A FAR UPSTREAM CIS-ELEMENT IS REQUIRED FOR WILMS TUMOR-1 (WT1) GENE-EXPRESSION IN RENAL-CELL CULTURE

To identify novel cis-regulatory elements responsible for the tissue-r estricted expression pattern of the Wihms' tumor-1 (WT1) gene, we mapp ed a total of 11 DNase I-hypersensitive sites in the 5'-flanking regio n and first intron of the human gene, six of which were specific for W T1 expressing cell lines. A 1.4-kilobase (kb) fragment from the mouse wt1 5'-flanking region contained cross-hybridizing sequence with signi ficant homology to a region of DNase I hypersensitivity in the human W T1 gene which bound to nuclear matrix in human fetal kidney 293 cells. None of the DNase I-hypersensitive sites/matrix attachment regions, e ither alone or in combination, were sufficient for tissue-specific WT1 expression in transient and stably transfected cell lines. However, s table transfection of an approximately 620-kb yeast artificial chromos ome (YAC) that carried the entire mouse wt1 locus into 293 cells resul ted in wt1 (trans)gene expression at a level of approximately 30% of t he endogenous human gene. Deletion of the 1.4-kb cross-hybridizing mou se fragment, located approximately 15 kb upstream of the transcription start site, caused complete loss of wt1 gene expression in the YAC-tr ansfected 293 cells. In summary, we have identified a far upstream ele ment that contains a region of DNase I hypersensitivity and that binds to nuclear matrix. This element includes phylogenetically conserved s equence and is required, although not sufficient, for mouse wt1 gene e xpression in human fetal kidney cells in culture.