Overlapping Sp1 and AP2 binding sites in a promoter element of the lens-specific MIP gene

The MIP gene, the founder of the MIP family of channel proteins, is specifically expressed in fiber cells of the ocular lens and expression is regulated temporally and spatially during development. We previously found that a DNA fragment containing 253 bp of 5'-flanking sequence and 42 bp of exon 1 of the human MIP gene contains regulatory elements responsible for lens-specific expression of the MIP gene, In this report we have analyzed the function of overlapping Spl and AP2 binding sites present in the MIP promoter, Using DNase I footprinting analysis we found that purified Spl and AP2 transcription factors interact with several domains of the human MIP promoter sequence -253/+42, Furthermore, addition of purified Spl to Drosophila nuclear extracts activates in vitro transcription from the MIP promoter -253/+42, This promoter activity is competed by oligonucleotides containing domains foot-printed with Spl, Using promoter-reporter gene (CAT) constructs we found that the sequence -39/-70 contains a cis regulatory element essential for promoter activity in transient assays in lens cells, EMSA analysis showed that lens nuclear extracts contain factors that bind to the MIP 5'-flanking sequence containing overlapping Spl and AP2 binding domains at positions -37/-65. Supershift experiments with lens nuclear extracts indicated that Sp3 is also able to interact with this regulatory element, suggesting that Spl and Sp3 may be involved in regulation of transcription of the MIP gene in the lens.