br PTOV directly induces the expression of ALDH A and
3.2. PTOV1 directly induces the expression of ALDH1A1 and CCNG2 in PCa cells
We next interrogated the mechanisms by which PTOV1 drives the transcription of ALDH1A1 and CCNG2. Prior work had shown that the engagement of two signaling networks, Wnt/β-catenin and Jun kinase (JNK), activate PTOV1-mediated functions [16,17]. Upon inhibition of these pathways by iCRT14 or JNK inhibitor II, specific inhibitors of Wnt and JNK signaling, respectively, the expression of ABCB1, driven by PTOV1, was abrogated (Supplementary Fig. S1). In contrast, the PTOV1-dependent expression of ALDH1A1 and CCNG2 was not af-fected, suggesting that PTOV1 induced their transcription in-dependently from these pathways (Supplementary Fig. S1C) [16,17]. Based on these observations, we hypothesized that PTOV1 mediates the transcription of ALDH1A1 and CCNG2 genes by direct association with their promoters. We thus performed chromatin immunoprecipitation (ChIP) in LNCaP cells, and observed a specific binding of PTOV1 to the chromatin of the ALDH1A1 and CCNG2 promoters (Fig. 2A). In con-trast, the binding of PTOV1 to the ABCB1 promoter resulted in unclear or non specific, suggesting that ABCB1 transcriptional activation occurs through other circuits promoted by PTOV1, as suggested above (Supplementary Fig. S1). As expected, no binding of PTOV1 was ob-served to internal sequences of the HES1 gene  .
3.3. PTOV1 directly binds to ALDH1A1 and CCNG2 promoters through a new motif in its A domain
AT-hook amino 6-NBDG motifs confer proteins the ability to bind DNA or RNA. The N-terminal region of PTOV1 contained an extended AT-hook (eAT-hook) motif . We tested diﬀerent regions of PTOV1, including the eAT-hook motif, the A domain, and the B domain for their ability to bind the promoter sequences of ALDH1A1 and CCNG2 genes using electrophoretic mobility shift assays (EMSA). We synthesized small DNA probes (32 nucleotides) from the ALDH1A1 and CCNG2 promoters containing putative AT-hook target sequences (Fig. 3), and from the HES1 intron-1 as negative control. These probes were labeled with [32P] and used in binding reactions with recombinant glutathione-S-transferase (GST), full-lenght GST-PTOV1 protein, GST-A domain, GST-B domain, and two short peptides containing the wild type eAT-hook or the mutated eAT-hook described previously  (Fig. 3). A specific shifted band is detected when recombinant GST-PTOV1 is in-cubated with either ALDH1A1 or CCNG2 probes (Fig. 3B). Surprisingly, the GST-A domain of PTOV1, but not the GST-B domain, showed a strong binding activity with both ALDH1A1 and CCNG2 probes. How-ever, either the wild-type or the mutated eAT peptides did not generate any shifted band with these probes, and no shifted band was visible with the HES1 negative control probe. (Fig. 3B).
The analysis of the amino acids sequence of the A domain revealed a motif with similarities to a ‘classic AT-hook’ (Fig. 3A) . This se-quence is not conserved in the corresponding homologous B domain of PTOV1. Therefore, we synthesized a short peptide of 15 amino acids that corresponds to the motif present in the A domain (Fig. 3A). In-terestingly, both labeled probes from ALDH1A1 and CCNG2 produced light shifted bands in the presence of this short peptide from the A domain (Fig. 3B), indicating that PTOV1 is able to bind to these pro-moters through a new amino acid sequence present in the A domain, but not with the eAT-hook previously described, nor with the B domain of the protein.
3.4. The new PTOV1 AT-hook-like motif is necessary to modulate ALDH1A1 and CCNG2 expression
In order to study the functional relevance of the KRRP sequence
Fig. 1. The ectopic expression of PTOV1 in prostate cancer cells promotes ALDH1A1 and CCNG2 ex-pression. (A) CRPC Du145 and PC3 cells transduced with a lentiviral vector HAPTOV1-ires-GFP, or a control lentivirus (GFP), were analyzed by real time qPCR for ALDH1A1 and CCNG2 expression. (B) Left, LNCaP cells transduced with the lentiviral vector HAPTOV1, or the control lentivirus (GFP), were analyzed by real time qPCR for gene expression. Right, LNCaP cells transduced with lentiviral vec-tors bearing shRNA sequences (sh1397 or sh1439) and a control shRNA (shCTL) for PTOV1 knock-down, were analyzed by real time qPCR.(C) Immunoblots of LNCaP cells as in B, identify the endogenous or the HAPTOV1 protein. Numbers in the right panel, express the reduction of PTOV1 protein levels were quantified by densitometric analysis of the signal with respect to actin using ImageJ software. p-value: * < 0.05; ** < 0.01; *** < 0.001.