ERV9 LTR-Containing Human Genes Alignment

1. Figure S1 AACCTGCTCAGGTCCCCTTCCATGCTGTGGAAGCTTTGTTTTTTTGCACTTT-GCAGTAA AATCTGTTCAGGTCGTTTTCCATAGTGTGGAAGCTTTGTTCTTTCGC-TCTTTGCAATAA ----CACTGAGGTCACCCTCCAGGCTGTGGAACCTTTGTTCTTTCACTCTTT-GCAATAA ----------------CTTCAACACTGTGGAAGCTTTGTTCTTTCGCTCTTTTGCAATAA AACTTGCTGGAGTCGCCTTCTGTGCTGTGGAAGTTTTGTTGTTTTGCTTGTTTGCAATAA ** . . ******* ****** *** .* ** ***.*** ATCTTGCTGCTGCTCATTCTTTGGGTCCACACTGCCTTTATGAGCTGTAACACTCACCG- ATCTTGCTGCTGCTCACTCTTTGGGTTCACACTGCCTTTATGAGCTGTAACACTCACCAC ATCTTGCTGCTGCTCACTCTTTGGGTCCACACTGCCTTTATGAGCTGTAACACTCACTGG ATCTTGCTACTGCTCACTCTTTGGGTCCACACTGCCTTTATGAGCTGTAACACTCACCGC CCGTTGCTGCTGCTTACTGTTTGGGTCCGCACTGCCTTTATGAGCTGTAACGCTCACCGT . *****.***** * * ******* *.**********************.***** . TGAAGGTCTGCAGCATCACTCCTGAAACCTGTGAAACAACGAACCCCCCGGGGAGAAACG GA-AGGTCTACAGCTTTACTCCTGAAGCCAGCGAGATCACGAACCCA-CTGGGAGAAACG GA-ATGTCTGCAGCTTCACTCCTGAAGCCAGCGAGACCACGAACCCA-CCAGGAGGAACA GAAAGGTCTGCAGCTTCACTCCTGAAGCCAGCGAGCCCACGAGCCCA-CAGGGAGGAATG GA-AGGTCTGCAGCTTCGTTCTTAAAGCCAGCCACATCACGGACCCA-CCGGGAGAAACG . * ****.****:* . ** *.**.**:* * . .***..***. * .****.** .  AACAACTCCAGACTCGCCGCCTGAAGAGCTGTAACTCTCACTTCGAAGGTCTGCAGCTTC AACAACTCCAGACGCACTACCTTAAGAGCTGTAACACTCACTGCGAAGGTCCGCAGCTGC AACAACTCCAGACGCGCAGCCTTAAGAGCTGTAACACTCACCGCGAAGGTCTGCAGCTTC AACAACTCCAGATGCACCGCCTTAAGAGCTGTAACACTCACGGCGAAGGTCTGCAGCTTC AATAACTCCAAACACGCCACCTTAAGAGCCGTAGCACTCATGATGAAGGTCCGTAGCTTC ** *******.* *.* .*** ****** ***.*:**** ******* * **** * GCTCCTGAG-TCAGTGAAACCACGAACCCACCGGAAGGAAGAAACTCTGAACACATCCAA ATTCCTGAG-CCAGCGAGACCACAAACCCACCAGAAGGAAGAAACTTCGAACGCATCCGA ACTCCTGAG-CCAGCCAGACCACGAACCCACCAGAAGGAAGAAACTCCAAACACATCCGA ACTCCTGAG-CCAGCGAGACCAGGAACCCACCAGAAGGAAGAAACTCCGAACACATCCGA ACTCCTGAAGTCAGCGAGATCACGAATCTGCCAGAAGGAAGAAACTCCGAACACATGCGA . ******. *** *.* ** .** * .**.************* .***.*** .* ACATCAGAAGGAACAAACTCCGGACACGCAGCCTTTAAGAATTGTAACACTCA-CCGCGA ACATCAGAAGGAACAAACTCCAGACACGCCGCCTTTAGGAACTGTAACACTCAACCGCGA ACATCAGAAGGAGCAAACTCCTGACACGCCACCTTTAAGAACCGTGACACTCA-ACGCTA ACATCAGAAGGAACAAACTCCAGACGGCGCCACCTTAAGAGCTGTAACACTCA-CCGCCA GCATCAGAAGAAACAAACTTGGGACACGCTGCCTTTAAAAACTGTAACACTCA-CCGCCA .*********.*.****** ***. ..* ****.*. **.******* .*** * GGGTCCGTGGCTTCATTCTTGAAGTCAGTGAGACGAAGAACCCACCAATTCCGGTAGG GGGTCCGCGGCTTCATTCTTGAAGTCAGTGAGACCAAGAACCCACCAATTCCGGACAC GGGTCCGCGGCTTCATTCTTGAAGTCAGTGAGACCAAGAACCCACCAATTCCGGACAC GGGTCCAGGGCTTCGTTCTTGAAGTCAGTGAGACCAAGAACCCACCAATTCCGGACGC GGGTCCGCGGCTTCGTTCTTGAAGTCAGTGAGACCAAGAATCCGCCGATTCTGGACAC ******. ******.******************* ***** **.**.**** **:.. 59 IRGM 59 CGREF1 55 GBP5 KCNN2 44 60 PGPEP1L Consensus 118 IRGM 119 CGREF1 115 GBP5 KCNN2 104 PGPEP1L 120 Consensus 178 IRGM CGREF1 177 173 GBP5 163 KCNN2 PGPEP1L 178 Consensus 238 IRGM 237 CGREF1 233 GBP5 223 KCNN2 238 PGPEP1L Consensus IRGM 297 CGREF1 296 GBP5 292 KCNN2 282 PGPEP1L 298 Consensus IRGM 356 CGREF1 356 351 GBP5 KCNN2 341 PGPEP1L 357 Consensus 414 IRGM CGREF1 414 GBP5 409 KCNN2 399 PGPEP1L 415 Consensus 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.

2. Figure S2 RACE_for1 RACE_for2 RACE_for3 RACE_for4 GH+TSA testis testis GH+TSA testis testis GH+TSA GH+TSA + - + - + - + - + - + - + - + - RT Primer Mix 3000 bp - 2000 bp - 1500 bp - 1031 bp - 700 bp - 500 bp - 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.

3. Figure S3 TNFRSF10B LTR-transcript 1: 4413 bp (splicing onto splice acceptor site within exon 1) ex1_rev LTR +339_for LTR ex1 CDS AACACTGTGGAAGCTTCCTTCTTTCCCTCTGCAATAAATCTTGCTACTGCTCACTCTTTGGGTCCACACTGCCTTTATGAGCTGTAACACTCACCTTGAAGGTCTGCAGCTTCACTCTTGAAGCCAGCGAGACCACGAGCCCACCAGGAGGAAAGAACAACTCCAGACCCACTGCCTTAAGAGCTGTAACACTCACTGGGAAGGTCTGCAGCTTCACTCCTGAGCCAGTGAGACCACGAACCCACCAGAAGGAAGAAACTCCGAACACATCCGAACATCAGAAGGAACAAACTCCAGACACGCCGCCTTTAAGAACTGTAACACTCACCGCGAGGGTCCGAGGCTTCATTCTTGAAGGCAGTGAGACCAAGAACCCACCAATTCCGGACACAGTACCATGAAGGAATGAAAATACATAACAATTTGCACATTGGATCTGATTCGCCCCGCCCCGAATGACGCCTGCCCGGAGGCAGTGAAAGTACAGCCGCGCCGCCCCAAGTCAGCCTGGACACATAAATCAGCACGCGGCCGGAGAACCCCGCAATCTCTGCGCCCACAAAATACACCGACGATGCCCGATCTACTTTAAGGGCTGAAACCCACGGGCCTGAGAGACTATAAGAGCGTTCCCTACCGCCATGGAACAACGGGGACAGAACGCCCCGGCCGCTTCGGGGGCCCGGAAAAGGCACGGCCCAGGACCCAGGGAGGCGC […] TATTTATGAATCCATGACCAAATTAAATATGAAACCTTATATAAAAA TNFRSF10B LTR-transcript 2: 4664 bp (splicing onto sequence upstream of exon 1) ex1 mid_rev LTR +1006_for LTR CDS ex1 AACACTGTGGAAGCTTCCTTCTTTCCCTCTGCAATAAATCTTGCTACTGCTCACTCTTTGGGTCCACACTGCCTTTATGAGCTGTAACACTCACCTTGAAGGTCTGCAGCTTCACTCTTGAAGCCAGCGAGACCACGAGCCCACCGGGAGGAAAGAGAAAGAGAGAAAGGAAGGAAAGAGAAAGCAGGAAGGACGGAAAGAAGACGAAAGAACGAAAGAAAACGAAAGAAAAAAGGAAAGAAGAGAGAAGGAGAGAACAGAAGGGGCAGGTGCCCCTGGGAAGGGGAGAAGATCAAGACGCGCCTGGAAAGCGGACTCTGAACCTCAAGACCCTGTTCACAGCCAAGCGCGCGACCCCGGGAGGCGTCAACTCCCCAAGTGCCTCCCTCAACTCATTTCCCCCAAGTTTCGGTGCCTGTCCTGGCGCGGACAGGACCCAGAAACAAACCACAGCCCGGGGCGCAGCCGCCAGGGCGAAGGTTAGTTCCGGTCCCTTCCCCTCCCCTCCCCACTTGGACGCGCTTGCGGAGGATTGCGTTGACGAGACTCTTATTTATTGTCACCAACCTGTGGTGGAATTTGCAGTTGCACATTGGATCTGATTCGCCCCGCCCCGAATGACGCCTGCCCGGAGGCAGTGAAAGTACAGCCGCGCCGCCCCAAGTCAGCCTGGACACATAAATCAGCACGCGGCCGGAGAACCCCGCAATCTCTGCGCCCACAAAATACACCGACGATGCCCGATCTACTTTAAGGGCTGAAACCCACGGGCCTGAGAGACTATAAGAGCGTTCCCTACCGCCATGGAACAACGGGGACAGAACGCCCCGGCCGCTTCGGGGGCCCGGAAAAGG […] TATTTATGAATCCATGACCAAATTAAATA TGAAACCTTATATAAAAA TNFRSF10B LTR-transcript 3: 4532 bp (splicing onto sequence upstream of exon 1) LTR +41_for upst ex1_rev LTR ex1 CDS 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.

4. Figure S4 A [...]

5. Figure S4, continued 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/).

6. Figure S5 A NCCIT cells Susa cells 1618-K cells * ** * * * * * 1 E-01 1 E-01 1 E-01 * * TNFRSF10Btotal vs. RPLP0 mRNA level (logarithmic scale) 1 E-02 1 E-02 1 E-02 1 E-03 1 E-03 1 E-03 1 E-04 1 E-04 1 E-04 D 0.5 1 2 D 0.5 1 2 1 5 D 0.5 1 2 1 5 TSA [µM] TSA [µM] SAHA [µM] TSA [µM] SAHA [µM] C D B TNFRSF10A (DR4) ** * 0.006 TNFRSF 10B total * 0.08 0.0015 * 0.004 TNFRSF10B vs. RPLP0 mRNA (linear scale) 0.06 TNFRSF 10B LTR transcript 2 Target vs. RPLP0 mRNA (linear scale) TNFRSF10A vs. RPLP0 mRNA (linear scale) 0.0010 * 0.04 0.002 * * 0.0005 0.02 CDKN1A 0.000 0.00 0.0000 SSC2 DR5_B DR5_A D0.5 1 2 DMSO 2µM 5µM 10µM TSA [µM] Nutlin 3a [24h] siRNA DR5_B E SSC2 DR5_A DMSO 0.5µM TSA [12h] 50ng/ml TRAIL [10h] 0.5µM TSA + 50ng/ml TRAIL 100 80 60 40 20 Cell confluency [%] 0 100 80 60 40 20 0 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5 day 0 1 2 3 4 5 F 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) PARP1 cleaved PARP 100 Caspase-3 35 cleaved Caspase-3 15 Beta-Actin 40

7. Figure S5, continued G GH cells 1618-K cells NaN control 25ng/ml TRAIL 50ng/ml TRAIL 0.57 90 90 0.61 NaN 0.66 Vviable cells [%] 60 60 0.62 0.58 Combination Index (CI) 30 30 0.52 0 0 DMSO 50nM 100nM DMSO 50nM 100nM TSA TSA TNFRSF10B expression sensitizes testicular cancer cells towards TRAIL. A. TSA treatment also increasedtheexpressionof 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 ofTNFRSF10AmRNAis not induced upon treatmentof GH cellswith TSA, asdeterminedbyqRT-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. Immunodetectionof 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. Cellviabilityassay in testicularcancercellstreatedfor 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).