Identification of p100 as a coactivator for STAT6 that bridges STAT6 with RNA polymerase II

Abstract

STAT6 is a central mediator of IL-4-induced gene responses. STAT6-mediated transcription is depend ent on the C-terminal transcription activation domain (TAD), but the mechanisms by which STAT6 activates transcription are poorly understood. Here, we have identified the staphylococcal nuclease (SN)-like domain and tudor domain containing protein p100 as a STAT6 TAD interacting protein. p100 was originally characterized as a transcriptional coactivator for Epstein-Barr virus nuclear antigen 2. STAT6 interacted with p100 in vitro and in vivo. The interaction was mediated by the TAD domain of STAT6 and the SN-like domain of p100. p100 did not affect the immediate activation events of STAT6, but enhanced STAT6-mediated transcriptional activation and the IL-4-induced Igepsilon gene transcription in human B-cell line. Finally, p100 associated with the large subunit of RNA polymerase II and was mediating interaction between STAT6 and RNA polymerase II. These findings identify p100 as a novel coactivator for STAT6 and suggest that p100 functions as a bridging factor between STAT6 and the basal transcription machinery.

Fig. 1. Physical interaction between STAT6-TAD and p100. (A and B) STAT6-TAD interacts specifically with several nuclear proteins. Aliquots of nuclear lysates from [³⁵S]methionine-labeled or unlabeled Ramos B lymphocytes were incubated with either GST alone or with GST–St6TAD. The bound proteins were subjected to SDS–PAGE and visualized by autoradiography (A) or silver staining (B). The band corresponding to the 100 kDa protein was cut out from silver-stained gel and subjected to trypsin digestion and MALDI-TOF mass spectrometry. The lower panel in (B) is the loading control of GST–St6TAD and GST proteins.

Fig. 2. p100 interacts with STAT6 in vivo and in vitro. (A) p100 co- immunoprecipitates with STAT6 in vivo. COS-7 cells were transfected with STAT6-HA and p100-Flag as indicated and immunoprecipitated with anti-HA antibody and blotted with anti-Flag antibody. Ten percent of total cell lysate (TCL) from p100-Flag-transfected cells was included as a control (upper panel). The same filter was stripped and re-blotted with anti-HA antibody for STAT6 (middle panel). The expression level of p100 from different lysates was detected by anti-Flag immunoblotting (lower panel). (B) STAT6 coimmunoprecipitates with p100. COS-7 cells were transfected as indicated and cell extracts were immunoprecipitated with anti-Flag and blotted with anti-HA antibody. Ten percent of TCL from STAT6-HA-transfected cells was included as a control (upper panel). The same filter was stripped and re-blotted with anti-Flag antibody (middle panel). The expression level of STAT6 from different lysates was detected by western blot analysis (lower panel). (C) Jak2 does not interact with p100. COS-7 cells were transfected with p100-Flag, HA-tagged Jak2 (Jak2-HA) or STAT6-HA expression plasmids. Cell extracts were immunoprecipitated with anti-HA and immunoblotted with anti-Flag antibody (upper panel). The same filter was stripped and re-blotted with anti-HA antibody (middle panel). The expression level of p100 in different lysates was detected by western blot analysis (lower panel). The positions of p100, STAT6 and Jak2 are indicated by arrows. (D) p100 interacts with STAT6-TAD in vitro. p100 was ³⁵S-labeled by in vitro translation and incubated with beads loaded with GST–St6TAD or GST. The bound proteins were subjected to SDS–PAGE and visualized by autoradiography. Twenty percent of the in vitro-translated p100 was included as a control (upper panel). p100-Flag was expressed in COS-7 cells and the lysates were incubated with GST–St6TAD or GST. The bound proteins were blotted with anti-Flag antibody. Twenty percent of TCL from p100-Flag transfected cells was included as a control (lower panel).

Fig. 3. p100 enhances STAT6-dependent transcriptional activation. (A) Overexpression of p100 enhances, while anti-sense p100 inhibits, transcriptional activation of STAT6. Ramos cells were transfected with Igε luciferase reporter construct (10 µg) and β-galactosidase vector (5 µg), together with increasing amounts (10, 20 and 30 µg) of p100 or anti-sense p100 expression plasmid, and treated with IL-4 as indicated. The normalized luciferase values are shown. The lower panels in (A) and (B) show p100 and STAT6 protein levels in the lysates. (B) p100 protein selectively enhances IL-4-induced transcriptional activation of STAT6. HeLa cells were transfected with STAT6 expression plasmid (1.0 µg), β-galactosidase vector (0.5 µg) and Igε luciferase reporter construct (1.0 µg) or C/EBP luciferase reporter constructs (1.0 µg), together with increasing amounts of p100 expression plasmid, and treated with IL-4. The normalized luciferase values are shown. (C) p100 enhances transcription of STAT6-TAD in a heterologous system. GAL4-St6TAD or GAL4-DBD alone (GAL4) expression plasmids (0.5 µg) were transfected into HeLa cells with reporter plasmid (GAL4)₃TK-luciferase (0.5 µg), together with increasing amounts of the p100 expression vector. β-galactosidase and luciferase values were measured 48 h after transfection. Mean normalized luciferase values of three independent experiments with standard deviations are shown.

Fig. 4. p100 enhances the transcription of Igε gene in BJAB cells. Real-time PCR analysis of Igε mRNA in BJAB and BJAB-p100 B-cell lines. Standard curves for Igε (A) and TBP (B) genes, plotting fractional cycle number at the fluorescent threshold on the y-axis and the logarithmic input cDNA concentration on the x-axis. (C) Relative expression of the Igε gene in untreated or IL-4-treated (100 ng/ml for 24 h) BJAB and BJAB-p100 B-cell lines. The results shown are representative of three experiments performed.

Fig. 5. Mapping the STAT6 interaction domain of p100. (A) STAT6 associates with the SN-like domain of p100 (p100-SN) in vitro and in vivo. Upper panel: p100-SN interacts with STAT6-TAD in vitro. p100-SN was [³⁵S]methionine labeled by in vitro translation and then incubated with beads loaded with GST or GST–St6TAD. After SDS–PAGE, the bound proteins were visualized by autoradiography. Lower panel: COS-7 cells were transfected with Flag-tagged p100-SN and STAT6-HA as indicated. Cell extracts were immunoprecipitated with anti-HA and blotted first with anti-Flag and then with anti-HA antibodies. (B) The TD of p100 (p100-TD) does not interact with STAT6. Upper panel: p100-TD was [³⁵S]methionine-labeled by in vitro translation and then incubated with beads loaded with GST or GST–St6TAD, and the bound proteins were visualized by autoradiography. Lower panel: COS-7 cells were transfected with Flag-tagged p100-TD and STAT6-HA expression plasmids as indicated. Cell extracts were immunoprecipitated with anti-HA and blotted first with anti-Flag and then with anti-HA antibodies. (C) IL-4-induced transcriptional activation of STAT6 is enhanced by p100-SN but not by the TD of p100. HeLa cells were transfected with Igε luciferase reporter construct (0.5 µg), β-galactosidase vector (0.5 µg) and the indicated amounts of p100-SN or p100-TD expression plasmids, and treated with IL-4 for 6 h. The normalized luciferase values are shown. The expression levels of p100-SN and p100-TD are shown in the anti-Flag blot.

Fig. 6. p100 interacts with RNA polymerase II in vitro and in vivo. (A) p100 interacts with RNA polymerase II in vivo. COS-7 cells were transfected with p100-Flag or STAT6-HA expression plasmids. Upper panel: cell extracts were immunoprecipitated with anti-RNA pol II antibody (lanes 3 and 6) or unrelated antibody (anti-myc) as control (lanes 2 and 5) and immunoblotted with anti-Flag antibody for p100 or anti-HA antibody for STAT6. Anti-RNA pol II blot of the immunoprecipitates in the lower panel shows typical laddering due to differential phosphorylation and ubiquitylation. (B) p100 interacts with RNA polymerase II in vitro. Upper panel: HeLa cell lysates were immunoprecipitated with anti-RNA pol II antibody (lanes 3 and 6) or unrelated antibody (anti-myc) as control (lanes 2 and 5), and after extensive washes the immunoprecipitates were incubated with in vitro-translated and [³⁵S]methionine-labeled p100 or STAT6. After washes, the bound proteins were subjected to SDS–PAGE and visualized by autoradiography. Lower panel: anti-RNA pol II blot of the immunoprecipitates. (C) HeLa cell lysates were immunoprecipitated with anti-RNA pol II antibody (lanes 3 and 5) or unrelated antibody (rabbit IgG) as control (lanes 2 and 4). After extensive washes, the immunocomplexes were incubated with in vitro-translated and [³⁵S]methionine-labeled STAT6 together with control lysates lacking p100 (lanes 2 and 3) or with p100 protein lysate (lanes 4 and 5). After washes, the bound proteins were subjected to SDS–PAGE and visualized by autoradiography. Lower panel: anti-RNA pol II blot of the immunoprecipitates.

Fig. 7. p100 co-precipitates in vivo with STAT6 and RNA pol II. (A) p100 co-precipitates with STAT6 in intact BJAB cells. BJAB cells were treated with vehicle or IL-4 (30 min), and the lysates were immunoprecipitated with anti-STAT6 or anti-myc (control) antibodies. Upper panel shows blotting with anti-STAT6, and lower panel shows blotting with anti-p100. (B) p100 co-precipitates STAT6 and RNA pol II in HeLa-p100-Flag cells. Total cell lysates from vehicle or IL-4-treated (30 min) HeLa-p100-Flag cells were immunoprecipitated with anti-STAT6, anti-Flag and anti-myc (control) antibodies and subjected to anti-STAT6 (upper panel), anti-Flag (middle panel) or anti-RNA pol II (lower panel) immunoblotting. (C) Expression level of p100 in HeLa and HeLa-p100-Flag cells. Lane 1: immunoprecipitated p100-Flag from transfected COS-7 cells; lane 2: 25 µg of HeLa cell lysate; lane 3: 25 µg HeLa-p100-Flag cell lysate blotted with anti-p100 antiserum. (D) Activation of Igε-luc reporter gene in HeLa and HeLa-p100-Flag cells in response to IL-4 stimulation. HeLa and HeLa-p100-Flag cells were transfected with Igε luciferase reporter construct (0.5 µg) and β-galactosidase vector (0.5 µg) and treated with IL-4 as indicated. The normalized luciferase values are shown. Mean normalized luciferase values of three independent experiments with standard deviations are shown.