Evolutionary conservation of zinc finger transcription factor binding sites in promoters of genes co-expressed with WT1 in prostate cancer-2

Hromatin from WT1 transfected 293 kidney cells verified that these TFBS were functional. Lanes 1 and 7 show the 1 Kb ladder, lane 2 shows the No DNA PCR control, and lane 3 shows PCR amplified input DNA. Lanes 4, 5, and 6 show PCR amplified DNA immunoprecipitated by IgG (no antibody control), WT1 or SP1 antibodies, respectively. ChIP analysis of chromatin from WT1 transfected LNCaP cells verified these TFBS were functional in prostate cancer cells as well. Lanes as described in section . Predicted TFBS are based on human sequences and marked by boxes as described in Figure 1. These functional WT1 (human 1755–1771), EGR1 (human 1717–1733) and SP1 (human 1721–1735) sites were conserved between primates (human, chimpanzee, and macaque) and dogs, but not in rodents; and the SP1 site overlapped with the EGR1 site.<p><b>Copyright information:</b></p><p>Taken from "Evolutionary conservation of zinc finger transcription factor binding sites in promoters of genes co-expressed with WT1 in prostate cancer"</p><p>http://www.biomedcentral.com/1471-2164/9/337</p><p>BMC Genomics 2008;9():337-337.</p><p>Published online 16 Jul 2008</p><p>PMCID:PMC2515153.</p><p></p

Evolutionary conservation of zinc finger transcription factor binding sites in promoters of genes co-expressed with WT1 in prostate cancer-0

By boxes: EGR1, dashed; SP1, dash-dotted; WT1, solid. Two separate WT1 sites in the promoter are conserved between multiple species and both overlap an EGR1 site, and one also overlaps an SP1 site. WT1 site (human 614–630) overlaps EGR1 site (human 608–624) and both sites are conserved between all eight species surveyed. The WT1 site (human 565–581) overlaps both an EGR1 site (human 563–575) and an SP1 site (human 563–577). The SP1 site is conserved between all eight species, the WT1 site is conserved between all but opossum and the EGR1 site is conserved between primates. Negative numbers in the chimpanzee promoter sequence indicate that the orthologous region was located 1,668 base pairs from the ATG site (further upstream than 1.5 kb analyzed for other species). Two overlapping WT1 sites (human 1127–1143 and human 1129–1145) overlap an SP1 site (human 1125–1139) in the gene promoter region. The WT1 sites are conserved between human, chimpanzee, and macaque, while the SP1 site is conserved between human, chimpanzee, macaque, and cow. Two WT1 and an SP1 TFBS in the promoter are conserved. The WT1 site (human 1444–1468) is conserved between human, chimpanzee, macaque, mouse, rat, and opossum. The WT1 site (human 1409–1425) that overlaps an SP1 site is conserved between human and chimpanzee only, while the SP1 site (human 1420–1434) is conserved between human, chimpanzee, macaque, mouse, and rat.<p><b>Copyright information:</b></p><p>Taken from "Evolutionary conservation of zinc finger transcription factor binding sites in promoters of genes co-expressed with WT1 in prostate cancer"</p><p>http://www.biomedcentral.com/1471-2164/9/337</p><p>BMC Genomics 2008;9():337-337.</p><p>Published online 16 Jul 2008</p><p>PMCID:PMC2515153.</p><p></p

Evolutionary conservation of zinc finger transcription factor binding sites in promoters of genes co-expressed with WT1 in prostate cancer-1

T1 binding sites (solid box), an EGR1 site (dashed box), an SP1 site (dash-dotted box), and an SP2 site (double dash-dotted box). WT1 sites (human 1332–1348 and 1336–1352) are conserved between human, chimpanzee, macaque, and cow and they overlap an SP2 site (human 1347–1361) conserved between human, chimpanzee, and cow. An EGR1 site (human 1400–1416) overlaps an SP1 site (human 1404–1418) and both are conserved between human, chimpanzee, macaque, and dog. The binding of WT1 and SP1 TFs to native chromatin obtained from WT1-transfected LNCaP cells was confirmed by ChIP. Lane 1 shows the no DNA PCR control and lane 2 shows PCR amplified input DNA. Lanes 3, 4, and 5 show PCR amplified DNA immunoprecipitated by IgG (no antibody control), SP1 or WT1 antibodies, respectively.<p><b>Copyright information:</b></p><p>Taken from "Evolutionary conservation of zinc finger transcription factor binding sites in promoters of genes co-expressed with WT1 in prostate cancer"</p><p>http://www.biomedcentral.com/1471-2164/9/337</p><p>BMC Genomics 2008;9():337-337.</p><p>Published online 16 Jul 2008</p><p>PMCID:PMC2515153.</p><p></p

Poly (A) Binding Protein Cytoplasmic 1 Is a Novel Co-Regulator of the Androgen Receptor

<div><p>The androgen receptor (AR) is a member of the steroid receptor superfamily that regulates gene expression in a ligand-dependent manner. The NTD of the AR plays a key role in AR transactivation including androgen-independent activation of the AR in castration-resistant prostate cancer (CRPC) cells. We recently reported that amino acids (a.a.) 50-250 of the NTD are capable of modulating AR nucleocytoplasmic trafficking. To further explore the mechanism associated with a.a. 50-250, GFP pull-down assays were performed in C4-2 CRPC cells transfected with GFP tagged a.a. 50-250 of the AR. Mass spectrometry analysis of the pulled down proteins identified poly (A) binding protein cytoplasmic 1 (PABPC1) interaction with this region of the AR. <i>In silico </i>analysis of gene expression data revealed PABPC1 up-regulation in prostate cancer tissue specimens and this up-regulation correlates to increased disease recurrence. Co-immunoprecipitation assays confirmed the association of PABPC1 with a.a. 50-250 of the NTD of the AR. Knockdown of PABPC1 decreased nuclear AR protein levels and inhibited androgen activation of the AR target PSA in LNCaP and C4-2 cells. Additionally, knockdown of PABPC1 inhibited transactivation of the PSA promoter by NAR (AR lacking the LBD) and attenuated proliferation of AR-positive prostate cancer cells. These findings suggest that PABPC1 is a novel co-regulator of the AR and may be a potential target for blocking activation of the AR in CRPC.</p></div

Knockdown of PABPC1 inhibits activation of the PSA promoter by NAR.

<p><b>(A)</b> Schematic diagram of GFP-NAR. <b>(B)</b> C4-2 cells were transfected with control siRNA (siC) or siPABPC1 (pool of 3 oligos) (40 pmol/mL) for 48 hours followed by transfection with PSA6.1Luc, pRL-CMV, and either GFP (control) or GFP-NAR in the presence of 5% charcoal stripped FBS RPMI for an additional 48 hours followed by luciferase assay. Experiments were repeated two times. Significance was determined by Student’s t-test (**p<0.01, ***p<0.001).</p

Knockdown of PABPC1 inhibits activation of the PSA promoter by NAR.

<p><b>(A)</b> Schematic diagram of GFP-NAR. <b>(B)</b> C4-2 cells were transfected with control siRNA (siC) or siPABPC1 (pool of 3 oligos) (40 pmol/mL) for 48 hours followed by transfection with PSA6.1Luc, pRL-CMV, and either GFP (control) or GFP-NAR in the presence of 5% charcoal stripped FBS RPMI for an additional 48 hours followed by luciferase assay. Experiments were repeated two times. Significance was determined by Student’s t-test (**p<0.01, ***p<0.001).</p

Knockdown of PABPC1 with siRNA decreases PSA luciferase activity and PSA protein levels.

<p><b>(A)</b> C4-2 cells were transfected with control siRNA (siC) (40 pmol/mL), siPABPC1 (pool of 3 oligos) (40 pmol/mL), pRL-CMV (0.03μg), and PSA6.1Luc (0.3μg) in OPTI-MEM. The media was changed the next day to 5% chS FBS RPMI for 24 hours followed by treatment with 0.1nM R1881for an additional 24 hours in 5% chS FBS RPMI. Luciferase assay was performed with pRL-CMV (Renilla) luciferase used as a normalizer. <b>(B)</b> C4-2 cells were transfected with control siRNA (siC) or siPABPC1 (40 pmol/mL) and treated as in (A) using increasing doses of R1881 (0, 0.1, and 1nM) followed by Western blot analysis. Blots were probed with antibodies specific for PABPC1 and PSA. GAPDH was used as a loading control. <b>(C)</b> C4-2 cells were transfected with control siRNA (siC) or siPABPC1 (pool of 3 oligos) (20 pmol/mL, 40 pmol/mL and 80 pmol/mL), in OPTI-MEM. Media was changed the next day to 10% FBS RPMI for 72 hours followed by Western blot analysis. Blots were probed as in (B). C4-2 cells were transfected with control siRNA (siC) or siPABPC1 (pool of 3 oligos) (40 pmol/mL), <b>(D)</b> or individual PABPC1 siRNA oligos <b>(E)</b>, pRL-CMV (0.03μg), and PSA6.1Luc (0.3μg) in OPTI-MEM. The media was changed the next day to 10% FBS RPMI for an additional 48 hours followed by luciferase assay. Experiments were repeated three times. Significance was determined by Student’s t-test (*p<0.05, **p<0.01, ***p<0.001).</p

Knockdown of PABPC1 with siRNA decreases nuclear AR protein levels and PSA mRNA expression levels.

<p><b>(A)</b> C4-2 cells were transfected with control siRNA (siC) (40 pmol/mL) or siRNA (40 pmol/mL) specific for PABPC1 (siPABPC1) for 48 hours followed by treatment with 0.1nM R1881 for 24 hours in the presence of 5% charcoal-stripped FBS RPMI. Nuclear and cytoplasmic extracts were analyzed by Western blot. GAPDH (Santa Cruz) and Lamin A/C (Genscript, Piscataway, NJ) were used as loading and cell compartment controls. PSA <b>(B-C)</b> and PABPC1 <b>(D-E)</b> mRNA expression levels were detected in LNCaP and C4-2 cells following siPABPC1 treatment as in (A). Expression was normalized to GAPDH. Experiments were reproduced twice. Significance was determined by Student’s t-test (**p<0.01, ***p<0.001).</p

Inhibition of PABPC1 decreases proliferation of AR positive prostate cancer cells.

<p>C4-2, LNCaP, PC3, and 22Rv1 cells were transfected with control siRNA (40 pmol/mL) or a pool of siPABPC1 oligos (40 pmol/mL) followed by BrdU assay (as described in the Methods section). <b>(A)</b> Shown are representative images from three individual experiments of cells treated with either control siRNA (siC) or siPABPC1. <b>(B)</b> BrdU positive cells were counted and compared between siC and siPABPC1 groups. Experiments were reproduced three times. Significance was determined by Student’s t-test. (*p<0.05, **p<0.01).</p

PABPC1 is expressed in prostate cancer cells.

<p><b>(A)</b> Prostate cancer cell lines (PC3, LNCaP, C4-2, and 22Rv1) and normal prostate cells (WPMY-1) were analyzed by Western blot for protein expression levels of PABPC1 (Santa Cruz). <b>(B)</b> Expression levels of the AR in C4-2, LNCaP, and 22Rv1 cell lines. GAPDH (Santa Cruz) was used as a loading control. The numbers indicate the relative expression of PABPC1 or AR quantitated using ImageJ software when normalized to GAPDH.</p