Fig. 2. A large N-terminal region in Gfi-1 is required for interaction with PIAS3. (A) Yeast two-hybrid assay in strain L40 with either a full-length LexA–Gfi-1 bait construct (Gfi-1 I) or the indicated mutants and a galactose-inducible construct encoding the full-length PIAS3 protein as a fusion with the transactivation domain VP16. Interaction depends on the presence of galactose (SG bars) and is only detectable with baits that bear the Gfi-1 region from amino acid 20 to 257. (B) Schematic representation of the Gfi-1 mutants that were expressed in the bait vector as fusions with the LexA DNA binding domain. The mutants that are able to interact with full-length PIAS3 protein are indicated. (C) Expression control of the LexA–Gfi-1 fusion protein and the Gfi-1 mutant baits Gfi-1 II and IV–X. Expression control (lane C) was a LexA fusion with the cytoplasmic domain of CD95. Yeast extracts were separated on SDS–PAGE and transferred on to a solid support by electroblotting. LexA–Gfi-1 proteins and the control protein were detected with an anti-LexA antibody.

Fig. 4. Endogenous PIAS3 and Gfi-1 proteins can be coprecipitated from human cells. (A and B) Nuclear and cytoplasmic extracts (see lanes marked N and C) were prepared from human KIT 225 T cells and immunoprecipitations (IP) were performed with anti-PIAS3c (Chung et al., 1997) or anti-PIAS1/3 (α-Pias3N) antibodies (Santa Cruz). To detect Gfi-1 (arrowheads) the precipitates were analyzed by SDS–PAGE and western blotting (We) using either the anti-Gfi-1 N20 antibody (Santa Cruz) or affinity-purified anti-Gfi-1 rabbit antibodies (Schmidt et al., 1998). (C) The expression level of endogenous PIAS3 was determined in nuclear and cytoplasmic extracts (lanes N and C) from human KIT 225 T cells, HepG2 cells and HeLa cells by western blotting using the anti-PIAS3c antibody. (D) Immunoprecipitations were performed with the nuclear extracts from (C) and the anti-PIAS3c antibody. The extracts and the precipitates were analyzed by SDS–PAGE and western blotting (We) using the anti-Gfi-1 N20 antibody. Gfi-1 could be precipitated only from KIT 225 extracts (arrowhead) but not from HepG2 or HeLa cell extracts. All data shown are representative of several independent experiments.

Fig. 6. Endogenous PIAS3 and transfected Gfi-1 colocalize in nuclear dots. (A) NIH 3T3 cells were transfected with GFP–Gfi-1 constructs, immunostained with anti-PIAS3c antibodies and a TRITC-labeled secondary antibody and analyzed by confocal microscopy. Shown are three independent sets of cells where the endogenous PIAS3 is found in nuclear dots and in the cytoplasm (red panels). GFP–Gfi-1 forms nuclear dots (green panels) and partly colocalizes with PIAS3 within the nuclear dots (see merged pictures, right). (B) As in (A) but instead of GFP–Gfi-1 a LTR–Gfi-1 construct was transfected that directs expression of an untagged Gfi-1 protein. Gfi-1 is detected by immunostaining with anti-Gfi-1 antibodies and a specific FITC-labeled secondary antibody (green panels). As demonstrated in (A), the endogenous PIAS3 (red panels) and the transfected Gfi-1 colocalize in nuclear dots (merged pictures, yellow areas) but regions are seen in which both proteins appear separate (merged pictures, green and red areas). (C) KIT 225 cells were immunostained with both anti-Gfi-1 and anti-PIAS3 antibodies and specific secondary antibodies labeled with FITC to detect Gfi-1 (green, left panel) or with TRITC to detect PIAS3 (red, middle panel). The merge of both pictures (right panel) demonstrates colocalization of both endogenous Gfi-1 and PIAS3 in KIT 225 cells (merged picture, yellow areas) but also reveals areas where both proteins do not form a complex.

Fig. 8. Gfi-1 regulates pACT and p600 c-fos promoters. (A) In comparison with full-length Gfi-1, the mutant Gfi-1 III, which contains only the zinc finger region, is unable to stimulate pACT transcription effectively in the presence of IL-6, regardless of whether a high exogenous PIAS3 level is reached by transfection or only the endogenous PIAS3 is present. Conditions of the experiment are as described for Figure 7. Numbers indicate the amount of plasmid transfected in µg. (B) Expression level of endogenous PIAS3 and transfected Flag-tagged PIAS3 is unaltered by co-expression of Gfi-1, Gfi-1 II or Gfi-1 III. PIAS3 protein is detected by immunoblotting with the anti-PIAS3c antibody in whole cell extracts of HepG2 cells that have been treated as indicated. (C) Gfi-1 can enhance IL-6/STAT3-mediated transcription of the c-fos promoter. Transcription of a reporter gene construct in which a 600 bp fragment of the c-fos promoter drives the luciferase gene can be stimulated in HepG2 cells with IL-6 ∼2.5-fold compared with values without IL-6. Cotransfection of constructs directing expression of Gfi-1 or Gfi-1 II lead to further stimulation of c-Fos reporter activity. SRE, serum response element; SIE, STAT-inducible element.

Fig. 1. Yeast two-hybrid screen identifies PIAS3 as a partner protein for Gfi-1. (A) Schematic representation of the LexA–Gfi-1 fusion protein used as the bait in a yeast two-hybrid screening and three LexA–Gfi-1 mutants. Only the full-length Gfi-1 and the N-terminal part spanning amino acids 1–257 were able to interact with the protein fragments expressed by clones 12 and 45, but not the SNAG domain comprising amino acids 1–20 or the zinc finger domain stretching from amino acid 257 to 423. NLS, nuclear localization signal. (B) Quantification of the Gfi-1–PIAS3 interaction by β-galactosidase assay in the yeast clones #12 and #45 (strain L40) transformed with the indicated LexA–Gfi-1 fusion constructs. (C) The sequences contained in the library plasmids of clone #12 and clone #45 were identical and encode the C-terminal part of the PIAS3 protein from amino acid 391 to 583.

Fig. 3. PIAS3 and Gfi-1 interact in vitro. (A) PIAS3 protein was produced and labeled with [³⁵S]methionine by in vitro transcription–translation (single right lane). The Gfi-1 mutant Gfi-1 II, which comprises the N-terminal region, amino acids 1–257, but lacks the zinc finger region, was produced in bacteria as a histidine-tagged protein (His–Gfi-1 II). As a control the histidine-tagged form of the N-terminal region of the POU factor Brn-3a was used. The ³⁵S-labeled PIAS3 protein could be precipitated by NTA beads from a mixture with His–Gfi-1 II but not from a mixture with the irrelevant protein His–Brn-3a (autoradiograph, upper panel). Both His–Gfi-1 II and His–Brn-3a were produced at equal levels (Coomassie staining, lower panel). (B) SDS–PAGE analysis of Gfi-1, PIAS3 and Brn-3a in vitro translation products directly from the transcription–translation reaction (input, lanes 1–3, right panel) and after incubation of the indicated in vitro translated proteins with GST–Gfi-1- or GST–PIAS3-coupled Sepharose beads (lanes 1–4, left panel). SDS–PAGE analysis after incubation of the indicated in vitro translated proteins with Sepharose beads coupled to GST alone (lanes 1–4, middle panel).

Fig. 5. Gfi-1 and PIAS3 are detected in nuclear dots. (A) NIH 3T3 cells transfected with GFP constructs were fixed in ethanol 24 h later and stained with propidium iodide. To detect Flag epitope-tagged PIAS3, live cells were treated after transfection with anti-Flag antibody and a TRITC-labeled secondary antibody. All preparations were then analyzed by laser scanning confocal microscopy. GFP–Gfi-1 fusion protein (top left), GFP–PIAS3 (top right and center left) as well as Flag epitope-tagged PIAS3 (center right) localize in the nucleus in a dotted structure. GFP–Gfi-1 II fusion proteins lacking an NLS were found in the cytoplasm (bottom left). The fusion protein between GFP and the Gfi-1 mutant Gfi-1 III (GFP–Gfi-1 III), which consists only of the zinc finger domain, resides in the nucleus still in a dotted structure (bottom right). (B) NIH 3T3 cells were cotransfected with constructs directing expression of Flag-tagged PIAS3 and either GFP–Gfi-1, GFP–Gfi-1 II (N-terminus only) or GFP–Gfi-1 III (zinc finger only) fusion proteins. Confocal microscopy revealed that GFP–Gfi-1 (top row, left) as well as Flag–PIAS3 (top row, middle) are localized in nuclear dots that perfectly overlap when the pictures are merged (top row, right). GFP–Gfi-1 II remains outside the nucleus and prevents the Flag–PIAS3 from entering the nucleus (middle row). Both GFP–Gfi-1 II and Flag–PIAS3 are perfectly colocalized outside the nucleus when both pictures are merged (middle row, right). GFP–Gfi-1 III (bottom row, left) and Flag–PIAS3 (bottom row, middle) enter the nucleus when the appropriate constructs are cotransfected, but do not colocalize (note red and green dots in bottom row, right) because the N-terminal interaction domain is lacking in GFP–Gfi-1 III.

Fig. 7. Gfi-1 relieves STAT3-mediated transcriptional transactivation from PIAS3 block. (A) HepG2 cells were transfected with the pACT reporter, a STAT3 construct and the indicated amounts of CMV–PIAS3. The structure of the α₁-antichymotrypsin promoter with the two STAT3 binding sites is given in the right panel. In the presence of STAT3 and IL-6, the pACT reporter could be activated ∼15-fold (black bars) compared with values obtained without IL-6 (open bars) and this stimulation could be inhibited by cotransfection of CMV–PIAS3 in a concentration-dependent manner. (B) Left panel: cotransfection of HepG2 cells with pACT, a STAT3 construct and a Gfi-1 expression construct (LTR–Gfi-1, amounts as indicated). Gfi-1 can further stimulate pACT transcription up to ∼6-fold in a concentration-dependent manner in the presence of IL-6 (black bars). Right panel: nuclear (N) and cytoplasmic (C) extracts were prepared from HepG2 cells that had been transfected with LTR–Gfi-1 or had been treated with IL-6 as indicated. The extracts were analyzed by SDS–PAGE and immunoblotting with anti-PIAS3 antibodies or antibodies that exclusively recognize STAT3 when phosphorylated on tyrosine 705. (C) Left panel: HepG2 cells were transfected with pACT, STAT3 and PIAS3 expression constructs and treated with IL-6. A marked repression of pACT reporter gene transcription is observed as in (B). This repression can be almost entirely relieved by co-expression of a Gfi-1 expression construct (LTR–Gfi-1) in a concentration-dependent manner. Right panel: a similar relief of pACT transcription could be reached in the presence of IL-6 with a construct expressing the Gfi-1 mutant Gfi-1 II, which lacks the zinc finger region but still interacts with PIAS3.

Fig. 9. Gfi-1 can enhance IL-6-dependent T-cell proliferation upon antigenic stimulation. (A) RNA expression levels of endogenous PIAS3 and Gfi-1 genes detected by northern blotting in extracts from isolated primary murine T cells after stimulation as indicated over time. (B) Schematic representation of the construct used to generate CD2–Gfi-1 transgenic mice. (C) Gfi-1 protein levels were analyzed by western blotting in extracts from thymocytes and T cells isolated from lymph nodes of animals representing the CD2–Gfi-1 transgenic lines tg1 and tg2. Extracts from non-transgenic age matched littermates (ntg) served as controls. (D) Peripheral T cells were prepared from either non-transgenic control mice (black bars) or CD2–Gfi-1 transgenic mice (open bars) and cultured under the conditions indicated. The cells were cultivated for 72 h and pulsed with [³H]thymidine for the last 12 h. The uptake of [³H]thymidine was measured and is given on the y-axis. All measurements were taken in triplicate. Given are average values with standard deviations representative of several independent experiments.