alignment of erv9-ltr sequences of human genes. blast search identified candidate genes that contain...
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Alignment of ERV9-LTR sequences of human genes.
Blast search identified candidate genes that contain ERV9 LTRs. The alignment shows the
LTR sequences of known human genes with the consensus sequence indicated below.
Consensus
AACCTGCTCAGGTCCCCTTCCATGCTGTGGAAGCTTTGTTTTTTTGCACTTT-GCAGTAAAATCTGTTCAGGTCGTTTTCCATAGTGTGGAAGCTTTGTTCTTTCGC-TCTTTGCAATAA ----CACTGAGGTCACCCTCCAGGCTGTGGAACCTTTGTTCTTTCACTCTTT-GCAATAA ----------------CTTCAACACTGTGGAAGCTTTGTTCTTTCGCTCTTTTGCAATAA AACTTGCTGGAGTCGCCTTCTGTGCTGTGGAAGTTTTGTTGTTTTGCTTGTTTGCAATAA ** . . ******* ****** *** .* ** ***.***
ATCTTGCTGCTGCTCATTCTTTGGGTCCACACTGCCTTTATGAGCTGTAACACTCACCG- ATCTTGCTGCTGCTCACTCTTTGGGTTCACACTGCCTTTATGAGCTGTAACACTCACCAC ATCTTGCTGCTGCTCACTCTTTGGGTCCACACTGCCTTTATGAGCTGTAACACTCACTGG ATCTTGCTACTGCTCACTCTTTGGGTCCACACTGCCTTTATGAGCTGTAACACTCACCGC CCGTTGCTGCTGCTTACTGTTTGGGTCCGCACTGCCTTTATGAGCTGTAACGCTCACCGT. *****.***** * * ******* *.**********************.***** .
TGAAGGTCTGCAGCATCACTCCTGAAACCTGTGAAACAACGAACCCCCCGGGGAGAAACGGA-AGGTCTACAGCTTTACTCCTGAAGCCAGCGAGATCACGAACCCA-CTGGGAGAAACG GA-ATGTCTGCAGCTTCACTCCTGAAGCCAGCGAGACCACGAACCCA-CCAGGAGGAACA GAAAGGTCTGCAGCTTCACTCCTGAAGCCAGCGAGCCCACGAGCCCA-CAGGGAGGAATG GA-AGGTCTGCAGCTTCGTTCTTAAAGCCAGCCACATCACGGACCCA-CCGGGAGAAACG . * ****.****:* . ** *.**.**:* * . .***..***. * .****.** .
AACAACTCCAGACTCGCCGCCTGAAGAGCTGTAACTCTCACTTCGAAGGTCTGCAGCTTC AACAACTCCAGACGCACTACCTTAAGAGCTGTAACACTCACTGCGAAGGTCCGCAGCTGC AACAACTCCAGACGCGCAGCCTTAAGAGCTGTAACACTCACCGCGAAGGTCTGCAGCTTCAACAACTCCAGATGCACCGCCTTAAGAGCTGTAACACTCACGGCGAAGGTCTGCAGCTTCAATAACTCCAAACACGCCACCTTAAGAGCCGTAGCACTCATGATGAAGGTCCGTAGCTTC ** *******.* *.* .*** ****** ***.*:**** ******* * **** *
GCTCCTGAG-TCAGTGAAACCACGAACCCACCGGAAGGAAGAAACTCTGAACACATCCAAATTCCTGAG-CCAGCGAGACCACAAACCCACCAGAAGGAAGAAACTTCGAACGCATCCGA ACTCCTGAG-CCAGCCAGACCACGAACCCACCAGAAGGAAGAAACTCCAAACACATCCGA ACTCCTGAG-CCAGCGAGACCAGGAACCCACCAGAAGGAAGAAACTCCGAACACATCCGA ACTCCTGAAGTCAGCGAGATCACGAATCTGCCAGAAGGAAGAAACTCCGAACACATGCGA . ******. *** *.* ** .** * .**.************* .***.*** .*
ACATCAGAAGGAACAAACTCCGGACACGCAGCCTTTAAGAATTGTAACACTCA-CCGCGA ACATCAGAAGGAACAAACTCCAGACACGCCGCCTTTAGGAACTGTAACACTCAACCGCGA ACATCAGAAGGAGCAAACTCCTGACACGCCACCTTTAAGAACCGTGACACTCA-ACGCTA ACATCAGAAGGAACAAACTCCAGACGGCGCCACCTTAAGAGCTGTAACACTCA-CCGCCA GCATCAGAAGAAACAAACTTGGGACACGCTGCCTTTAAAAACTGTAACACTCA-CCGCCA.*********.*.****** ***. ..* ****.*. **.******* .*** *
GGGTCCGTGGCTTCATTCTTGAAGTCAGTGAGACGAAGAACCCACCAATTCCGGTAGGGGGTCCGCGGCTTCATTCTTGAAGTCAGTGAGACCAAGAACCCACCAATTCCGGACACGGGTCCGCGGCTTCATTCTTGAAGTCAGTGAGACCAAGAACCCACCAATTCCGGACACGGGTCCAGGGCTTCGTTCTTGAAGTCAGTGAGACCAAGAACCCACCAATTCCGGACGCGGGTCCGCGGCTTCGTTCTTGAAGTCAGTGAGACCAAGAATCCGCCGATTCTGGACAC******. ******.******************* ***** **.**.**** **:..
IRGM
CGREF1GBP5KCNN2PGPEP1LConsensus
IRGMCGREF1GBP5KCNN2PGPEP1LConsensus
IRGMCGREF1
GBP5KCNN2PGPEP1LConsensus
IRGMCGREF1GBP5KCNN2PGPEP1LConsensus
IRGMCGREF1GBP5KCNN2
PGPEP1LConsensus
IRGMCGREF1GBP5KCNN2PGPEP1L
Consensus
IRGMCGREF1GBP5KCNN2
PGPEP1L
297296292282298
356356351
341357
238237233223238
414
414
409399
415
178
177173163
178
118
119115104
120
59
5955
4460
Figure S1
testis
1031 bp -
2000 bp -
1500 bp -
500 bp -
3000 bp -
+ - + - + - + - + - + - + - + - RT
GH+TSA
700 bp -
testis GH+TSA testis GH+TSA testis GH+TSA
RACE_for1 RACE_for2 RACE_for3 RACE_for4
Primer Mix
3’RACE PCR products.
Pools of PCR-products obtained by the 3’RACE protocol using 4 LTR-specific forward
oligonucleotides and the reverse Universal Primer Mix, visualized by agarose gel
electrophoresis and ethidium bromide staining. Similar products were obtained using the 4
degenerated forward oligonucleotides.
Figure S2
ex1_revLTR +339_for
CDSex1LTR
LTR +1006_for ex1 mid_rev
CDSex1LTR
TNFRSF10B LTR-transcript 1: 4413 bp (splicing onto splice acceptor site within exon 1)
TNFRSF10B LTR-transcript 2: 4664 bp (splicing onto sequence upstream of exon 1)
AACACTGTGGAAGCTTCCTTCTTTCCCTCTGCAATAAATCTTGCTACTGCTCACTCTTTGGGTCCACACTGCCTTTATGAGCTGTAACACTCACCTTGAAGGTCTGCAGCTTCACTCTTGAAGCCAGCGAGACCACGAGCCCACCAGGAGGAAAGAACAACTCCAGACCCACTGCCTTAAGAGCTGTAACACTCACTGGGAAGGTCTGCAGCTTCACTCCTGAGCCAGTGAGACCACGAACCCACCAGAAGGAAGAAACTCCGAACACATCCGAACATCAGAAGGAACAAACTCCAGACACGCCGCCTTTAAGAACTGTAACACTCACCGCGAGGGTCCGAGGCTTCATTCTTGAAGGCAGTGAGACCAAGAACCCACCAATTCCGGACACAGTACCATGAAGGAATGAAAATACATAACAATTTGCACATTGGATCTGATTCGCCCCGCCCCGAATGACGCCTGCCCGGAGGCAGTGAAAGTACAGCCGCGCCGCCCCAAGTCAGCCTGGACACATAAATCAGCACGCGGCCGGAGAACCCCGCAATCTCTGCGCCCACAAAATACACCGACGATGCCCGATCTACTTTAAGGGCTGAAACCCACGGGCCTGAGAGACTATAAGAGCGTTCCCTACCGCCATGGAACAACGGGGACAGAACGCCCCGGCCGCTTCGGGGGCCCGGAAAAGGCACGGCCCAGGACCCAGGGAGGCGC […] TATTTATGAATCCATGACCAAATTAAATATGAAACCTTATATAAAAA
AACACTGTGGAAGCTTCCTTCTTTCCCTCTGCAATAAATCTTGCTACTGCTCACTCTTTGGGTCCACACTGCCTTTATGAGCTGTAACACTCACCTTGAAGGTCTGCAGCTTCACTCTTGAAGCCAGCGAGACCACGAGCCCACCGGGAGGAAAGAGAAAGAGAGAAAGGAAGGAAAGAGAAAGCAGGAAGGACGGAAAGAAGACGAAAGAACGAAAGAAAACGAAAGAAAAAAGGAAAGAAGAGAGAAGGAGAGAACAGAAGGGGCAGGTGCCCCTGGGAAGGGGAGAAGATCAAGACGCGCCTGGAAAGCGGACTCTGAACCTCAAGACCCTGTTCACAGCCAAGCGCGCGACCCCGGGAGGCGTCAACTCCCCAAGTGCCTCCCTCAACTCATTTCCCCCAAGTTTCGGTGCCTGTCCTGGCGCGGACAGGACCCAGAAACAAACCACAGCCCGGGGCGCAGCCGCCAGGGCGAAGGTTAGTTCCGGTCCCTTCCCCTCCCCTCCCCACTTGGACGCGCTTGCGGAGGATTGCGTTGACGAGACTCTTATTTATTGTCACCAACCTGTGGTGGAATTTGCAGTTGCACATTGGATCTGATTCGCCCCGCCCCGAATGACGCCTGCCCGGAGGCAGTGAAAGTACAGCCGCGCCGCCCCAAGTCAGCCTGGACACATAAATCAGCACGCGGCCGGAGAACCCCGCAATCTCTGCGCCCACAAAATACACCGACGATGCCCGATCTACTTTAAGGGCTGAAACCCACGGGCCTGAGAGACTATAAGAGCGTTCCCTACCGCCATGGAACAACGGGGACAGAACGCCCCGGCCGCTTCGGGGGCCCGGAAAAGG […] TATTTATGAATCCATGACCAAATTAAATATGAAACCTTATATAAAAA
LTR +41_for upst ex1_rev
CDSex1LTR
TNFRSF10B LTR-transcript 3: 4532 bp (splicing onto sequence upstream of exon 1)
AACACTGTGGAAGCTTCCTTCTTTCCCTCTGCAATAAATCTTGCTACTGCTCACTCTTTGGGTCCACACTGCCTTTATGAGCTGTAACACTCACCTTGAAGGTCTGCAGCTTCACTCTTGAAGCCAGCGAGACCACGAGCCCACCAGGAGGAAAGAACAACTCCAGACCCACTGCCTTAAGAGCTGTAACACTCACTGGGAAGGTCTGCAGCTTCACTCCTGAGCCAGTGAGACCACGAACCCACCAGAAGGAAGAAACTCCGAACACATCCGAACATCAGAAGGAACAAACTCCGGACAGGACCCAGAAACAAACCACAGCCCGGGGCGCAGCCGCCAGGGCGAAGGTTAGTTCCGGTCCCTTCCCCTCCCCTCCCCACTTGGACGCGCTTGCGGAGGATTGCGTTGACGAGACTCTTATTTATTGTCACCAACCTGTGGTGGAATTTGCAGTTGCACATTGGATCTGATTCGCCCCGCCCCGAATGACGCCTGCCCGGAGGCAGTGAAAGTACAGCCGCGCCGCCCCAAGTCAGCCTGGACACATAAATCAGCACGCGGCCGGAGAACCCCGCAATCTCTGCGCCCACAAAATACACCGACGATGCCCGATCTACTTTAAGGGCTGAAACCCACGGGCCTGAGAGACTATAAGAGCGTTCCCTACCGCCATGGAACAACGGGGACAGAACGCCCCGGCCGCTTCGGGGGCCCGGAAAAGG […] TATTTATGAATCCATGACCAAATTAAATATGAAACCTTATATAAAAA
Novel TNFRSF10B LTR-transcripts .
Sizes and sequences of the newly identified TNFRSF10B transcripts expressed from the
upstream ERV9-LTR are shown. Alternative splicing results in three novel transcripts that are
predicted to encode the same TNFRSF10B protein, due to use of the same start codon.
Binding sites for the oligonucleotides used for specific transcript amplification are indicated by
small arrows. The ERV9-LTR-sequences are marked in grey, and exon 1 of TNFRSF10B is
marked in red.
Figure S3
B
Insertion of the ERV9-LTR-sequence upstream of TNFRSF10B in primates.
A. Alignment of genomic sequences of diverse primate species including hominids containing
the upstream region of each TNFRSF10B gene, using the ClustalX algorithm. The ERV9-LTR
(yellow shading) is only inserted from Homo sapiens to Hylobatidae.
B. Sequence comparison between ERV9 subfamily sequences by the ClustalX algorithm
reveals that the TNFRFS10B-associated ERV9 LTR belongs to subfamily IX of ERV9 [47]. The
radial alignment tree was generated using FigTree v1.4.0
(http://tree.bio.ed.ac.uk/software/figtree/).
Figure S4, continued
A
F
NCCIT cells
DMSO 50ng/ml TRAIL 0.5µM TSA TRAIL + TSA SSC2 DR5_A DR5_B SSC2 DR5_A DR5_B SSC2 DR5_A DR5_B SSC2 DR5_A DR5_B
MW (kDa)
PARP1cleaved PARP 100
Beta-Actin40
35 Caspase-3
cleavedCaspase-315
E
1 E-01
1 E-02
1 E-03
TSA [µM]
TN
FR
SF
10
Bto
tal v
s.
RP
LP
0 m
RN
A le
vel
(lo
gari
thm
ic s
cale
)
**
B
TN
FR
SF
10
A
vs.
RP
LP
0 m
RN
A
(lin
ear
scal
e) 0.0015
0.0010
0.0005
0.0000
TSA [µM]
C D0.006
0.004
0.002
0.000TN
FR
SF
10
B v
s.
RP
LP
0 m
RN
A
(lin
ear
scal
e)
DR5_BDR5_A
SSC2
siRNA
Ta
rge
t vs
. RP
LP0
mR
NA
(lin
ear
sca
le)
DMSO 2µM 5µM 10µM
Nutlin 3a [24h]
TNFRSF10B total
CDKN1A
**
TNFRSF10B LTR transcript 2
1 E-04
Figure S5Susa cells 1618-K cells
D 0.5 1 2
1 E-01
1 E-02
1 E-03
1 E-04
D 0.5 1 2 1 5
TSA [µM] SAHA [µM]
*
**
***
D 0.5 1 2 1 5
TSA [µM] SAHA [µM]
1 E-01
1 E-02
1 E-03
1 E-04
**
0.06
0.04
0.02
0.00
0.08
**
*
*
**TNFRSF10A (DR4)
D 0.5 1 2
day 0 1 2 3 4 5 0 1 2 3 4 50 1 2 3 4 5 0 1 2 3 4 5
Cel
l co
nflu
en
cy [
%]
8060
40200
8060
40
200
100
100
SSC2 DR5_A DR5_B
DMSO 50ng/ml TRAIL [10h] 0.5µM TSA + 50ng/ml TRAIL0.5µM TSA [12h]
A. TSA treatment also increased the expression of total TNFRSF10B in NCCIT, Susa and
1618-K testicular cancer cells, as determined by qRT-PCR (mean values of three
independent experiments). p-values were calculated using Student’s t-test (ns: p>0.05, *:
p<0.05, **: p<0.01, ***: p<0.001).
B. Expression of TNFRSF10A mRNA is not induced upon treatment of GH cells with TSA, as
determined by qRT-PCR (mean values of three independent experiments).
C. The MDM2 inhibitor Nutlin 3a does not activate transcription of TNFRSF10B from the ERV9-
LTR-promoter. Total mRNAs isolated from GH cells treated with increasing concentrations of
Nutlin 3a or the solvent DMSO for 24 hrs were subjected to quantitative real-time-RT-PCR.
Expression levels of total TNFRSF10B, TNFRSF10B LTR-transcript 2, or CDKN1A were
normalized to RPLP0 (mean values of three independent experiments). Transcription of
CDKN1A was induced as a consequence of p53 activation.
D. Knockdown of TNFRSF10B expression by specific siRNAs. GH cells were transfected with
two different siRNAs against the TNFRSF10B gene product DR5 (DR5_A and DR5_B) or a
control scrambled SSC2 siRNA and harvested after 36 hrs for quantification of TNFRSF10B
mRNA levels by real-time RT-PCR. RPLP0 served as a reference gene.
E. siRNA-mediated depletion of TNFRSF10B expression in GH cells rescued cell survival upon
combined treatment with TRAIL and TSA. Cell confluency was measured prior to treatment
(t=0) or at different time points after treatment over 5 days (t=2-5). The experiment was
performed thrice; the first experiment is shown in Fig. 5D.
F. Immunodetection of PARP1 as well as full-length or cleaved caspase-3 in GH cells depleted
of TNFRSF10B after treatment with TRAIL (16 hrs) or TSA (18 hrs) alone, or both. Combined
treatment resulted in nearly complete cleavage of PARP1 and caspase-3, which was partially
rescued by knocking down TNFRSF10B expression.
G. Cell viability assay in testicular cancer cells treated for 24h with TRAIL or TSA alone, or a
combination of both, using ATP luminometry. For combined treatment the combination index
(CI) was calculated using the Chou and Talalay method. Experiments were performed thrice.
In cases where combined treatment resulted in an inhibitory effect less than 25% of control
levels, CI scores were not determined (NaN) (Miller et al., Sci Signal 2013; 6(294): ra85).
TNFRSF10B expression sensitizes testicular cancer cells towards TRAIL.
Figure S5, continued
G
Vvi
able
cel
ls [
%]
GH cells 1618-K cells
TSA
60
30
0
90
DMSO 50nM 100nM
60
30
0
90
TSA
DMSO 50nM 100nM
control
25ng/ml TRAIL
50ng/ml TRAIL
Combination Index (CI)
0.62
0.660.61
0.52
0.57
0.58
NaN
NaN
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