Sp1 Specifically Binds to an Evolutionarily Conserved DNA Segment within a Region Necessary for Podocyte-Specific Expression of Nephrin

Abstract

We have analyzed a conserved 237-bp segment located in a 1.9-kb upstream region of the nephrin gene, previously shown to contain kidney specific enhancer element(s). Electromobility shift assay was used to identify a 20-nucleotide region specifically recognized and bound by protein factors in nuclear extracts from immortalized podocyte and human embryonic kidney cell lines. The region was further narrowed down by competition assays to a stretch of 6 consecutive guanines, which are conserved at this location in multiple species. Introduction of mutations in this sequence abolished all protein binding activity whereas mutations in the flanking nucleotides did not. By means of gel supershift and chromatin immunoprecipitation assays we have shown that the protein factor from podocyte nuclear extracts able to recognize and bind the target sequence is the Sp1 zinc-finger protein.