A second transcriptionally active DNA-binding site for the Wilms tumor gene product, WT1.

Powell

The Journal of Immunology

et al. 2000

232

175

IL-10 is an 18-kDa cytokine with a key role in homeostatic control of inflammatory and immune responses. We have investigated how transcription of the IL-10 gene is regulated, so as to be able to understand the circumstances of IL-10 expression in both health and disease. In the mouse, IL-10 gene expression is regulated by a TATA-type promoter with a critical cis-acting element containing GGA repeats located at −89 to −77. Its complementary sequence is similar to the cis-acting elements (TCC repeats) in the promoters of genes encoding epidermal growth factor receptor and CD58. All these elements comprise a common CCTCCT sequence with less conserved C + T-rich sequences. Eliminating this CCTCCT sequence results in a marked reduction in promoter activity, suggesting a necessary role in IL-10 gene expression. Despite its dissimilarity to the G + C-rich Sp1 consensus sequence (GC box), Sp1 and Sp3 transcription factors could be shown to bind to this motif. The requirement for Sp1 and Sp3 in transcription of IL-10 was confirmed using Drosophila SL2 cells, which lack endogenous Sp factors. These results suggest that the transcription of IL-10 is positively regulated by both Sp1 and Sp3.

Section: Il-10 Gene Expression Is Regulated By the Transcription Factmentioning

confidence: 99%

IL-10 Gene Expression Is Controlled by the Transcription Factors Sp1 and Sp3

Powell

The Journal of Immunology

et al. 2000

232

175

“…The second alternative splice site inserts (+) or removes (7) 3 amino acids, (+) KTS, between zinc ®ngers III and IV and alters the DNA binding speci®city of the protein (Rauscher et al, 1990). The WT1 isoforms recognize the GC-rich motif, 5'-GCGGGGGCG-3', as well as a (TCC) n motif, albeit with di erent a nities, and can a ect expression of a number of genes involved in the regulation of cell proliferation or di erentiation (Reddy and Licht, 1996;Rauscher et al, 1990;Wang et al, 1993). These include insulin-like growth factor II, insulin-like growth factor 1 receptor (IGF-IR), platelet-derived growth factor Achain, colony stimulating factor-1, transforming growth factor-b1, retinoic acid receptor-a, Pax-2, c-myb, epidermal growth factor receptor, bcl-2, c-myc, syndecan-1, and the WT1 gene itself (Reddy and Licht, 1996).…”

Section: Introductionmentioning

confidence: 99%

The DNA binding domains of the WT1 tumor suppressor gene product and chimeric EWS/WT1 oncoprotein are functionally distinct

1998

The t(11; 22)(p13; q12) translocation associated with desmosplastic small round cell tumor results in a chimeric molecule fusing the amino terminal domain (NTD) of the EWS1 gene to three of the four carboxyterminal zinc ®ngers of the WT1 tumor suppressor gene. Since the DNA binding domains of WT1 and EWS/WT1 are structurally di erent, we have assessed the functional consequences of the EWS/WT1 fusion. We ®nd that the EWS/WT1 protein has a higher binding a nity for a given recognition target than the WT1 product. This is unlike other fusion products involving translocation of the NTD of EWS to DNA binding domains in which DNA binding speci®city and a nity is not changed. We demonstrate that EWS/WT1 is a nuclear protein and that the NTD of EWS contains (a) nuclear localization signal(s). We also ®nd that the integrity of a domain within the WT1 zinc ®ngers, responsible for mediating interaction between WT1 and the transcriptional repressor par-4, is disrupted in the EWS/WT1 fusion product. Deletion analysis of the NTD of EWS indicated that integrity of the entire domain was necessary to achieve full transactivation potential.

“…WT1 has several key structural features of a transcription factor such as nuclear localization, a proline/glutamine N-terminal rich region, and a DNA-binding domain consisting of four zinc ®ngers. Three of the four zinc ®ngers (II, III and IV) share extensive homology with those of the early growth response (EGR) family of transcription factors and consequently, WT1 can bind to, and regulate, the expression of numerous genes through the GC-rich egr-1 consensus binding site (5'GCGGGGGCG3'), as well as through a TC-rich site (5'TCC3') n (Rauscher et al, 1990;Wang et al, 1993). Genes harboring these elements are often involved in the regulation of cellular proliferation and di erentiation (Reddy and Licht, 1996).…”

Section: Introductionmentioning

confidence: 99%

Activation of the wt1 Wilms' tumor suppressor gene by NF-κB

1998

The Wilm's tumor suppressor gene, wt1, is expressed in a very de®ned spatial-temporal fashion and plays a key role in development of the urogenital system. Transacting factors governing wt1 expression are poorly de®ned. The presence of putative kB binding sites within the wt1 gene prompted us to investigate whether members of the NF-kB/Rel family of transcription factors are involved in regulating wt1 expression. In transient transfection assays, ectopic expression of p50 and p65 subunits of NF-kB stimulated wt1 promoter activity 10 ± 30-fold. Deletion mutagenesis revealed that NF-kB responsiveness is mediated by a short DNA fragment located within promoter proximal sequences of the major transcription start site. Two kB-binding sites are present in this region and form speci®c complexes with puri®ed NF-kB proteins, as revealed by electrophoretic mobility gel shift assays. Ectopic expression of p50 and p65 resulted in increased transcription of the endogenous wt1 gene, as revealed by nuclear run-on experiments. Taken together, these results indicate that members of the NF-kB/Rel family are important for activating expression of wt1 and reside upstream of the regulatory cascade leading to wt1 activation.