S Kim1, H Ge, C Ohtaka-Maruyama, A B Chepelinsky. 1. Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Abstract
PURPOSE: To characterize the cis regulatory elements and their interaction with transcription factors responsible for the lens specific expression of the MIP gene, which encodes the Major Intrinsic Protein of the lens fiber membranes. METHODS: Study interaction of factors present in newborn mouse lens nuclear extracts with DNA fragments corresponding to mouse MIP gene 5' flanking sequence by electrophoresis mobility shift assay (EMSA) and DNase I footprinting. RESULTS: We found a high degree of identity in the first 100 bp of 5' flanking sequence of mice and humans, however, a lower degree of conservation is observed further upstream. We have found by DNase I footprinting analysis that lens specific factors may interact with the first 100 bp of 5' flanking sequence. A domain containing an E box, conserved in mouse and human, may interact with a lens specific factor. However, general factors may interact with a NF-1 binding site. An overlapping GC and CT box is present in the mouse MIP gene. In the human MIP gene GC and CT boxes are found in different domains of the MIP gene promoter. Both CT boxes interact with factors present in lens nuclear extracts including Sp3. They are able to interact with purified Sp1but not with Sp1 present in mouse lens nuclear extracts. CONCLUSIONS: The transcription factor Sp3 may play an important role in regulating MIP gene expression in the lens.
PURPOSE: To characterize the cis regulatory elements and their interaction with transcription factors responsible for the lens specific expression of the MIP gene, which encodes the Major Intrinsic Protein of the lens fiber membranes. METHODS: Study interaction of factors present in newborn mouse lens nuclear extracts with DNA fragments corresponding to mouseMIP gene 5' flanking sequence by electrophoresis mobility shift assay (EMSA) and DNase I footprinting. RESULTS: We found a high degree of identity in the first 100 bp of 5' flanking sequence of mice and humans, however, a lower degree of conservation is observed further upstream. We have found by DNase I footprinting analysis that lens specific factors may interact with the first 100 bp of 5' flanking sequence. A domain containing an E box, conserved in mouse and human, may interact with a lens specific factor. However, general factors may interact with a NF-1 binding site. An overlapping GC and CT box is present in the mouseMIP gene. In the humanMIP gene GC and CT boxes are found in different domains of the MIP gene promoter. Both CT boxes interact with factors present in lens nuclear extracts including Sp3. They are able to interact with purified Sp1but not with Sp1 present in mouse lens nuclear extracts. CONCLUSIONS: The transcription factor Sp3 may play an important role in regulating MIP gene expression in the lens.