Sp1 Is a Critical Regulator of the Wilms' tumor-1 Gene

Abstract

We performed deletion analysis of WT1-reporter constructs containing up to 24 kilobases of 5-flanking and first intron WT1 sequence in stably transfected cultured cells as an unbiased approach to identify cis elements critical for WT1 transcription. Although not a tissuespecific element, a proximate 9-base pair CTC repeat accounted for ϳ80% of WT1 transcription in this assay. Enhancer activity of the element and mutated versions correlated completely with their ability to form a DNAprotein complex in gel shifts. Antibody supershift, oligonucleotide competition, and Southwestern studies indicated that the CTC-binding factor is the transcriptional activator Sp1. Sp1 binds the CTC repeat with an affinity, K D ‫؍‬ 0.37 nM, at least as high as the consensus GC box. Similar CTC repeats are found in promoters of other growth-related genes. Because Sp1 is important for WT1 expression, we examined Sp1 immunohistochemistry in fetal and adult kidney. In a pattern that precedes that of WT1 message, Sp1 immunostaining was highest in uninduced mesenchyme, early tubules, developing podocytes, and mature glomeruli, but was minimal in mature proximal tubules. This work suggests abundant Sp1 may be a prerequisite for WT1 expression, and that Sp1 may have a wider role in nephrogenesis.

Mutations in the promoter reveal a cause for the reduced expression of the human manganese superoxide dismutase gene in cancer cells

“…SP-1 binds to GC box promoter element and selectively activates mRNA synthesis by RNA polymerase II Tjian, 1983, 1985). A number of closely regulated genes have been reported to possess promoters with a high frequency of G and C residues suggesting that SP-1 plays a key role in transcription of these genes (Baker et al, 1996;Mo at et al, 1996;Cohen et al, 1997;Dharmavarm et al, 1997). However, SP-1 binding a nity and transcription properties can be altered by interaction with other co-factors.…”

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

“…Two developmentally regulated paired-box family members, Pax-2 and Pax-8, have been shown to activate wt1 expression through a 38 bp conserved motif present upstream of the ®rst transcription start site . In addition, multiple sites for Sp1 are present throughout the wt1 promoter, and this factor has been shown to transactivate wt1 expression (Ho man et al, 1993;Cohen et al, 1997). The wt1 promoter is also responsive to its own gene product, and this is thought to down-regulate the wt1 response in vivo (Rupprecht et al, 1994).…”

“…Such cooperativity has recently been described by Perkins et al (1993), who demonstrated that activation of the HIV-1 LTR depends on an interaction between p65 and Sp-1. Transactivation of the wt1 promoter is also positively regulated by Sp-1 (Cohen et al, 1997;Ho man et al, 1993). Whether transactivation of wt1 by NF-kB complexes is Sp-1 dependent remains to be determined.…”

“…The upstream regulatory regions of the human and mouse wt1 genes are GC-rich and do not contain a CAAT or a TATA box (Pelletier et al, 1991c, Ho mann et al, 1993. Several ubiquitous and tissue-speci®c transcription factors, such as Sp1, WT1, GATA-1, Pax-2 and Pax-8, have been shown to modulate the expression of the wt1 promoter in transient transfection assays (Rupprecht et al, 1994;Wu et al, 1995;Cohen et al, 1997).…”

Upregulation of Wilms' tumor gene 1 (WT1) in desmoid tumors

“…Transcription factor Sp1 fulfills an important role in controlling WT1 gene expression. 23 Additional transcription factors involved in activation of the WT1 gene are nuclear factor-B, PAX2, and PAX8. 24 -26 Furthermore, WT1 is capable of autoregulating its own expression in a negative manner.…”