Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Summary

Here we employ FISH methodology to track LINE-1 retrotransposition at the single nuclei level in chromosome spreads of HepG2 cell lines stably expressing synthetic LINE-1.

Abstract

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are inactive due to 5' truncations, ~80-100 of these elements remain retrotransposition competent and propagate to different locations throughout the genome via RNA intermediates. While older L1s are believed to target AT rich regions of the genome, the chromosomal targets of newer, more active L1s remain poorly defined. Here we describe fluorescence in situ hybridization (FISH) methodology that can be used to track patterns of L1 insertion and rates of ectopic L1 incorporation at the single nucleus level. In these experiments, fluorescein isothiocyanate/cyanine-3 (FITC/CY3) labeled neomycin probes were employed to track L1 retrotransposition in vitro in HepG2 cells stably expressing ectopic L1. This methodology prevents errors in the estimation of rates of retrotransposition posed by toxicity and account for the occurrence of multiple insertions into a single nucleus.

Introduction

Human Long Interspersed Nuclear Element-1 (Line-1 or L1) is an autonomous mobile element that propagates within the genome through a "copy and paste" retrotransposition mechanism. A typical human L1 is ~6 kb long and consists of a 5'UTR (Untranslated Region) that serves as a promoter, two open reading frames (ORFs): L1-ORF1 and L1-ORF2, and a 3'UTR with a polyA tail. L1-ORF1 protein has three distinct domains: a coil-coil domain, RNA recognition Motif and C-terminal domain, while L1-ORF2 protein has endonuclease, reverse transcriptase and cysteine rich domains^1,2,3,4,5. L1-ORF1 exhibits nucleic acid chaperone activities, while L1-ORF2 provides enzymatic activities, with both proteins required for retrotransposition^1,2,6.

A cycle of L1 retrotransposition starts with transcription from the 5'UTR of L1 bicistronic mRNA by RNA polymerase II, translocation into the cytoplasm, and translation. In the cytoplasm, L1-ORF1 exhibits either cis-binding where it packages its own RNA or trans-binding where it packages other RNAs (i.e., SINE/SVAs/pseudogenes) to form a ribonucleoprotein particle (RNP) with L1-ORF2p^7,8. RNPs translocate into the nucleus where the endonuclease activity of L1-ORF2p nicks genomic DNA (gDNA) to expose an OH^- group⁹, that is in turn used by reverse transcriptase to prime and reverse synthesize RNA to DNA from the 3' end. During reverse synthesis, the second strand of DNA is nicked 7-20 bp from the original nick site to create staggered breaks which are filled to form the signature L1 insertion sequences (e.g., TTTTAA) called target site duplications (TSD)^9,10. This process is known as target prime reverse transcription (TPRT) and leads to insertion of full or truncated copies of L1/other DNAs with TSDs at both ends of the inserted sequence. L1 retrotransposition has also been shown to be mediated through TPRT-like non-homologous end-joining in some cells types¹¹.

The L1 retrotransposition vector employed in our studies is non-episomal and consists of tagged L1-ORF1&2p driven by combined CMV-L1-5'UTR promoters (Figure 1)¹². Earlier versions of this construct have been described in studies using yeast and human cell cultures^13,14,15. Two distinct CMV promoters located at the 5' and 3' ends of the vector, with the 3' end placed in reverse orientation to drive expression of neomycin after splicing and integration. The retrotransposition indicator cassette at the 3' end consists of a neomycin gene inserted antisense to L1-ORFs and rendered inactive by separation into two halves by a globin intron with spliced donor (SD) and spliced acceptor (SA) sites (Figure 1). Upon integration into a chromosome, L1 is transcribed from the common promoter to produce an mRNA that consists of a bicistronic mRNA and inactive neomycin mRNA. During RNA processing, the globin intron is spliced out of the neomycin gene to restore a fully functional neomycin gene. The hybrid mRNA is packaged into a RNP in cis and translocated into the nucleus where it is integrated into the genome as either full length or truncated insertions using TPRT.

Here we describe a methodology that uses fluorescence in situ hybridization (FISH) with probes specifically directed at the spliced neomycin gene (SNeo) to track L1-retrotransposiition patterns and insertion rates at the single nucleus level. The efficiency and specificity of detection was confirmed using retrotransposition competent and incompetent constructs and probes to detect SNeo or the neomycin and globin intron junction¹⁶. This methodology accounts for some of the shortcomings of cell culture based retrotransposition assays, such as multiple insertions, colony resistance and favorable clone expansion.

Protocol

NOTE: All steps should be carried out at room temperature unless otherwise specified. Please refer to Reagents section for details on how to prepare individual reagents.

1. Labeling L1 Probes

NOTE: Probes can be labeled by chemical or PCR labeling.

1. Chemical labeling
   1. Make 0.7-1% agarose gel in Tris-acetate-EDTA (TAE) buffer, heat in a microwave until melted, allow the gel to cool, and then add ethidium bromide (0.5 µg/ml). Pour into gel tray, add combs and allow the gel to solidify at room temperature.
   2. Label the SNeo probe (1-2 µg) with CY3 or FITC at 37 °C for 1 hr according to the manufacturer's protocol.
      NOTE: SNeo denotes the Spliced Neomycin gene expressed after a full cycle of retrotransposition. The probe is ~1,000 bp in size. SNeo can be PCR amplified from any vector or from genomic DNA. See Table of Materials and Reagents for information on Streptavidin-CY3/FITC labeling reagents.
   3. Mix the labeled SNeo probe solution with 1x DNA loading buffer, load into each well and run at 75-100 volts for 15-20 min.
   4. Visualize the probe band using a Ultra-Violet (UV) Illuminator. Note that the size of the SNeo probe can be adjusted and that Lock nuclei acid (LNA) can be also be added (Figure 2).
   5. Cut labeled SNeo probe from the gel with a clean blade, solubilize and purify the SNeo probe from the gel using a PCR Clean-Up kit according to manufacturer's protocol.
      CAUTION: Cover every exposed part of the body with protective shields (i.e., laboratory coats and UV clear face mask) when viewing and cutting the gel. See Table of Material and Reagents for information to gel purify PCR products.
   6. Re-run 5 µl of SNeo labeled probe with an un-labeled SNeo probe on a 0.7% agarose gel (see 1.1.1) to confirm size increase and loss of signal intensity (Figure 2).
   7. Quantify the amount of labeled SNeo probe by measuring the absorbance at 260 nm and dilute the SNeo probe to 10 ng/µl aliquots in nuclease free H₂O. Store aliquots at -20 °C.
2. PCR Labeling (Alternative method for probe labeling)
   1. Combine SNeo specific primers (5'-ggatagcattgggagatatacct-3' and 5'-attgaacaagatg gattgcacgc-3') with PCR reagents into a single tube: 13.6 µl nuclease free H₂O; 0.1 µl dATP, dCTP, dGTP (10 nM); 0.05 µl dTTP (10 nM); 0.05 µl dUTP-biotin/dUTP-FITC/CY3 (10 nM); 4 µl Go Tag 5x buffer; 0.4 µl Go Tag Polymerase; 1 µl SNeo template (10 ng). See Table of Materials and Reagents for information on PCR reagents.
   2. Adjust the amount of each reagent by multiplying by the total number of samples.
   3. Use the following PCR cycling parameters to amplify and label SNeo probes: 95 °C for 2 min; 35 cycles of 95 °C for 30 sec, 62 °C for 30 sec, 72 °C for 60 sec; 72 °C for 2 min; Hold at 4 °C indefinitely.
      NOTE: The annealing temperature can be adjusted or gradient PCR can be completed to determine optimal annealing temperatures for other probes.
   4. Make 0.7-1% gel as in section 1.1.1, load and visualize the probe band as in section 1.1.4.
   5. Cut and purify the labeled SNeo probe as in section 1.1.5.
   6. Re-run 5 µl of SNeo labeled probe with un-labeled SNeo probe to confirm size increase and loss of signal intensity (see Figure 2).
   7. Quantify the amount of labeled SNeo probe by measuring absorbance at 260 nm and dilute SNeo probe to 10 ng/µl aliquots in nuclease free H₂O.

2. Preparing Chromosome Spreads

1. Generate stable clones expressing vector backbone (control) or retrotransposition competent L1 using standard selection methodologies.^12,16 While non-episomal reporters were used to correlate findings with studies of transcription, episomal vectors could also be used. Grow cells stably expressing control or L1 vector (1 x 10⁶ cells per 10 cm plate, with adjustments in plate size made as needed) in complete growth media. For HepG2 cells, use RPMI-1600, 10% FBS and 200 µg/ml of hygromycin. Grow cultures to 70% confluence at 37 °C and 5% CO₂.
   NOTE: The choice of growth medium is dependent on cell type. Given that the plasmids used here carry selection cassettes for both hygromycin and neomycin, stable selection of clones could also be done with neomycin after selection on hygromycin, but the amount of neomycin needs to be optimized to establish tolerance levels for each cell type.
2. Add colcemid (0.4 µg/ml) to culture medium and incubate for 90 min to arrest cells at metaphase. If using different cell types, determine the optimal concentration of colcemid and time of exposure (60, 90, and 120 min) for optimal metaphase arrest empirically.
3. Wash cells 2x with 10 ml of 1x Dulbecco's PBS (DPBS), trypsinize by adding 3-5 ml of 0.25% trypsin solution to the cells and incubate for 5 min to detach the cells. Inactivate trypsin with an equal amount of media containing 10% FBS.
4. Centrifuge the cells for 2 min at 1,000 x g at 4 °C, aspirate the medium from the cell pellet and wash the cells with DPBS. Aspirate all DPBS, leaving 200 µl of DPBS to re-suspend the cells. Ensure cells are mixed well and that all clumps are dispersed. Use flickering or gentle pipetting to mix and disperse clumps and avoid vortexing.
5. Add 5 ml of hypotonic solution (i.e., 75 mM KCl) that is pre-warmed to 37 °C drop-wise while rotating the 15 ml tube horizontally and incubate at 37 °C for 20 min. See Table of Materials and Reagents for information on how to obtain and make hypotonic solution.
6. Centrifuge cells at 120 x g for 5 min at 4 °C and repeat steps 2.4 - 2.5 3x leaving approximately 200 µl of hypotonic solution to re-suspend the cells after each wash. Ensure that at the end of the 3 washes, the pellet is visibly white and swollen.
7. Re-suspend the pellet in 200 µl of Carnoy Fixative solution and make 1: 2, 1: 4, 1: 8, 1:16 dilutions in Carnoy Fixative solution. Drop 10 µl of each dilution onto a dry clean slide from approximately 1 cm above and immediately expose the spread-free side to hot steam from boiling water for 30 sec. See Table of Materials and Reagents for information on how to make Carnoy Fixative solution.
8. Dry the spreads at room temperature, stain with 0.1 µg/ml Hoechst-33342 by immersion for 15-20 min in Coplin Jar and wash 3x with DPBS. See Table of Materials and Reagents for information on how to make the Hoechst-33342 solution.
9. View spreads on fluorescence/phase-contrast microscope at 40X magnification. After optimizing spread preparation, spreads need not be stained with Hoechst-33342. View spreads on phase-contrast microscope at 10X magnification to determine spread quality and separation as outlined in the Results section (see Figure 3).
10. Select and circle good spreads with a Diamond Point Marker on the opposite side of the slide (i.e., spread-free side).
11. Store spreads at -20 °C for up to a month. Avoid exposure to moisture.

3. Fluorescence In Situ Hybridization (FISH)

1. Stabilizing and dehydrating spreads
   NOTE: Equilibrate metaphase chromosome spreads to room temperature if stored at -20 °C. See Table of Materials and Reagents for information on how to obtain and make reagents.
   1. Incubate metaphase chromosome spreads with 200 µl RNase A for 1 hr at 37 °C.
   2. Wash slides in 2x-SSC buffer twice for 5 min each followed by rinsing with 10 mM HCl solution.
   3. Incubate spreads with 1% pepsin for 10 min at 37 °C, rinse with deionized H₂O and wash twice with 2x-SSC buffer for 5 min each.
   4. Incubate spreads with 4% paraformaldehyde for 10 min and wash in 2x SSC buffer twice for 5 min each.
   5. Dehydrate spread by incubating for 2 min in ethanol series: 70%, 80%, and 95% ethanol.
   6. Air-dry slides.
2. Hybridization Spreads with L1 -labeled retrotransposition probes (i.e., SNeo)
   CAUTION: For direct-labeled probes, keep away from light at all times. See Table of Materials and Reagents for information on how to obtain and make reagents.
   1. Add 30 ng of SNeo to hybridization buffer, heat at 72 °C for 10 min and cool for 2 min at room temperature.
   2. Add 30 µl of SNeo probe solution to each spread, cover with coverslip and seal the edges with rubber cement. Ensure no bubble formation.
   3. Heat the slide at 72 °C for 5 min on a heat block, gradually drop the temperature to 37 °C, and incubate overnight in a dark humidified chamber at 37 °C.
3. Washing and Viewing (or Addition of Secondary Antibody)
   CAUTION: For direct-labeled probes, keep away from light at all times.
   NOTE: See Table of Materials and Reagents for information on how to prepare. Use of sufficient volume to cover the entire chromosome spread is recommended. Remember that excess volume will be lost when coverslip is added.
   1. Immerse slides in 2x SSC buffer to remove coverslips.
   2. Wash slides by immersion in 2x-SSC buffer at 45 °C for 5 min.
   3. Wash slides by immersion in wash buffer at 45 °C for 5 min, 2x.
   4. Wash slides by immersion in 0.1x SSC buffer at 45 °C for 10 min.
   5. Wash slides by immersion in 2x SSC buffer at 45 °C for 10 min.
      NOTE: Place a Coplin Jar containing recommended buffer in a water bath, adjust temperature to 45 °C.
   6. Cool slides to room temperature and equilibrate slides in detection buffer.
   7. For direct labeled probes, skip to step 3.4.10.
   8. For indirect labeled probes, block in blocking buffer for 20-30 min and wash 3x in DPBS.
   9. Incubate with 50 µl of secondary antibody (e.g., 5 µg/ml streptavidin-FITC, or αCY3 in blocking buffer) for 1 hr.
   10. Wash slides in 2x SSC for 5 min twice.
   11. Counterstain with Hoechst-33342 solution (0.1 µg/ml) for 10 min.
       NOTE: this staining is done to stain chromosomes for co-localization with probe.
   12. Wash in DPBS 3x, add a drop of mounting medium, place a coverslip and seal the edges with nail polish.
   13. Analyze L1 retrotransposition using fluorescence microscope at 40X magnification.

Results

A schematic diagram of the L1 retrotransposition vector is presented in Figure 1. The vector consists of a neomycin gene in antisense orientation to L1 ORFs that is interrupted by a globin intron in sense orientation and sandwiched by SD and SA sites. When stably integrated into a chromosome, L1 mRNA is transcribed from the combined CMV and L1-5'UTR promoter (Figure 1). During RNA processing, the globin intron is spliced out...

Discussion

Methodologies such as whole genome sequencing, inverse PCR and southern blotting have been employed to study L1 retrotransposition. Although these methodologies are extremely valuable in locating where L1 insertions occur within genomes, a confounding challenge for all of them is the need for in-silico programing to reassemble sequences. The FISH methodology described here is designed to complement these methods, especially in the case of studies requiring analyses of ectopic L1 retrot...

Materials

Name	Company	Catalog Number	Comments
Labeling Probes
Go Tag DNA polymerase	Promega	M3178
GO Tag 10X colorless buffer	Promega	M3178
Individual dNTP	Sigma	DNTP10	Adjust the concentration of dATP, dGTP, dCTP to 2mM and dTTP to 1.5mM  in nuclease free water.
dUTP-16-Biotin (0.5mM)	Roche	11093070910
dUTP-FITC (0.5mM)	Thermo Scientific	R0101
dUTP-CY3 (0.5mM)	Sigma	GEPA53022
MIRUS FISH Labeling Kit	MIRUS	MIR3625
MIRUS FISH Labeling Kit	MIRUS	MIR3225
NucleoSpin PCR Clean-Up kit	Qiagen	1410/0030	Any PCR clean reagent can be used in place of NucleoSpin PCR Clean Up Kit
PCR Machine			Any Thermocycler machine can be used to amplify the label product.
Agarose	Sigma	A9539
Name	Company	Catalog Number	Comments
Preparing chromosome Spreads.
Colcemid	Life Technologies	15212-012	Add Colcemid directly to growth media  to a final concentration of 0.4 µg/mL
1M KCl	Sigma	P9541	Dilute 1M KCl solution to 75 mM solution to make Hypotonic Solution
Carnoy Fixative Solution			Mix 3 volumes of methanol and 1 volume of acetic acid to make Carnoy Fixative Solution. Make Fresh everytime.
Methanol	Sigma	322415
Acetic acid	Sigma	320099
Hoechst-33342	Thermo Scientific	62249	Dissolve Hoechst-33342 in PBS to final concentration of 1 mg/mL.
Diamond Point Marker	Thermo Scientific	750
Frosted Slides	Thermo Scientific	2951-001
Trypsin-EDTA (0.25%)	Life Technologies	R001100	To detech cells, incubate cell in Trypsin for 5 mins and inactive with equal volume of complete media
Cover slides	VWR	48366067
Coplin Jars	Thermo Scientific	107
Heat Block			Any heat block can be used for this purpose, though blocks fitted for heating slides are recommended.
Beaker (600ml)	Sigma	CLS1003600	Fill the beaker with water, heat to boil, use the hot steam to burst chromosomes.
Nikon Microscope	Nikon	125690	Any microscope can be used to look at spread quality.
Name	Company	Catalog Number	Comments
Reagents and Materials for FISH
Trisodium citrate	Sigma	S1804
Sodium Chroride	Sigma	S3014
Formamide	Sigma	F9037
Tween-20	Sigma	F1379
Detran Sulfate	Sigma	D8906
SDS	Sigma	L3771
Salmon Sperm DNA	Life Technologies	15632-011
Bovine Serum Albumin (BSA)	Sigma	A8531
HCl (N)	Sigma	38283
Ethyl Alcohol	Sigma	459844
Methanol	Sigma	322415
DPBS	Life Technologies	14190-250
NaOH	Sigma	S8045
Rnase A	Sigma	R4642
Pepsin	Sigma	P6887
Paraformaldehyde (PFA)	Sigma	P6148
Hoechst-33342	Thermo Scientific	62249	Dissolve Hoechst-33342 in PBS to final concentration of 1 mg/mL.
Rubber Cement/Cytobond Sealant		2020-00-1
Seven Coplin Jars	Thermo Scientific	107
Dark Humidified chamber	Sigma	CLS2551
Cover slides	VWR	48366067
Dry Digital Heat Block	VWR	13259
Fluorescence Microscope
20X Saline-Sodium Citrate (20X SSC)			Combine 175 g of NaCl, and 88.2 g of trisodium citrate with 800 mL of molecular grade H2O. Stir while adjusting to pH 7. Once the all salts dissolve, adjust the volume to 1 L and filter through 0.22 µm Filter paper. Store at 4 °C.
1mg/mL of Rnase A			Dilute 20X SSC buffer to 2X SSC buffer. Dissolve RNase A in 2X SSC buffer to a final concentration of 1 mg/mL. Make fresh solution every time.
1% Pepsin			Dissolve  pepsin (W/V) in 10 mM HCl to a final concentration of 1 % solution. Make fresh every time.
4% Paraformaldehyde (4% PFA)			Weigh 4.0 g of PFA in fume hood, add 50 mL of 1xPBS, heat to 60 °C while stirring and adjust the pH with drops of NaOH until all PFA dissolves and the solution becomes clear. Adjust the volume to 100 mL, filter through 0.22 µm Filter paper and store 5 mL aliquots at -20 °C. Thaw aliquot of PFA at 37 °C for 10-15 min for subsequent uses.  Adjust filter size depending on amount of paraformaldehyde.
Wash Buffer			Combine 100 mL of Formamide (20%) with 2.5 mL of 20X SSC, adjust to pH 7 with 1.0 M HCl and bring the volume to 500 mL with molecular grade H2O.
Hybridization Buffer			Combine 50% formamide, 10% dextran sulfate, 0.1% SDS, and 300 ng/mL Salmon Sperm DNA in 2X SSC buffer.  Amount of Formamide determine how focused chromatids are, titrate the amount
Detection Buffer			Dilute 20X SSC buffer to 4X and add 0.2% Tween-20 to make detection buffer.
Blocking Buffer			Combine 5% bovine serum albumin (BSA) with 0.2% Tween-20 in 4X SSC buffer.
Dehydrating Solution			Make 70%, 80%, and 95% ethanol solutions.