Control bacteria, or bacteria that express SUMO-3, were transformed with a plasmid driving expression of his-tagged BASP1 or, as a control, GST

Control bacteria, or bacteria that express SUMO-3, were transformed with a plasmid driving expression of his-tagged BASP1 or, as a control, GST. expression during differentiation. == INTRODUCTION == The Wilms tumour suppressor protein WT1 was identified on the basis of its frequent mutation in Wilms tumours, a paediatric malignancy of the kidneys (1,2). WT1 plays a central role in nephrogenesis and WT1 null mice diein utero, displaying complete agenesis of the kidneys and also several other organs (3,4). WT1 is a nuclear protein containing FOS a zinc finger nucleic acid-binding domain that is alternatively spliced, leading to insertion of an additional three amino acids (KTS) between zinc fingers 3 and 4. This insertion produces a form of WT1 (+KTS) that has a high affinity for RNA, while the KTS form binds DNA. These and other observations have led to the proposal that the KTS form of WT1 acts as a transcriptional regulator, while the +KTS form plays a role in RNA processing. Mice that express exclusively either the +KTS or KTS forms of WT1 show that WT1 KTS plays a greater role in nephrogenesis and podocyte differentiation (5). The transcriptional regulatory properties of WT1 are highly context-specific and can manifest as both transcriptional activation and repression (6). Candidate target genes of WT1 include signalling-related factors such as amphiregulin (7,8), sprouty (9), platelet-derived growth factor-A (PDGFA; 10,11), colony-stimulating factor-1 (CSF-1; 12) and Wnt 4 (13,14), regulators of cell growth/survival such as Bcl2 (15), Bak (16,17), c-Myc (18,19) and p21 (20) RAF265 (CHIR-265) and also podocyte-specific proteins such as nephrin (21,22) and podocalyxin (23,24). Studies of these putative target genes in physiologically relevant cells are few and thus we have very little understanding of how WT1 regulates its target genes during RAF265 (CHIR-265) development. Several interaction partners have been described for WT1 (6). It is likely that they provide specificity to WT1 function and determine whether WT1 acts as a transcriptional activator or repressor. WT1 contains a suppression domain at its N-terminus that inhibits the function of the transcriptional activation domain (25). We previously identified Brain Acid Soluble Protein 1 (BASP1) as a WT1 transcriptional cosuppressor that mediates this inhibition (26). RAF265 (CHIR-265) Very little is known of BASP1 function or structure, but it exists as multiple forms from 30 to 150 kDa that are derived from post-translational modifications and proteolytic processing (26,27). BASP1 is present in the nephrogenic intermediates of the embryonic kidney RAF265 (CHIR-265) coincident with WT1 and, like WT1, BASP1 becomes restricted to the podocyte cells of the adult kidney. In the present study we analyse WT1 and BASP1 in a cell line model of podocyte differentiation. Our results demonstrate occupancy by WT1 and BASP1 of the promoters of the endogenous podocalyxin, Bak and c-myc genes. During podocyte differentiation BASP1 dissociates from the podocalyxin promoter by a mechanism involving BASP1 sumoylation and coincident with the transcriptional upregulation of podocalyxin. Conversely, WT1 and BASP1 remain bound to the Bak and c-myc promoters, both of which show differentiation-dependent repression. == MATERIALS AND METHODS == == Cell lines, immunofluorescence and transfection == MPC5, HEK 293 and K562 cells were cultured as described previously (26,28,29). Transfection was performed using Lipofectamine 2000 or calcium phosphate as previously (25,30). Luciferase assays were performed as described before (30). Immunofluorescence was performed as described previously (26). Antibodies against WT1 were the C19 antibody (Santa Cruz), PML was H238 (Santa Cruz), tubulin was DM1A (Sigma) and synaptopodin was P19 (Santa Cruz). The affinity-purified rabbit anti-BASP1 antibodies have been described before (26). Sheep anti-BASP1 antibodies were generated against full-length BASP1 by Diagnostics Scotland and affinity purified. == Plasmids and proteins == pcDNA3 BASP1 has been described before (26). BASP1 K78R:K83R RAF265 (CHIR-265) was generated using the Quickchange mutagenesis kit (Stratagene). pRSET BASP1, producing 6-Histidine-tagged BASP1.