Fig. 1. Nucleotide and predicted amino acid sequence of hZimp10. (A) Diagram of the hZimp10 transcript. Both 5′ and 3′ untranslated regions are shown as a solid line and the coding region is displayed as an open box. The translation start site (ATG) and stop codon are labeled with nucleotide numbers. (B) The pcDNA3 constructs containing full-length hZimp10 cDNA, either with or without Flag-epitope tag, were transcribed and translated in vitro. Proteins were labeled and separated on an SDS–PAGE. (C) The full-length cDNA and predicted amino acid sequences of hZimp10 are shown (DDBJ/EMBL/GenBank accession No. AY235683). The putative functional domains are colored: a nuclear localization signal (green), a Miz domain (red) and two proline-rich regions (pink). (D) Alignment of the Miz domain of hZimp10 with those of other PIAS proteins is shown. Identical or similar amino acids are shaded with either blue or orange.

Fig. 2. Specific interaction between hZimp10 and AR. (A) cDNA fragments containing different portions of the human AR were fused to GAL4DBD in the pGBT9 vector. Numbers correspond to amino acid residues. The AR vectors were co-transformed with the pVP16 vector alone or the pVP16-Zimp10 construct. Three independent colonies were inoculated from each transformation experiment for a liquid β-gal assay. The data are shown as the mean ± SD. (B) Different truncation mutants of hZimp10 were generated by fusing fragments of hZimp10 to the TAD of VP16, and co-transformed with the pGBT9-AR/pTAD1. Transformants were selected and analyzed. (C) CV-1 cells were transiently co-transfected with AR and Flag-tagged hZimp10. Equal amounts of whole-cell lysates were blotted with AR or Flag antibodies to detect expression of the two proteins (input) and subjected to immunoprecipitation with normal mouse IgG or anti-Flag monoclonal antibody. The precipitated fractions were then resolved by SDS–PAGE and analyzed by western blotting using anti-Flag antibody or anti-AR antibody (IP).

Fig. 3. Expression of hZimp10 in cell lines and human tissues. (A) A schematic representation of the hZimp10 protein is shown with the locations of three probes indicated. The numbers correspond to the amino acid sequence of the protein. (B) Northern blots containing poly(A)⁺ RNA samples from various cell lines were hybridized with the three hZimp10 probes and a probe derived from human β-actin cDNA. (C) Relative densities (signals of probe 1 divided by those of β-actin) were used to measure the expression levels. (D) The blot with multiple human tissues was probed with probe 1 and the fragment of β-actin.

Fig. 4. Detection of intrinsic transcriptional activity of hZimp10. Full-length or truncated fragments of hZimp10 were fused to GAL4 DBD in the pM vector. Numbers correspond to amino acid residues. The pM constructs were co-transfected with luciferase reporter constructs containing the chicken myelomonocytic growth factor gene minimal promoter (–41 to +61) into CV-1 cells. Data are presented in relative light units (RLU), which were obtained by normalizing the activities of luciferase to those of β-gal. The results are reported as the mean ± SD from representative experiments.

Fig. 5. hZimp10 selectively enhances AR-mediated transcription. (A) CV-1 cells were transfected with a luciferase reporter driven by the 7 kb PSA promoter (100 ng), pcDNA3-β-gal (25 ng), pSV-hARo (5 ng) and different amounts of the pcDNA3-Flag-hZimp10 as indicated. Twenty-four hours after transfection, cells were incubated with or without 10 nM DHT for 24 h. Cell lysates were prepared for assessment of luciferase and β-gal activities. (B) Similar to (A), except that a 2×ARE-luc reporter (100 ng) was used. (C) LNCaP cells were transfected with the PSA7kb-luc reporter (100 ng), pcDNA3-β-gal (25 ng) and the pcDNA3-Flag-hZimp10 as indicated. Twenty-four hours after transfection, cells were treated with or without 10 nM DHT for 24 h. Cell lysates were measured for luciferase and β-gal activities as described above. (D) One hundred nanograms of luciferase reporters driven by different response elements, as labeled in the figure, were co-transfected with 50 ng of pSV40-β-gal and 10 ng of different receptors into CV-1 cells. Either 0, 5 or 20 ng of pcDNA3-Flag-hZimp10 was co-transfected. Cells were cultured either in the presence or absence of the specific ligands to each receptor, including 10 nM dexamethasone (DEX), 10 nM progesterone, 100 nM β-estradiol, 10 nM tri-iodothyronine and 10 nM 1α, 25-dihydroxyvitamin D3.

Fig. 6. The AR and hZimp10 co-localize in prostate epithelial cells. Four pairs of human prostate tissue samples (A–H) were stained either with anti-AR (left panel) or anti-hZimp10 (right panel) antibody. Color was developed with DAB in PBS. All sections used for immunohistochemistry were lightly counterstained with 5% (w/v) Harris hematoxylin.

Fig. 7. Immunofluorescent localization of hZimp10 at replication foci. The pcDNA3-Flag-hZimp10 construct was transfected into CV-1 cells. Cells were synchronized with 0.5 mM mimosine (see Materials and methods). Representative confocal laser scanning microscopy images of nuclei from cells expressing Flag-tagged hZimp10 proteins and pulsed with BrdU are shown. Color was developed with either FITC- conjugated monoclonal anti-BrdU antibody (green) or the anti-Flag primary antibody followed by secondary antibodies conjugated with rhodamine (red). Merge (right panel) of left and middle panels indicates areas of co-localization (yellow).

Fig. 8. Immunofluorescent co-localization of hZimp10 and AR. CV-1 cells co-transfected with pcDNA3-Flag-hZimp10 and pSVARo were synchronized with mimosine. Double immunostaining was conducted with anti-Flag and anti-AR antibodies followed by the secondary antibodies conjugated with FITC (green) and rhodamine (red), respectively. Representative confocal laser scanning microscopy images of nuclei from cells expressing Flag-tagged hZimp10 and AR proteins are shown. Merged images demonstrating co-localization of proteins are shown on the right panels (yellow). Staining was performed at different time points, including late G₁ phase before entering S phase (0 h, A), early S phase (4 and 6 h, B and C), mid-S phase (8 and 12 h, D and E) and late stages of S phase (18 h, F).

Fig. 9. Immunofluorescent co-localization of GFP–SUMO-1 and hZimp10 at DNA replication foci throughout S phase. CV-1 cells co-transfected with pcDNA3-Flag-hZimp10 and pGFP-SUMO-1 were synchronized with mimosine. Immunostaining was conducted with anti-Flag monoclonal antibody followed by secondary antibodies conjugated with rhodamine (red) to contrast with the GFP proteins. Merged images demonstrating co-localization of proteins are shown in the right panels (yellow). Staining was conducted at different time points as described in Figure 8.

Fig. 10. Immunofluorescent localization of SUMO-1 at replication foci. CV-1 cells were transfected with GFP–SUMO-1 plasmid, and then synchronized as described in Materials and methods. Representative confocal laser scanning microscopic images of nuclei from cells expressing GFP–SUMO-1 proteins and pulsed with BrdU are shown. Color was developed by either FITC-conjugated monoclonal anti-BrdU antibody (red) or fluorescent images of GFP proteins (green). Merge (right panel) of left and middle panels indicates areas of co-localization (yellow).

Fig. 11. hZimp10 enhances the sumoylation of AR and augments AR-mediated transcription. (A) COS-1 cells were co-transfected with 200 ng of pSG5-AR in the presence and absence of 200 ng of pFlag-PIAS1 or 360 ng of pcDNA3-Flag-hZimp10 along with 40 or 100 ng of pSG5-His-SUMO-1 as indicated. The cells were collected 48 h after transfection and the lysates were immunoblotted with the AR antibody. The slower migrating AR bands (sumoylated) are labeled with solid arrows. (B) CV-1 cells were transfected with a luciferase reporter driven by the 7 kb PSA promoter (100 ng), pcDNA3-β-gal (25 ng), pcDNA-hARwt or mut (5 ng) and pcDNA3-Flag-hZimp10 (10 ng). Cell lysates were prepared for assessment of luciferase and β-gal activities.