Sp1 is a critical regulator of the Wilms' tumor-1 gene.
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 tissue-specific element, a proximate 9-base pair CTC repeat accounted for approximately 80% of WT1 transcription in this assay. Enhancer activity of the element and mutated versions correlated completely with their ability to form a DNA-protein 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, KD = 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.
Cohen
H T
HT
Renal Division, Beth Israel Hospital and Harvard Medical School, Boston, Massachusetts 02215, USA.
Bossone
S A
SA
Zhu
G
G
McDonald
G A
GA
Sukhatme
V P
VP
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D004331:Drosophila melanogaster; D004742:Enhancer Elements, Genetic; D005333:Fetus; D018507:Gene Expression Regulation, Developmental; D016162:Genes, Wilms Tumor; D015698:Genomic Library; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D007668:Kidney / Q000196:embryology / Q000254:growth & development / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012689:Sequence Homology, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000096:biosynthesis*; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D018911:Trinucleotide Repeats; D025721:WT1 Proteins; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D025721:WT1 Proteins; D015500:Chloramphenicol O-Acetyltransferase