FIGURES AND TABLES
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hnRNP-U directly interacts with WT1 and modulates WT1 transcriptional activation
L Spraggon, T Dudnakova, J Slight, O Lustig-Yariv, J Cotterell, N Hastie and C Miles
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Figure 1.
Generation of WT1-null ES cells lines. (a) Induction of WT1 protein was confirmed in ATRA-treated monolayer E14 (iv) ES cells by Western blot. E14 (iv) ES cells were differentiated with 1 
M all-trans retinoic acid ATRA for 96 h as previously described (Scharnhorst et al., 1997). Nuclear protein (40 g) from 24, 48, 72 and 96 h of differentiation was resolved and probed with anti-WT1 rabbit polyclonal antibody (C-19; Santa Cruz) or anti-p116 rabbit polyclonal (gift of Dr A Luhrmann) to control for loading. Strong induction of WT1 is seen after 72 and 96 h. (b) Western blot analysis confirms LK1 as being a WT1-null ES cell line. Western Blot of 40 g of nuclear protein from monolayer cultures of E14 and LK1 ES cells undifferentiated and differentiated for 96 h with 1
M ATRA. Proteins were resolved and probed with anti-WT1 rabbit polyclonal antibody (C-19; Santa Cruz) or anti-p116 rabbit polyclonal (gift of Dr A Luhrmann) to control for loading. WT1 is only detectable in wild-type ES cells induced with ATRA.
Figure 2.
Identification of WT1 interacting proteins. (a) Immunoprecipitation of endogenous WT1 complexes. Nuclear extracts were prepared from ATRA-differentiated monolayer cultures of E14 (iv) and LK1 embryonic stem cells and pre-cleared with sepharose beads, then incubated with immobilized anti-WT1 rabbit polyclonal antibody (C-19; Santa Cruz) overnight at 4°C. Equivalent amounts of WT1 immunoprecipitates from E14 (iv) and LK1 were resolved and probed with anti-WT1 mouse monoclonal antibody (6F-H2; Dako) for the presence of WT1. T, total nuclear extract; UN, unbound fraction; E1, first elution; E2, second elution. (b) Identification of WT1 associated protein by mass spectrometry. WT1 immunoprecipitates (20 g) from E14 (iv) and LK1 were resolved on a gradient NuPAGE 4–12% Bis-Tris Gel (Invitrogen) along with a Mark12-unstained standard marker (Invitrogen). Proteins were visualized by Coomassie Blue stain (Genomic Solutions). Protein bands annotated as L1–L4 were excised from the gel along with the corresponding regions from the LK1 lane and peptide fingerprint analysis was carried out using an Applied Biosystems Voyager DE™ STR Maldi-TOF instrument. Interpretation of data was preformed using the Voyager V5 Data Explorer software and database searching performed using the Protein Prospector MS-Fit program (http://prospector.ucsf.edu/). Table 2.1 shows the results from the MS-Fit program.
Figure 3.
Endogenous WT1 and hnRNP-U interact. (a–d) Reciprocal endogenous immunoprecipitation of WT1 and hnRNP-U. Nuclear extracts from 1
M ATRA-treated monolayer cultures of E14 (iv) ES cells (a and c) or M15 (b and d) were immunoprecipitated with anti-WT1 rabbit polyclonal antibody (C-19; Santa Cruz), anti-hnRNP-U mouse monoclonal antibody (3G6; gift of Dr G Dreyfuss) or pre-immune IgG antibody (Sigma). WT1 immunoprecipitates were resolved and probed with anti-WT1 mouse monoclonal antibody and anti-hnRNP-U mouse monoclonal antibody (A, ES cells; B, M15 cells), while hnRNP-U immunprecipitations were resolved and probed with anti-WT1 rabbit polyclonal antibody (C, ES cells; D, M15 cells). (e) Colocalization of WT1 and hnRNP-U proteins. Endogenous expression of WT1 protein (red) and hnRNP-U protein (green) was determined by indirect immunofluoresence in M15 cell lines as previously described (Ladomery et al., 1999), using C-19 and 3G6 antibodies and visualized with deconvolution fluorescence microscopy. Three-dimensional (3D)-immunolocalization of the endogenous proteins was preformed by the acquisition of Z-axis stacks (0.2 mcm) using the Delta Vision Soft Worx software. The deconvolved two-channel 3D image was exported to ImarisColoc for quantitative analysis. The calculated colocalization channel was built using surface rendering techniques of ImarisSurpass, with colocalization volume displayed in white. The percent of the red channel volume colocalized with green was calculated for n=5 nuclei and is given as a mean value 
s.d. in the upper left corner. The Pearson channel correlation in colocalized volume is given in the upper right corner (1=perfect colocalisation, 0=no correlation). (f) Colocalization of WT1 and hnRNP-U during nephrogenesis. Endogenous expression of WT1 protein (green) and hnRNP-U protein (red) was determined by indirect immunofluoresence in mouse E16.5 fetal kidneys using the C-19 and 3G6 antibodies.
Figure 4.
Domains of association and affects of hnRNP-U on WT1 transcriptional activation. (a) Schematic representation of the GST-WT1 expression constructs. Construction and use of GST-WT1 bacterial expression vectors has been previously described (Lee et al., 2002). GST-WT1 (FL) represents full-length WT1 protein (amino acids 1–446) including exon 5 insert and excluding the KTS insert. GST-WT1 (N) represents the N-terminal region of WT1 protein (amino acids 1–242). GST-WT1 (C) represents the C-terminal region of the WT1 protein (amino acids 297–446) which covers the zinc-finger region (excluding KTS insert). (b) hnRNP-U interacts with the zinc-finger domain of WT1. GST and GST-WT1 recombinant proteins were purified as described previously (Lee et al., 2002). 35S-methionine-labelled hnRNP-U or Luciferase was prepared using the TNT-coupled Transcription/Translation system (Promega) as per the manufacturer's instructions. 35S-methionine-labelled hnRNP-U or Luciferase were incubated with GST or GST-WT1 recombinant proteins. Captured proteins were resolved and visualized by autoradiography. (c) Schematic representation of HA-tagged hnRNP-U expression constructs. Construction of the HA-tagged hnRNP-U constructs were generated as previously described (Kim and Nikodem, 1999). HA-hnRNP-U (FL) represents full-length hnRNP-U (amino acids 1–806), HA-hnRNP-U (N) represents the N-terminal domain (amino acids 1–286), HA-hnRNP-U (M) represents the middle domain (amino acids 239–536) and HA-hnRNP-U (c) represents C-terminal domain (amino acids 537–806). (d and e) WT1 interacts with the middle domain of hnRNP-U. HA-tagged hnRNP-U constructs along with T7-tagged full-length WT1 were transfected in Cos-7 cells using Lipofectamine 2000 (Invitrogen) as per the manufacturer's instructions. (d) [i] Total cell extracts from transfected Cos-7 cells were resolved and probed with the anti-HA mouse monoclonal antibody (Calbiochem). (d) [ii] Nuclear extracts prepared from transfected Cos-7 cells were immunoprecipitated with anti-WT1 rabbit polyclonal antibody (C-19; Santa Cruz). WT1 immunoprecipitations were resolved and probed with an anti-HA mouse monoclonal antibody (Calbiochem). (e) Indirect immunofluorescence for WT1 (red) and hnRNP-U (green) was carried out on transiently transfected Cos-7 cells as previously described (Ladomery et al., 1999; Niksic et al., 2004). 3D-immunolocalization of the transfected proteins was preformed by the acquisition of Z-axis stacks (0.2 mcm) using Delta Vision Soft Worx software. The deconvolved two-channel 3D image was exported to ImarisColoc for quantitative analysis. The calculated colocalization channel was built using surface rendering techniques of ImarisSurpass with colocalization volumes displayed in white (a–d). The percent of the red channel volume colocalized with green was calculated for n=5 nuclei and is given as a mean values.d. in the upper left corner (a–d). The Pearson channel correlation in colocalized volume is given in the upper right corner (1=perfect colocalization, 0=no correlation). (f) hnRNP-U represses WT1 transcriptional activity. NIH3T3 cells were transfected with the amphiregulin promoter reporter construct containing or lacking the WRE-binding site (Amphiregulin-LucWRE), along with (1) WT1 () isoforms (blue) or WT1 (-/-) (red), (2) hnRNP-U (FL), (3) hnRNP-U (FL) and WT1 () or hnRNP-U (FL) and WT1 (-/-) or (4) empty vector (pcDNA3.1, Invitrogen). NIH3T3 cells were transfected with the various DNA vectors using Lipofectamine 2000, following the manufacturer's instructions (Invitrogen). All transfections were carried out using same amount of DNA, with the addition of empty expression vector (pcDNA3.1; Invitrogen) to equalize all transfections. Experiments were carried out in triplicate. [i] Luciferase activity was assayed after 48 h using the Promega Luciferase Assay Kit (Promega) as per the manufacturer's instructions. The error bars represent the s.d. of three independent experiments. The results were analysed using Student's t-test. P-values <0.05 were considered significant. [ii] NIH3T3 whole-cell extracts were immunoblotted with both anti-WT1 antibody and anti-hnRNP-U antibody to show the levels of expression of transfected WT1 and hnRNP-U.
