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.
Subject(s)
Cell Nucleus/metabolism , Conserved Sequence/physiology , DNA/physiology , Evolution, Molecular , Membrane Proteins/metabolism , Podocytes/physiology , Sp1 Transcription Factor/metabolism , Animals , Cell Line , Humans , Mice , Protein BindingABSTRACT
We have generated transgenic mice harboring the murine matrix metalloproteinase 9 (MMP-9) promoter cloned in front of human TIMP-1 cDNA. The transgenic mice were viable and fertile and exhibited normal growth and general development. During wound healing the mice were shown to express human TIMP-1 in keratinocytes that normally express MMP-9. However, the healing of skin wounds was significantly retarded with slow migration of keratinocytes over the wound in transgenic mice. In situ zymography carried out on wound tissues revealed total blockage of gelatinolytic activity (i.e., MMP-9 and MMP-2). The results confirm studies with MMP-9 knockout mice showing that MMP-9 is not essential for general development, but they also demonstrate an important role of keratinocyte MMP-9, as well that of other keratinocyte MMPs that are inhibited by TIMP-1, in wound healing. The transgenic mice generated in this study provide a model for the role of MMPs in MMP-9-producing cells in other challenging situations such as bone fracture recovery and cancer invasion.
Subject(s)
Gene Expression Regulation , Matrix Metalloproteinase 9/genetics , Promoter Regions, Genetic , Tissue Inhibitor of Metalloproteinase-1/genetics , Wound Healing/genetics , Animals , Genes, Reporter , Humans , Immunohistochemistry , In Situ Hybridization , Keratinocytes/metabolism , Mice , Mice, Transgenic , Tissue Inhibitor of Metalloproteinase-1/immunology , beta-Galactosidase/geneticsABSTRACT
Nephrin, an essential component of the glomerular ultrafilter, the slit diaphragm, has also been found to be expressed in the central nervous system and pancreas. This study examined the regulation of the nephrin gene by analyzing the expression of different length nephrin promoter-lacZ reporter constructs in transgenic mice. An upstream segment between -4 kb and -4 bp was shown to be sufficient for driving expression in all three tissues. Surprisingly, a 5.7-kb construct lacking the transcription initiation site and the immediate upstream region of the gene could drive expression in the central nervous system. This led to the identification of a novel, alternatively used exon 1B located 1871 bp upstream of the ATG codon of the previously known first exon, now termed exon 1A. The existence and functionality of exon 1B was verified in nephrin knockout mice in which exon 1A is deleted. Deletion of exon 1B and its immediate surrounding sequence, introduced in the 4-kb promoter-lacZ reporter construct, abolished the expression of the transgene in pancreas and spinal cord but not in kidney and brain in transgenic mice. Analysis of five promoter-reporter gene constructs showed that regulatory elements driving expression encoded by exon 1A in kidney and brain are localized in the region between -4 kb and 2.1 kb.
