., Takagi, N. Tetraploid embryos rescue embryonic lethality caused by an additional maternally inherited X chromosome in the mouse. Development. 125, 3353-3363 (1998).
  12. Csankovszki, G., Nagy, A., Jaenisch, R. Synergism of Xist RNA, DNA methylation, and histone hypoacetylation in maintaining X chromosome inactivation. J Cell Biol. 153, 773-784 (2001).
  13. Minajigi, A., et al. Chromosomes. A comprehensive Xist interactome reveals cohesin repulsion and an RNA-directed chromosome conformation. Science. 349, (2015).
  14. McHugh, C. A., et al. The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3. Nature. 521, 232-236 (2015).
  15. Bhatnagar, S., et al. Genetic and pharmacological reactivation of the mammalian inactive X chromosome. Proc Natl Acad Sci U S A. 111, 12591-12598 (2014).
  16. Minkovsky, A., et al. The Mbd1-Atf7ip-Setdb1 pathway contributes to the maintenance of X chromosome inactivation. Epigenetics Chromatin. 7, 12(2014).
  17. Minkovsky, A., et al. 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. Epigenetics Chromatin. 8, 42(2015).

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