Alignment of ERV9-LTR sequences of human genes. Blast search identified candidate genes that contain ERV9 LTRs. The alignment shows the LTR sequences of.

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Presentation transcript:

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-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 GGGTCCAGGGCTTCGTTCTTGAAGTCAGTGAGACCAAGAACCCACCAATTCCGGACGC GGGTCCGCGGCTTCGTTCTTGAAGTCAGTGAGACCAAGAATCCGCCGATTCTGGACAC ******. ******.******************* ***** **.**.**** **:.. IRGM CGREF1 GBP5 KCNN2 PGPEP1L Consensus IRGM CGREF1 GBP5 KCNN2 PGPEP1L Consensus IRGM CGREF1 GBP5 KCNN2 PGPEP1L Consensus IRGM CGREF1 GBP5 KCNN2 PGPEP1L Consensus IRGM CGREF1 GBP5 KCNN2 PGPEP1L Consensus IRGM CGREF1 GBP5 KCNN2 PGPEP1L Consensus IRGM CGREF1 GBP5 KCNN2 PGPEP1L Figure S1

testis 1031 bp bp bp bp bp RT GH+TSA 700 bp - testisGH+TSA testis GH+TSA testisGH+TSA RACE_for1RACE_for2RACE_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_rev LTR +339_for CDS ex1LTR LTR +1006_for ex1 mid_rev CDS ex1 LTR 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) AACACTGTGGAAGCTTCCTTCTTTCCCTCTGCAATAAATCTTGCTACTGCTCACTCTTTGGGTCCACACTGCCTTTATGAGCTGTAACACTCACCTTGAAGGTCTGCAGCT TCACTCTTGAAGCCAGCGAGACCACGAGCCCACCAGGAGGAAAGAACAACTCCAGACCCACTGCCTTAAGAGCTGTAACACTCACTGGGAAGGTCTGCAGCTTCACTCCTG AGCCAGTGAGACCACGAACCCACCAGAAGGAAGAAACTCCGAACACATCCGAACATCAGAAGGAACAAACTCCAGACACGCCGCCTTTAAGAACTGTAACACTCACCGCGA GGGTCCGAGGCTTCATTCTTGAAGGCAGTGAGACCAAGAACCCACCAATTCCGGACACAGTACCATGAAGGAATGAAAATACATAACAATTTGCACATTGGATCTGATTCG CCCCGCCCCGAATGACGCCTGCCCGGAGGCAGTGAAAGTACAGCCGCGCCGCCCCAAGTCAGCCTGGACACATAAATCAGCACGCGGCCGGAGAACCCCGCAATCTCTGCG CCCACAAAATACACCGACGATGCCCGATCTACTTTAAGGGCTGAAACCCACGGGCCTGAGAGACTATAAGAGCGTTCCCTACCGCCATGGAACAACGGGGACAGAACGCCC CGGCCGCTTCGGGGGCCCGGAAAAGGCACGGCCCAGGACCCAGGGAGGCGC […] TATTTATGAATCCATGACCAAATTAAATATGAAACCTTATATAAAAA AACACTGTGGAAGCTTCCTTCTTTCCCTCTGCAATAAATCTTGCTACTGCTCACTCTTTGGGTCCACACTGCCTTTATGAGCTGTAACACTCACCTTGAAGGTCTGCAGCT TCACTCTTGAAGCCAGCGAGACCACGAGCCCACCGGGAGGAAAGAGAAAGAGAGAAAGGAAGGAAAGAGAAAGCAGGAAGGACGGAAAGAAGACGAAAGAACGAAAGAAAA CGAAAGAAAAAAGGAAAGAAGAGAGAAGGAGAGAACAGAAGGGGCAGGTGCCCCTGGGAAGGGGAGAAGATCAAGACGCGCCTGGAAAGCGGACTCTGAACCTCAAGACCC TGTTCACAGCCAAGCGCGCGACCCCGGGAGGCGTCAACTCCCCAAGTGCCTCCCTCAACTCATTTCCCCCAAGTTTCGGTGCCTGTCCTGGCGCGGACAGGACCCAGAAAC AAACCACAGCCCGGGGCGCAGCCGCCAGGGCGAAGGTTAGTTCCGGTCCCTTCCCCTCCCCTCCCCACTTGGACGCGCTTGCGGAGGATTGCGTTGACGAGACTCTTATTT ATTGTCACCAACCTGTGGTGGAATTTGCAGTTGCACATTGGATCTGATTCGCCCCGCCCCGAATGACGCCTGCCCGGAGGCAGTGAAAGTACAGCCGCGCCGCCCCAAGTC AGCCTGGACACATAAATCAGCACGCGGCCGGAGAACCCCGCAATCTCTGCGCCCACAAAATACACCGACGATGCCCGATCTACTTTAAGGGCTGAAACCCACGGGCCTGAG AGACTATAAGAGCGTTCCCTACCGCCATGGAACAACGGGGACAGAACGCCCCGGCCGCTTCGGGGGCCCGGAAAAGG […] TATTTATGAATCCATGACCAAATTAAATA TGAAACCTTATATAAAAA LTR +41_for upst ex1_rev CDS ex1LTR TNFRSF10B LTR-transcript 3: 4532 bp (splicing onto sequence upstream of exon 1) AACACTGTGGAAGCTTCCTTCTTTCCCTCTGCAATAAATCTTGCTACTGCTCACTCTTTGGGTCCACACTGCCTTTATGAGCTGTAACACTCACCTTGAAGGTCTGCAGCT TCACTCTTGAAGCCAGCGAGACCACGAGCCCACCAGGAGGAAAGAACAACTCCAGACCCACTGCCTTAAGAGCTGTAACACTCACTGGGAAGGTCTGCAGCTTCACTCCTG AGCCAGTGAGACCACGAACCCACCAGAAGGAAGAAACTCCGAACACATCCGAACATCAGAAGGAACAAACTCCGGACAGGACCCAGAAACAAACCACAGCCCGGGGCGCAG CCGCCAGGGCGAAGGTTAGTTCCGGTCCCTTCCCCTCCCCTCCCCACTTGGACGCGCTTGCGGAGGATTGCGTTGACGAGACTCTTATTTATTGTCACCAACCTGTGGTGG AATTTGCAGTTGCACATTGGATCTGATTCGCCCCGCCCCGAATGACGCCTGCCCGGAGGCAGTGAAAGTACAGCCGCGCCGCCCCAAGTCAGCCTGGACACATAAATCAGC ACGCGGCCGGAGAACCCCGCAATCTCTGCGCCCACAAAATACACCGACGATGCCCGATCTACTTTAAGGGCTGAAACCCACGGGCCTGAGAGACTATAAGAGCGTTCCCTA CCGCCATGGAACAACGGGGACAGAACGCCCCGGCCGCTTCGGGGGCCCGGAAAAGG […] 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

[...] A Figure S4

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 ( Figure S4, continued

A F NCCIT cells DMSO 50ng/ml TRAIL0.5µM TSATRAIL + 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 Beta-Actin Caspase-3 cleaved Caspase-3 15 E 1 E-01 1 E-02 1 E-03 TSA [µM] TNFRSF10Btotal vs. RPLP0 mRNA level (logarithmic scale) * * B TNFRSF10A vs. RPLP0 mRNA (linear scale) TSA [µM] CD TNFRSF10B vs. RPLP0 mRNA (linear scale) DR5_B DR5_A SSC2 siRNA Target vs. RPLP0 mRNA (linear scale) DMSO 2µM 5µM 10µM Nutlin 3a [24h] TNFRSF 10B total CDKN1A ** TNFRSF 10B LTR transcript 2 1 E-04 Figure S5 Susa cells1618-K cells D E-01 1 E-02 1 E-03 1 E-04 D TSA [µM]SAHA [µM] * * * * ** D TSA [µM]SAHA [µM] 1 E-01 1 E-02 1 E-03 1 E-04 * * ** * * * * TNFRSF10A (DR4) D day Cell confluency [%] SSC2DR5_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 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 Vviable cells [%] GH cells1618-K cells TSA DMSO 50nM 100nM TSA DMSO 50nM 100nM control 25ng/ml TRAIL 50ng/ml TRAIL Combination Index (CI) NaN