ERG transcription factor is required to maintain high <i>TDRD1</i> expression.

<p>(A) <i>ERG</i> and <i>TDRD1</i> mRNA expression levels in VCaP cells measured 72 h after gene silencing with siRNAs. Three independent experiments were performed in triplicate. (B) ERG and TDRD1 protein expression in VCaP cells 72 h after gene silencing with siRNAs.</p

<i>TDRD1</i> does not control LINE1 activity in <i>TMPRSS2:ERG</i>-positive VCaP cells.

<p>(A) mRNA expression analysis of <i>PIWIL</i> genes in prostate cell lines by qRT-PCR. Testis RNA was used as a positive control. (B) mRNA expression analysis of LINE1 ORF2 in VCaP cells following 5 days of treatment with 5-aza-2′-deoxycytidine. (C) mRNA expression analysis of LINE1 ORF2 in VCaP cells following prolonged (8 days) <i>ERG</i> or <i>TDRD1</i> silencing. (D) Metabolic viability assay of VCaP cells treated with siRNAs. One (B), two (A) or three (C, D) independent experiments were performed in triplicate.</p

<i>TDRD1</i> promoter associated CpG island is hypomethylated in <i>TMPRSS2:ERG</i>-positive prostate cancer.

<p>(A) Analysis of <i>TDRD1</i> promoter methylation in prostate tumors by MeDIP-Seq. The values represent the average degree of DNA methylation of the 500-bp bins. (B) Correlation analysis of <i>TDRD1</i> promoter methylation and <i>TDRD1</i> mRNA expression in prostate cancer. The Spearman correlation coefficient is shown.</p

<i>TDRD1</i> is co-expressed with <i>ERG</i> but not with <i>ETV1</i> in prostate cancer.

<p>(A) Correlation analysis of mRNA levels measured by qRT-PCR in <i>TMPRSS2:ERG</i>-negative (ERG-, n = 30) and <i>TMPRSS2:ERG</i>-positive (ERG+, n = 17) prostate cancers as well as adjacent benign prostate tissue (n = 46). Pearson correlation coefficient is shown. (B) Analysis of mRNA expression in prostate cell lines by qRT-PCR. Two independent experiments were performed in triplicate. Human testis RNA was used as positive control for <i>TDRD1</i> expression. (C) Analysis of protein expression in prostate cell lines by western blotting. (D) Analysis of mRNA expression in hematopoietic cancer cell lines by qRT-PCR. Two independent experiments were performed in triplicate.</p

ERG-induced loss of epigenetic repression at the <i>TDRD1</i> promoter is a major mechanism of <i>TDRD1</i> overexpression.

<p>(A) DNA methylation analysis of the <i>TDRD1</i> promoter-associated CpG island in prostate cell lines by bisulfite sequencing. Average methylation level of the whole CpG island calculated from five sequenced colonies is shown (%). (B) Analysis of mRNA expression in LNCaP cells after treatment with the demethylating agent 5-aza-2′-deoxycytidine. Insert: analysis of protein expression in LNCaP cells after treatment with 5-aza-2′-deoxycytidine. 75µg of protein lysate from LNCaP cells was used per lane. (C) Analysis of mRNA expression in stable LNCaP clones overexpressing <i>ERG</i>. Two independent experiments were performed in triplicate. Insert: ERG expression analysis at 48 h in LNCaP clones by western blotting. (D) Bisulfite sequencing of the <i>TDRD1</i> promoter-associated CpG island in LNCaP cells 48 h after induction of ERG expression with doxycycline. (E) Bisulfite sequencing of the <i>TDRD1</i> promoter-associated CpG island 96 h after silencing of <i>ERG</i> in VCaP cells. The data shown in (D) and (E) are mean % of methylation of the entire CpG island calculated from 11–12 sequenced clones.</p