supporting online material for -...

www.sciencemag.org/cgi/content/full/science.1193004/DC1

Supporting Online Material for

PiggyBac Transposon Mutagenesis: A Tool for Cancer Gene Discovery in Mice

Roland Rad, Lena Rad, Wei Wang, Juan Cadinanos, George Vassiliou, Stephen Rice, Lia S. Campos, Kosuke Yusa, Ruby Banerjee, Meng Amy Li, Jorge de la Rosa,

Alexander Strong, Dong Lu, Peter Ellis, Nathalie Conte, Fang Tang Yang, Pentao Liu, Allan Bradley*

*To whom correspondence should be addressed. E-mail: [email protected]

Published 14 October 2010 on Science Express

DOI: 10.1126/science.1193004

This PDF file includes

Materials and Methods Figs. S1 to S14 Tables S1 to S7 References

Supplementary Methods.

ATP constructs. ATP1/2/3 transposons are shown in figure 1B. All transposons have

both PB and SB inverted terminal repeats and can therefore be mobilized with both

transposon systems. ITRs have been cloned into pBlueScript and the following genetic

elements have been introduced in between the ITRs for all 3 types of transposons: Carp

β-actin splice acceptor (CβASA); En2SA, splice acceptor from exon 2 of the mouse

Engrailed-2 gene, Lun-SD from exon 1 of the mouse Foxf2 gene and two bidirectional

SV40 polyAs. Promoter elements carried by the transposons were unique to individual

transposons: CAG (CMV immediate early enhancer and chicken beta-actin gene

promoter) for ATP1; MSCV (murine stem cell virus long terminal repeat) for ATP2; and

PGK (phosphoglyeratekinase promoter) for ATP3. Detailed protocols of individual

cloning steps are available upon request.

Mouse strains. ATP transposons were cut out of pBlueScript using FspI/AflIII or

FspI/DraIII digestion and prepared for pronuclear injection using standard techniques in

house or at Polygene Inc. Transgenic lines were generated in the FVB (ATP1-S1/S2,

APT2-S1/S2, ATP3-S1/S2) or C57BL/6 (all other lines) background. Tail DNA from

founder animals was screened by Southern blotting using an En2SA probe or by PCR

(primers are listed in table S6). Metaphases from blood were prepared to identify

transposon donor loci and quantitative PCR was used to determine the transposon copy

number in founder mice. Founder mice were then crossed to C57BL/6 and offspring were

genotyped by PCR using primers listed in table S6. In founders with multiple transposon

integrations, the alleles were bred apart and separate transgenic lines were established.

Generally, F2 mice were used to establish transgenic lines and the transposon integration

site was confirmed in these animals after death by FISH on metaphases from spleen

preparations.

The RosaPB knock-in allele was established in AB1 embryonic stem cells as described

earlier (1). RosaPB knock-in mice were generated by blastocyst injection using standard

techniques. Mice were genotyped by PCR with primers listed in table S6. RosaSB mice

expressing the SB11 transposase have been described earlier (2, 3).

To generate the Hprt-Cag-DsRed knock-in reporter mouse line, an expression cassette

containing the DsRed-Monomer-N1 cDNA (Clontech) under the control of the CAG

promoter (4) was cloned together with a G418 selection cassette into a backbone

containing LoxP and Lox511 sites. Using RMCE (5), the CAG-DsRed cassette was

introduced intro the hprt locus of hprttm(rmce1)Brd mouse ES cells, which were

subsequently used to generate Hprt-Cag-DsRed mice. Animal experiments were

performed in accordance with the Animal Scientific Procedures Act 1986.

Analysis of DsRed expression in Hprt-Cag-DsRed mice. Tissues from 3 wild-type and

3 Hprt-Cag-DsRed male mice were dissected and DsRed expression was analyzed by

fluorescence detection using a Xenogen IVIS system (Caliper Life Sciences). The same

tissues were then homogenized in RIPA buffer supplemented with 1% SDS and 4 ug

(Hprt-Cag-DsRed heart) or 20 ug (all other tissues) of protein from them were analyzed

by Western blot using anti-DsRed polyclonal (632496; Clontech), anti-β-actin

monoclonal (A5441, Sigma) and anti-α-tubulin monoclonal (CP06, Calbiochem)

antibodies.

Comparative Genomic Hybridization. CGH array was executed using Agilent 244K

mouse whole genome arrays. DNA was labeled with Cy3 or Cy5 according to BioPrime

array CGH genomic labeling protocol (Invitrogen, Carlsbad, CA) and cleaned using

purelink PCR purification kit (Invitrogen). Hybridization was performed using mouse

genome CGH microarray244K from Agilent Technologies (Santa Clara, CA) according

to the manufacturer’s protocol. Slides were hybridized for 48 hours, washed, and scanned

with an Agilent microarray scanner; the data were analyzed using Feature Extraction

(Agilent Technologies), aCGH Spline (Tomas Fitzgerald, http://cran.r-

project.org/web/packages/aCGH.Spline/aCGH.Spline.pdf) and Genomic workbench

software packages (Agilent Technologies). Data normalization was done using the R

Package aCGH.Spline. This algorithm allows robust spline interpolation normalization

on dual color aCGH data. CGH calls were made with Genomic workbench software

using the ADM2 algorithm (6.0 threshold), with a minimum of 4 probes in the region as a

filter.

Quantitative PCR (qPCR) and quantitative reverse transcription PCR. (qRT-PCR).

qPCR on DNA from ear biopsies was used to determine the transposon copy number of

individual mouse lines. En2SA DNA amounts were quantified and normalized to beta-

actin DNA levels. Transposon copy numbers were determined by normalizing values

obtained from ATP mice to those from wild-type mice that only possessed endogenous

En2SA (2 copies) but not transposon-derived En2SA. Primer and probe sequences are

shown in table S6. qRT-PCR was used to analyze Nras and Spic expression in

hematopoietic tumors. Results were normalized to Gapdh expression. Primer and probe

sequences are listed in table S6. Experiments were performed as described earlier (6) on

the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) as described

earlier.

Flourescence in situ hybridization (FISH). Probes, specific for transposons, were

amplified using WGA 2-50 RXN (Whole Genome Amplification, Sigma). Amplified

DNA probes were directly labelled with Chromatide Texas Red dUTP (Invitrogen) using

WGA3 – 50 RXN (Genome Plex Reamplification Kit, Sigma). Probes were denatured at

65°C for 10 mins and preannealed at 37°C for approximately 30 – 45 mins. Slides were

prepared approximately a week in advance, to age metaphases at RT with a dessicant.

Slides were then pretreated in prewarmed (37°C) 2XSSC and Pepsin (75µl of 1% Pepsin

in 50 ml of 0.01M HCl) for 5 mins each. Pepsin treatment helps in the removal of

cytoplasmic proteins. After washing slides in 3 washes of 2XSSC for 3 mins each, slides

were treated in formaldehyde fixative (1.25 mls of formaldehyde, 40% w/v and 2.5mls of

1M MgCl2, 50mM MgCl2 in PBS) for 10 mins at RT. Slides were then washed in 3

washes of 2XSSC for 3 mins each, passed through an ethanol series and air dried. Slides

were then denatured at 63°C in 70% formamide, 2 XSSC for 1 min 30 secs , passed

through an ethanol series and air dried. Denatured and pre-annealed probe mixtures were

added to the slides, overlayed with coverslips, sealed with fixogum and incubated at 37°C

overnight. To further remove unbound probes, slided were subjected to posthybridysation

washes as follows: Fixogum was removed from the slides and coverslips soaked off in

2XSSC at RT. Slides were passed through the following stringency washes of 2XSSC,

50% formamide (2XSSC), 50% formamide (2XSSC) and 2XSSC respectively, for 5mins

each, prewarmed at 43°C. Slides were finally mounted in SlowFade (Invitrogen) with

DAPI (4,6 diamidino-2-phenylindole), overlayed with coverslip and sealed with nail

varnish. FISH’d metaphases were scanned in an epifluorescence microscope fitted with a

CCD camera and narrow band-pass filters. Images were captured using the Digital

Scientific, Smartcapture software. Metaphase images captured with SmartCapture were

karyotyped using the Digital Scientific Smarttype software.

Multicolor FISH. Multicolour FISH (M-FISH) was performed using mouse paints

prepared with flow sorted mouse chromosomes. The ‘mouse paint mix’ was prepared

using whole chromosome paints labelled with different combinations of 4 fluorochromes

(CY5, Cy3.5, Cy3 and FITC) and one hapten (Biotin). In total, 21 combinations were

obtained; no more than 3 fluorochromes for 20 combinations and all 5 for 1 combination

(Y chromosome). This resulted in a unique colour for each chromosome. After metaphase

slides were treated with pepsin to remove cytoplasmic proteins. Slides were then baked at

65°C for an hour before denaturation at 63°C in 70% formamide (in saline sodium

citrate) and dehydrated. Mouse paints were denatured at 65°C for 10 mins and a measure

of 10µl of paint was applied to each slide. This was followed by incubation, of the slides,

at 37°C for 48hrs to allow the probes to hybridise to the chromosomes. Following

hybridisation, slides were washed in 50% formamide (in saline sodium citrate) post-

hybridisation washes at 43°C and detected with Streptavidin Dylight 680 conjugated for

Biotin. Slides were then counterstained with 10µl of 4,6 diamidino-2-phenylindole

(DAPI) counterstain in antifade. Images of metaphase spreads were captured using an

epifluorescent microscope equipped with a 6 position filter wheel and a cooled charge

coupled device (CCD) camera, Leica DFC 350 FX. For each metaphase spread, six

images were captured using filter combinations specific for each of the five

fluorochromes and DAPI. A minimum of 10 metaphases were captured from each cell

line and analysed with Leica CW 4000 CytoFISH.

Detection of transposon mobilization. To analyze whether transposition indeed occurs

in double-transgenic mice, we performed PCR-based excision assays to determine

mobilized or non-mobilized transposons. At their chromosomal donor loci, transposons

are flanked by short sequences which are originating from the cloning vector. Primers for

PCR assays to detect mobilization (red arrows in figure 2D) were set into those flanking

regions. Lack of excision can be detected by PCR assays that detect non-mobilized

transposons (green arrows in figure 2D). Assays were performed on tail DNA from 6

weeks old mice. Primer sequences are listed in table S6.

Detection of transposon-Nras and transposon-Spic and Pten-transposon fusion

transcripts. Fusion transcripts were detected in tumors by RT-PCR using a MSCV-

LTR/LunSD-specific sense primer and exon 3 specific anti-sense primer in the Nras and

Spic genes or a Pten exon-1 specific forward primer and a Lun splice donor specific

reverse primer. Primer sequences and PCR product sizes are shown in table S6.

Sequencing of PCR products was performed to analyze whether fusion transcripts indeed

started from the 5’LTR of MSCV and were spliced into exon 2 of the Nras or Spic genes

through the transposon’s splice donor.

Splinkerette PCR for the amplification of transposon integration sites. PB transposon

integration sites were determined by splinkerette PCR as described earlier (1). After

digestion of genomic DNA with MboI, DNA was ligated with splinkerette adaptors. Both

ends of the transposon were used as a tag for first round PCRs using HmSp1/PB-L-Sp1

or HmpSp1/PB-R-Sp1 primer pairs (table S6). Second round PCRs were performed with

HmSp2/PB-LSp2 or HmSp2/PB-R-Sp2 primers pairs. PCR products were cloned into

pGEM-T Easy vector and transformed into E. coli. Approximately 50.000 E. coli

colonies were picked and grown from 63 tumors. Sequencing was performed after DNA

preparation using Sp6 and T7 primers.

Mapping of insertion sequences to the mouse genome and identification of common

integration sites. For mapping integrations to the mouse genome, we used ssaha2. Query

sequences were filtered to contain splinkerette primer sequences which lie in the

transposon ITRs. Redundant sequences that arose from the same tumor and mapped to

the same genomic location were "collapsed" to one integration. To identify regions in the

genome that are more frequently hit by transposons than would be expected by chance

(common insertion sites; CISs), non-redundant insertions were finally subjected to

statistical analysis using a frame-work based on Gaussian Kernel Convolution as

described earlier (7, 8). The analysis was performed at different Kernel sizes (10Kb,

30Kb, 60Kb, 100Kb). The number of CISs identified in individual windows was similar

and ranged between 50 and 59. There was a considerable overlap, with most of the CISs

being detected in several windows. The combined analysis identified 72 unique CISs at

67 independent loci.

Histology and immunohistochemistry. Staining of mouse hematopoietic tumors was

performed on paraffin-embedded sections for T, B and myeloid makers using rabbit anti-

mouse CD3 (Abcam; UK), rat anti-mouse B220 (CD45R; R&D systems) and rabbit anti-

human myeloperoxidase (Dako). Biotin-conjugated donkey anti-rat IgG and Biotin-

conjugated donkey anti-rabbit IgG (both from Jackson ImmunoResearch) were used as

secondary antibodies.

Generation of mouse lines

RosaPB

19 PB/SBATP lines

Characterizationof mice

Transposon location & copy number

in ATP lines

Establishment and comprehensive functional analysis of the PB cancer gene discovery platform in vivo

EFFECTS OF: - Transposon type- Transposon donor locus- Transposon copy number ON: - Embryonic lethality- Tumor development- Tumor latency/spectrum

Characteristics of PB transposition in vivo

- Mobilization efficiency/dynamics- Integration pattern: local hopping

vs. genome-wide mutagenesis

- Molecular validation of transp. effects - Clonality of integrations- Genomic stability

- Cloning of transposon integrations- Identification of novel CIS

RosaPB x 14 ATP lines

RosaSB

RosaSB x 3 ATP lines

Analysis of hematopoetictumours

Identification of new candidate cancer genes

Supplementary figure S1

ATP1-H10

ATP1-H12

Chr10 A1

Chr18 E3-4

ATP1-H5

Chr6 F1-2

ATP1-S2

ATP1-H36

Chr16 E1-2

ChrX F1-2

ATP1-H37

Chr5 A2-3


ATP1-H8

ATP1-H39

ATP1-H44

Chr5 A2-3

Chr13 C3-D1

Chr5 C3D

ATP2-S2

Chr12 A1.1

ATP2-S1

Chr17 A1

ATP2-H27

Chr4 A2-3

Supplementary figure S2 (cont)

ATP2-H28

Chr19 A

ATP2-H32

Chr2 E2

ATP2-H31

Chr2 E2

ATP2-H33

Chr7 E3-F1

ATP3-S2

Chr8 A2

Supplementary figure S2 (cont)

0

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21 53 4

CβA SApb sb sb pb

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ATP1-S2R26PBase/+

ATP1-S2No PBase

B

PBase - + - + - + - +

ATP1-S1

ATP1-S2

ATP2-S1

ATP3-S2C

TS TS/TP

trans

poso

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tp/ts

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RosaPB/ATP3-S1 median survival = 656d

RosaPBATP3-S1RosaPB/ATP3-S1


A CB

D E F

G H I J



B

A

Chr17 del (0.7Mb)


1 2 3 4 5 6 7 8 9 Ros

aPB

ATP2

ATP2

ATP2

tail tumorRosaPB/ATP2

3Kb

2Kb1.5Kb1.0Kb

10Kb

1 2 3 3 4 4 5 6 7 8 9s s s t s L s s s s s

2.6 Kb1.3 Kb

En2SA

BamHIBamHI

En2SA

BamHIBamHIEn2SA

probe


B

A

0

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ST S L S LSS S S S S S T S LTSS S

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Hprt

Tumor samples from RosaPB/ATP2 mice controls

H2O

S spleenL lymph nodeT thymus

% tr

ansp

oson

cop

ies

of

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TP m

ice

ATP2-H32founder: ATP2-S1

Chr 17 A1.1

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XC

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Chr

Y

RP23-146J17.3 Farsb1 Ptprc Sel

Pou2f1

Pip4k2a Notch1

Zeb2Bcl2l1

Ptpn1*

Evi1

Ecm1

NrasCdc14aSlc35a3EG381438

Bach2

E130308A19Rik

Prdm16Pex14

Cnr2

Plac8

AB041803mmu-mir-29b-1

Tax1bp1

Foxp1

Ppfibp1

CebpaCebpg

Cbfa2t3Wwp2Ell

Tcf12CblFli1

Rasgrf1

Syne1 Spic Rassf3

Ikzf1

Meis1Csf2

Tmem49Pik3R5

Asxl2

*

Bcl9Mef2c

2010111/01Rik

Acin1

Ndrg1

Il2rbAsap1Hdac7

Tmprss7

Tiam2

Bat1a

Chrd

Erg5830404H04Rik

*

Eml4

Pkd2l2

Pten

Gnaq

RP23-198C2.5

RP23-426M5.1

Etv6

Supplementary Figure S12


Hdac7 (ENSMUST00000116408)

Ex1

CIS

Ex8

Evi1 (ENSMUST00000029230)

Ex15

44bp

CIS 1CIS 2 CIS 1

CIS 2

kernel smoothed estimatespecificity pattern – densitysignificance thresholdsignificance threshold after multiple testing correctionpeaks locationinsertion locations

B

A

C

15

101520

rel N

ras

expr

. ***

Control tumorsNras tumors

MSCV-SD Ex2 untranslated Ex2 transl

Ex3

Transcription/Splicing

Nras

MSCV-Nras

Gapdh

Tu1

Tu2

Tu3

Tu4

Tu5

ATP

ATP

PBas

eW

TH2

O

C

A

Pten

B

Ex4 PB5 En2SA


Ex1 Ex2 Ex3

Ex4

Pten


Ex2 PB5 En2SA

500

300

pb sb sb pbEx1 Ex2 Ex3 Ex4 Ex5

321 21

3

Ex1 Ex2 Ex3 Ex4 Ex5

Tu6aLN

Tu6bThymus

1 2 3 4 5 6 C

intron 2 intron 4

500

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tive

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sion 1 tranposon integration in Pten

>1 transposon integration in Pten

pb sb sb pb

654 54

6

Tu1-

ThTu

2-Th

Tu3a

-LN

Tu4-

ThTu

3b-S

plTu

5-Th

Tu6a

-Th

Tu6b

-LN

Tu7-

Th

500

300


Supplementary figure legends:

Figure S1: Summary of the main aims, steps and results of the study. Mice carrying

the individual genetic components of the transposon system are: PB transposase knock-in

mice (RosaPB), SB transposase knock-in mice (RosaSB) and 19 ATP mice carrying

different types of transposons, which can be mobilized by both, PB and SB. Mouse lines

were characterised and systematically intercrossed to generate 17 different

transposase/transposon double-positive colonies. RosaSB/ATP mice were used to confirm

that transposition can be induced by SB. The RosaPB/ATP mice were used to characterize

PB transposition in vivo. Tumors from these animals were used to identify candidate

cancer genes.

Figure S2: Analysis of transposon donor loci in individual ATP mouse lines using

fluorescence in situ hybridization. The analysis was performed on metaphases from

blood or spleen samples. The chromosomal region of the transposon array is indicated for

each line.

Figure S3: Determination of transposon copy numbers in individual ATP mouse

lines. Transposon copy numbers were determined using qPCR on tail DNA as described

in materials and methods.

Figure S4. Effects of transposon mobilization in vivo. (A) Embryonic lethality in high-

copy transposon lines. In crosses of RosaPB to high-copy ATP1-H39 mice, no double-

transgenic mice were born, but double-transgenic embryos (TS/TP; E11.5) were

identified. (B) Evidence for transposon mobilization in RosaPB;ATP mice but not single-

transgenic ATP mice. At donor loci transposons are flanked by short plasmid sequences.

PCR assays to detect mobilization (red arrows) or lack of excision (green arrows) on tail

DNA from 6-week-old mice are indicated. (C) Overall transposon copy number in tail

DNA of ATP mice that were positive or negative for PB transposase as detected by qPCR

in tail DNA from 6-week-old mice. Transposon copy number loss in double-transgenic

mice as compared to corresponding single-transgenic ATP animals is consistent with the

fact that only 50-60% of mobilization events result in reintegration for each transposition

cycle. Transposon loss was particularly pronounced in "high copy" ATP3-S2 mice. Only

3 out of 110 mice born in the RosaPB;ATP3-S2 colony were double-transgenic and all 3

had drastically reduced transposon copy numbers, possibly reflecting selective pressure

for loss of a large part of the transposon array at an early stage of embryonic

development.

Figure S5: RosaPB;ATP2 mice develop aggressive leukemias/lymphomas. Animals

presented usually with marked splenomegaly with or without hepatomegaly,

lymphadenopathy and thymic enlargement. (A-D) Examples of severe splenomegaly (A),

hepatomegaly (B), thymus enlargement (C) and cervical as well as axillary

lymphadenopathy (D) in RosaPB;ATP2 mice. Haematoxylin and Eosin (H&E) stained

sections from mouse 1230 show extensive infiltration of the kidney (E), liver (F),

epididymus (G) and cord (H) by an aggressive lymphoma. Sections from mouse 1021

show infiltration of the myocardium by malignant cells (I) and large amounts of

leukaemic cells in the left ventricular cavity (J). The same mouse has an extensive

infiltrate in the parenchyma and vessels of the liver (K) and lung (L). Sections from

mouse 999 show a massive lymphoid infiltrate in the liver (M). HE sections from mouse

1429 reveal infiltration of the myocardium (N), liver (O) and lung (P) by an aggressive

lymphoma/leukaemia (note that the lung infiltrate involves both the parenchyma and the

blood vessels). In mouse 1032 there is extensive kidney (Q) and lung (R) involvement by

lymphoma. In mouse 1248 the pancreas (S) and the lamina propria of the stomach (T)

contain lymphoma. Scale bars, 100μm. Tp/ts, transposon-transposase double-transgenic;

tp, transposon-positive; ts, transposase-positive;

Figure S6. RosaPB;ATP1 mice develop solid tumors. H&E stained sections showing

examples of solid tumors: squamous cell carcinoma of the skin (A), intestinal adenoma

with high grade dysplasia/intramucosal carcinoma (B), salivary gland adenocarcinoma

(C), a small bowel adenocarcinoma showing moderately differentiated areas (D) and

poorly differentiated areas (E), hepatocellular carcinoma (F), lung adenocarcinoma (G),

angiosarcoma (H), metastatic adenocarcinoma in a lymph node (I), poorly differentiated

carcinoma in a cervical mass (J), moderately differentiated squamous cell carcinoma in

lymph nodes (K), metastasis of a poorly differentiated carcinoma or sarcoma in

intraabdominal adipose tissue (L). Scale bars, 100μm.

Figure S7. Kaplan-Meier survival curves of RosPB/ATP3 mice. Results are shown for

the 2 copy mouse line ATP3-S1. Survival was slightly, but significantly reduced in

transposase/transposon double-transgenic animals as compared to single transgenic mice

(p<0.05). Results for another ATP3 mouse line (ATP3-S2; 60 transposon copies) are not

presented as a graph, because only 3 RosaPB;ATP3-S2 mice were born. The median

survival of these mice was 501 days.

Figure S8. Immunohistochemical staining of hematopoetic tumors. (A-G) Myeloid

leukaemia, negative for CD3 (A, D) and B220 (B, E) in the liver (A,B) and lymph node

(D,E). Myeloperoxidase-positive leukaemic cells in the liver (C), lymph node (F) and

spleen (G) are shown. Scale bars, 100μm. (H-J) CD3 positive T cell lymphoma with

massive infiltration of the kidneys (H), liver (I) and spleen (J). The analysis of 52 tumors

revealed that 21% were CD3-positive T cell lymphomas and 35% were MPO-positive

myeloid leukaemias (Figure S5). The remaining heamatopoetic tumors could not be

classified as they were negative for CD3, MPO and the B cell marker B220. They might

reflect "primitive" forms of hematopoietic tumors. Less than 10% of RosaPB;ATP2 mice

had solid tumors, including two meningeomas, one liver adenoma, one lung adenoma and

one squamous cell carcinoma of the uterine cervix.

Figure S9. PiggyBac-induced tumors do not show gross genomic instability. (A) High

resolution CGH revealed that deletions and amplifications were rare events in PB-

induced hematopoietic tumors. In more than half of the tumors however rearrangements

at transposon donor loci were observed. The figure shows the genome-wide CGH plot of

a RosaPB;ATP2-S1 tumor. A deletion at the transposon donor locus is indicated. (B)

Multicolour FISH was performed on 5 haematopoietic tumors after in vitro culture of

cancer cells to screen for chromosomal abnormalities, such as balanced/reciprocal

translocations. The analysis showed that PB-induced tumors had a normal karyotype.

Figure S10. Clonality of integration sites in PiggyBac-induced tumors. Southern

blotting of BamHI-digested tail and/or tumor DNA from RosaPB, ATP2 and

RosaPB;ATP2 mice. The 9.9Kb endogenous En2 fragment (orange arrow) and the

smallest detectable fragment size (1.3 Kb; blue arrow) are indicated. The 2.6 Kb

concatemer band (green arrow) is present in ATP2 mice, but not in RosaPB;ATP2 mice,

indicating that all transposons have been mobilized.

Figure S11. Transposase expression and transposon copy numbers in PiggyBac-

induced tumors. (A) PB transposase expression was analyzed in 28 PB-induced tumor

samples from RosaPB;ATP2 mice. Spleen samples from RosaPB mice were used as

controls. Robust expression was found in all samples, indicating that transposase

silencing does not occur in tumors. (B) Transposon copy numbers in tail and tumor

samples from RosaPB;ATP2 mice. There was consistently transposon copy number loss

in all tail samples of transposon/transposase double-positive mice as compared to

"transposon-only" mice, reflecting the fact that not every mobilization event results in

reintegration. Most of the tumors also had various degrees of copy number loss, which

however was in all cases less pronounced than in the tail samples. This might reflect

selective pressure to keep tumor-causing integrations in cancer samples. In 3 out of 15

mice copy number gain rather than loss was found. (C) CHG analysis for one of these

samples revealed that rearrangements at the donor locus on Chromosome 17 can lead to

amplification of the transposon array, resulting in transposon copy number gain. Copy

number gain might be selected for as it might be advantageous for a cell during

malignant transformation (increased mutagenicity), or might just reflect clonal

expansion of random events.

Figure S12. Genome-wide distribution of transposon integration sites identified in

63 hematopoetic tumors. A total of 5590 unique transposon insertion sites were

identified in 63 hematopoetic tumors from 3 cohorts of RosaPB;ATP mice. The absolute

number of integrations per Mb is shown for each chromosome. Significant CISs in

individual 1Mb intervals are indicated. Asterisks above bars indicate transposon donor

loci of individual ATP lines on Chr2 (ATP1-H32), Chr12 (ATP1-S2), Chr17 (ATP1-S2).

Figure S13. Transposon integration pattern in Nras, Evi1, and Hdac7. (A) Nras was

hit in 6 tumors. All Nras insertions were in a narrow region 5´of the gene´s translation

start site. RT-PCR and sequencing confirmed the presence of MSCV-Nras fusion

transcripts starting from the 5’LTR of MSCV and splicing into Nras exon 2 through the

transposon’s splice donor. qRT-PCR showed Nras overexpression in these tumors as

compared to control tumors. (B) All Evi1 insertions were located either upstream of the

gene or in the first two introns, thus in front of the translation start site, which is in exon

3. In all cases transposons integrated in the sense orientation. Two significant CIS regions

were identified, as indicated in the Kernel plot. (C) Hdac7 integrations were identified in

5 tumors. All integrations were in intron 1, upstream of the translation start site and were

sense oriented.

Figure S14. Transposon integrations in the Pten gene. (A) Pten was hit in tumors from

11 mice. 6 of the animals had >1 transposon integration. Pten expression is shown for a

subset of tumors with one or more than one transposon integration as analyzed by

quantitative RT-PCR. Some, but not all tumors with >1 transposon integration had

drastically reduced Pten expression as compared to tumors with only one integration. (B)

PCR analysis confirmed the integration sites in intron 2 and 4 of Pten as well as presence

of the wild-type alleles. Primer locations for the individual assays are indicated in green

(to prove insertion) and red (only amplifying the wild-type allele). (C) RT-PCR and

sequencing confirmed Exon-4-transposon fusion transcripts as well as Exon2-transposon

fusion products. Note that the 5´PB ITR has a cryptic splice acceptor and splice donor,

confirming our previous observations that this ITR can trap genes by itself. All together

these data suggest that in some tumors both Pten alleles are disrupted by transposon

integration.

Supplmentary tables Table S1: Characteristics of ATP mouse lines and their phenotypes upon induction of PB transposon moblization

Mouse line Promoter carried by transposon

Transposon copy number

Median survival of RosPB/ATP mice1 Tumour type

ATP2-S2 MSCV 10 261 predominantly hematopoietic

ATP2-S1 MSCV 15 305 predominantly hematopoietic

ATP2-H32 MSCV 25 374 predominantly hematopoietic

ATP2-H27 MSCV 20 324 predominantly hematopoietic

ATP1-S1 CAG 3 594 predominantly solid

ATP1-S2 CAG 20 514 predominantly solid

ATP1-H5 CAG 20 n.a. predominantly solid

ATP3-S1 PGK 2 656 mixed

ATP3-S2 PGK 60 5012 mixed 1 Mice were sacrificed at signs of sickness or if visible tumours appeared; 2 Only 3 RosaPB/ATP3-S2 mice were born; n.a. median survival cannot be calculated, as most of the RosaPB/ATP1-H5 mice are still alive.

Table S2: Tumour spectrum in RosaPB/ATP1 mice.

Mouse ID ATP line Transposon coyp number Age at death Tumour type

GONE1.1c ATP1-S1 3 582 angiosarcoma

GONE1.1d ATP1-S1 3 651 sebaceous carcinoma

GONE1.1e ATP1-S1 3 644 thymoma

GONE1.1f ATP1-S1 3 187 small bowel adenocarcinoma, lung metastasis

GONE1.1j ATP1-S1 3 498 lung adenocarcinoma, cervical cancer, lymph node metastasis

GONE1.2j ATP1-S1 3 582 small bowel adenoma

GONE1.2f ATP1-S1 3 594 lymphoma

GONE1.3b ATP1-S1 3 463 skin sqamous cell carcinoma salivary gland adenocarcinoma

GONE1.3c ATP1-S1 3 449 small bowel adenoma

GONE1.4a ATP1-S1 3 564 hepatocellular carcinoma

GTWO1.1a ATP1-S2 20 231 salivary gland adenocarcinoma

GTWO1.1g ATP1-S2 20 563 small bowel adenoma

GTWO1.2c ATP1-S2 20 505 small bowel adenoma

GTWO1.2e ATP1-S2 20 309 salivary gland adenocarcinoma

GTWO1.5i ATP1-S2 20 514 lymph node metastasis of a small cell carcinoma

GTWO1.6i ATP1-S2 20 496 angiosarcoma

GTWO2.2b ATP1-S2 20 343 skin squamous cell carcinoma

GTWO2.2g ATP1-S2 20 410 skin squamous cell carcinoma

PSEA1.4c ATP1-S2 20 349 Small bowel adenoma

PSEA2.5j ATP1-S2 20 129 lymph node metastasis of adenocarcinoma

PAEA1.1d ATP1-H5 10 323 lung adenoma

PAEA1.2a ATP1-H5 10 186 skin pilomatrixoma

PAEA1.2f ATP1-H5 10 301 salivary gland adenocarcinoma, lymph node metastasis

PAEA1.6a ATP1-H5 10 196 skin pilomatrixoma

PAEA1.9g ATP1-H5 10 285 metastasised soft tissue sarcoma

PAEA1.9a ATP1-H5 10 303 hepatocellular carcinoma

PAEA1.9b ATP1-H5 10 121 skin pilomatrixoma

Table S3. Analysis of location and orientation of transposon integrations in tumours induced with the ATP1 and the ATP2 transposons. The analysis was performed separately for intragenic integrations with or without inclusion of 2Kb or 5Kb of the 5ÚTR of genes that were listed in the COSMIC database as candidate cancer genes. Sense-oriented integrations upstream of the translation start site can lead to overexpression of genes and are more likely to occur in oncogenes than integrations downstream of ATG (even though, less frequently oncogene activation can also occur as a consequence of disruption of the ORF). P values relate to the comparison of sense-oriented integrations in ATP1 and ATP2 tumors (X2 test).

Integrations analyzed Location Orientation ATP1

integrations (%) ATP2

integraions (%) P

value* Upstream ATG sense 21 (9.5) 325 (12.2) Upstream ATG antisense 22 (9.9) 244 (9.2)

Downstream ATG sense 97 (43.7) 1059 (39.9) Intragenic only

Downstream ATG antisense 82 (36.9) 1027 (38.7)

0.22

Upstream ATG sense 29 (12.2) 440 (15.5) Upstream ATG antisense 29 (12.2) 320 (11.3)

Downstream ATG sense 97 (40.9) 1058 (37.2) Intragenic plus

2Kb 5ÚTR


0.18

Upstream ATG sense 37 (14.6) 582 (19.1) Upstream ATG antisense 41 (16.1) 436 (14.3)

Downstream ATG sense 96 (37.8) 1034 (33.9) Intragenic plus 10Kb 5ÚTR


0.08

Table S4: Distribution of transposon integration sites (percent hits) across chromosomes in tumours from RosaPB/ATP2-S1 mice (n=50), RosaPB/ATP2-S2 mice (n = 7) and RosaPB/ATP2-H32 mice (n = 6). For each chromosome the relative size (percent genome) and the relative frequency of TTAA sites (percent TTAA) compared to the whole genome is shown. The transposon donor chromosome is indicated in red for each line.

Chromosome Percent genome Percent TTAA Percent hits ATP2-S1

Percent hits ATP2-S2

Percent hits ATP2-H32

1 7.43 7.74 7.41 6.51 6.96 2 6.85 6.94 8.54 7.35 10.03 3 6.01 6.61 5.94 5.68 5.66 4 5.86 5.75 5.69 8.85 5.02

5 5.75 5.61 4.91 4.34 6.15 6 5.63 5.80 6.39 7.18 5.18 7 5.75 4.94 5.09 5.01 5.02 8 4.96 4.73 4.16 4.67 3.56 9 4.67 4.52 5.23 5.34 4.85 10 4.90 5.15 5.14 4.17 6.80

11 4.59 4.21 6.35 6.01 8.74 12 4.57 4.61 3.63 4.84 1.94 13 4.53 4.59 5.00 5.51 4.37 14 4.72 4.88 3.68 3.84 4.21 15 3.90 3.90 3.45 4.17 3.88 16 3.70 3.95 3.79 3.84 4.21

17 3.59 3.41 5.69 3.84 2.43 18 3.42 3.50 2.95 2.17 3.07 19 2.31 2.19 3.22 2.84 3.72 X 6.28 6.86 3.61 2.84 4.05 Y 0.60 0.11 0.14 1.00 0.16

Table S5. Gene density on individual chromosomes.

Chromosome Chromosome size (bp)

Percent genome

Number of genes

Percent of all genes

Gene density (genes/Mb)

1 197195432 7.43 1262 5.56 6.4 2 181748087 6.85 1932 8.51 10.6 3 159599783 6.01 1066 4.69 6.7 4 155630120 5.86 1406 6.19 9.0 5 152537259 5.75 1332 5.86 8.7

6 149517037 5.63 1182 5.20 7.9 7 152524553 5.75 2009 8.85 13.2 8 131738871 4.96 1117 4.92 8.5 9 124076172 4.67 1285 5.66 10.4

10 129993255 4.90 1039 4.57 8.0 11 121843856 4.59 1736 7.64 14.2

12 121257530 4.57 724 3.19 6.0 13 120284312 4.53 881 3.88 7.3 14 125194864 4.72 871 3.83 7.0 15 103494974 3.90 830 3.65 8.0 16 98319150 3.70 703 3.10 7.2 17 95272651 3.59 1085 4.78 11.4

18 90772031 3.42 527 2.32 5.8 19 61342430 2.31 755 3.32 12.3 X 166650296 6.28 958 4.22 5.7 Y 15902555 0.60 12 0.05 0.8

Table S7. Primers used in this study Chromosome Product size Sequence

Primer for genotyping ATP and RosaPB mice ATP F 808 bp CTCGTTAATCGCCGAGCTAC ATP R GCCTTATCGCGATTTTACCA Rosa 5F 250 bp CCAAAGTCGCTCTGAGTTGTTATCAG Rosa 3R GGCGGATCACAAGCAATAATAACCTGTAGTTT BpA 5F 450 bp GCTGGGGATGCGGTGGGCTC Rosa 3R GGCGGATCACAAGCAATAATAACCTGTAGTTT Primer for detection of mobilized and non-mobilized transposons Mob1 F 253 bp GGCCTCTTCGCTATTACGC Mob1 R TCAAACGAAGATTCTATGACGTG Mob2 F 220 bp GGGCCTCTTCGCTATTACG Mob2 R GGTCGAGTAAAGCGCAAATC Mob3 F 182 bp GTGCTGCAAGGCGATTAAGT Mob3 R GGTCGAGTAAAGCGCAAATC Non-mob1 F 274 bp GGGCCTCTTCGCTATTACT Non-mob1 R CCGATAAAACACATGCGTCA Non-mob2 F 423 bp AACAAGCTCGTCATCGCTTT Non-mob2 R GGTCGAGTAAAGCGCAAATC Primer to detect MSCV-Nras and MSCV-Spic and Pten-En2SA fusion transcripts. MSCV F 233 bp GCCCATCAAGCTTGCTACTA Nras Ex3 R AGTATGTCCAGCAGGCAGGT MSCV F 247 bp GCCCATCAAGCTTGCTACTA Spic Ex3 R TCCAGCTGATTGTTGGATCA Pten Ex1 F variable* CCATCTCTCTCCTCCTTTTTCTTCA En2SA R CGCTTGTCCTCTTTGTTAGGGTTCT Oligonucleotides used for splinkerette PCR

HMSpAa - CGAAGAGTAACCGTTGCTAGGAGAGACCGTGGCTGAATGAGACTGGTGTCGACACTAGTGG

HMSpBb - GATCCCACTAGTGTCGACACCAGTCTCTAATTTTTTTTTTCAAAAAAA HMSp1 - CGAAGAGTAACCGTTGCTAGGAGAGACC HMSp2 - GTGGCTGAATGAGACTGGTGTCGAC PB-L-Sp1 - CAGTGACACTTACCGCATTGACAAGCACGC PB-L-Sp2 - GAGAGAGCAATATTTCAAGAATGCATGCGT PB-R-Sp1 - CCTCGATATACAGACCGATAAAACACATGC PB-R-Sp2 - ACGCATGATTATCTTTAACGTACGTCACAA Oligonucleotides used for quantitative PCR and RT-PCR Gapdh F - TGTGTCCGTCGTGGATCTGA Gapdh R - CACCACCTTCTTGATGTCATCATAC Gapdh probe - FAM-TGCCGCCTGGAGAAACCTGCC-TAMRA Nras F - AGAGGAGTACAGTGCCATGAGAGA Nras R - CATACAACCTTGAGTGCCATCGT Nras probe - FAM-TACGCCAGTACCGAATGAAAAAGCTCAACA-TAMRA Spic F - AGAGGCAACGCTAACTACTATGGAA Spic R - AGGTCTGCAGAACCATTTGTTACA Spic probe - FAM-CCACAGAGAACCCCCTCTATGACTGGAGA-TAMRA Pten F - ACGATCTTGACAAAGCAAACAAAG Pten R - TGGCTCCTCTACTGTTTTTGTAAAGTAT Pten probe - FAM-CAAGGCCAACCGATACTTCTCTCCAAATTT-TAMRA En2Sa F - TACTGGTTGCTGGAGTCTAGCTACTT En2SA R - CCCCAGTATCTGCAACCTCAA En2SA probe - FAM-TCCACAACCAACGCACCCAAGCT-TAMRA Actb F# - TTCAACACCCCAGCCATGTA Actb R# - TGTGGTACGACCAGAGGCATAC Actb probe# - FAM-TAGCCATCCAGGCTGTGCTGTCCC-TAMRA * PCR product size depends on the location of the transposon integration (Pten intron2: 333bp; intron 4:422bp) # Assay to quantify an intronic Actb sequence (control PCR for normalization of DNA amounts)

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