Methoctramine br Fig PTOV is associated to
Fig. 2. PTOV1 is associated to the chromatin of ALDH1A1 and CCNG2 promoters. (A) Sheered chromatin from LNCaP cells transduced with a lentivirus encoding the fusion protein HAPTOV1 was immunoprecipitated with rabbit antibodies to PTOV1, total and phosphorylated polymerase II, and rabbit or mouse IgGs as controls. Co-immunoprecipitated DNA fragments were analyzed by PCR with specific primers for ALDH1A1,CCNG2, ABCB1 and HES1 promoter regions. (B) Graphical representation of primers localization and length of the amplified sequences on the promoters of the indicated genes. TSS: transcription start site.
resembling the core AT-hook (Fig. 3A) present in the peptide that binds to ALDH1A1 and CCNG2 promoters, we changed the motif to EGGP, by means of site-directed mutagenesis. This mutant was created in the GFP-full-length PTOV1 plasmid, so as to study the function of the
protein as a transcription activator. As it is shown in Fig. 4, both wild-type or mutant GFP-PTOV1 were eﬃciently expressed in transfected cells. Importantly, the expression of the mutant caused a moderate but significant decrease in ALDH1A1 and CCNG2 transcript levels when
Fig. 3. EMSA identify a new AT-hook-like motif in the A domain of PTOV1. (A) Schematic of the protein structure of PTOV1 identifying the AT-hook domains present at the N-terminal (eAT-hook) and within the A domain (AT-hook-like) (not in scale). Bottom, eAT-hook, the first 43 amino acids from the N-terminal of PTOV1; A domain, the partial amino Methoctramine sequence (85–125) of the A domain; B domain, the partial amino acid sequence of the B domain (amino acid 249 to 292); A domain peptide, the amino acid sequence of the AT-hook-like motif peptide used for EMSA assays (amino acid 100 to 114); AT-hook consensus, the described AT-hook of HGMA1 (amino acid 21 to 31). Bold characters identify amino acids in the ‘core’ of AT-hook motifs. (B) Drawings show the localization of the DNA sequence probes with respect to the transcription start sites (TSS). Bottom panels: two gel shift assays are shown for each ALDH1A1 and CCNG2 probe: in the left gels, labeled sequences from ALDH1A1 promoter were incubated with recombinant GST-PTOV1 (PTOV1), GST-A domain (A dom), GST-B domain (B dom), eAT-hook wild type peptide (eAT-WT pept) and mutated eAT-hook peptide (eAT-mut pept). In the right gels, labeled sequences from the CCNG2 promoter were incubated with the GST-A domain (A dom), GST-B domain (B dom), and the A domain AT-hook-like peptide (A dom pept). Arrows indicate the shift provoked by the binding of protein domains or peptides to labeled DNA. A gel shift assay performed with the HES1 probe is shown on the right panel as control.
compared to wild-type PTOV1 (Fig. 4B). The GFP-PTOV1 plasmids express comparable levels of the exogenous protein, indicating similar levels of transfection in these experiments (Fig. 4C). Together with the above demonstration of direct binding to ALDH1A1 and CCNG2 promoter sequences, these results lend support to the hypothesis that the AT-hook-like motif in the A domain of PTOV1 is critical for the PTOV1 promoted activation of transcription of these genes.
3.5. PTOV1, ALDH1A1 and CCNG2 expression levels are associated with aggressiveness in prostate and colon carcinomas
To understand the significance of the ability of the oncoprotein
PTOV1 to directly bind and activate the expression of ALDH1A1 and CCNG2 genes in tumor cells, we interrogated several publicly available databases containing expression data, clinical and pathological in-formation of untreated patients with prostate cancer for the association of expression of these genes. Data derived from micro-dissected un-treated prostate tumors specimens  show that PTOV1, ALDH1A1 and CCNG2 transcript levels are significantly increased in patients with high Gleason Score (> 8) in comparison to patients with low Gleason score (< 7) (Fig. 5A) (GSE97284)34. The transcript levels of these genes are also significantly higher in prostate adenocarcinomas of patients who developed regional or distal metastasis after radical prostatectomy, suggesting their relationship with metastatic progression . In addi-tion, the expression levels of PTOV1 significantly correlated with the