Identification and functional analysis of the mouse lens filensin gene promoter.
Filensin (also called CP94; CP95; CP97; 115kDa protein) is a component of the lens-specific beaded filament which is believed to be functionally important in lens fiber cell differentiation and in maintaining lens fiber cell conformation and transparency. A 17.2kb fragment containing the 5'-upstream sequence of the filensin gene was isolated. S1-mapping analysis determined the transcription start point (tsp; +1) which locates at 94base pairs upstream from the initiating ATG on the filensin gene. In addition to a major tsp, a minor tsp (-136) was observed. DNA sequence of the fragment around the tsp (-2144 to +155) was identified. Analysis of the DNA sequence of the promoter region around tsp revealed two motifs with sequence homology to Sox2 and Maf recognition sequences in addition to one GATA-1 site, two Sp1 binding sites, and three AP-2 binding motifs. No TATA-box or CCAAT-motif was found around the tsp region. A series of sequentially deleted fragments of (-2144 to +40) were fused to firefly luciferase reporter plasmid pGL2 and tested for activity in chicken embryonic lens explants. A minimal promoter region for mouse filensin of (-70 to +40) was identified. The lens-specific promoter activity was detected using lens explants cultured within 12h after dissection. The activity was remarkably enhanced by culture in the presence of 5ng/ml of basic fibroblast growth factor. Each one of the Sp1 and AP-2 binding motifs was localized to the fragment of (-27 to +40) using electrophoretic mobility shift assays. These are the first data to identify the basic elements to the 5'-upstream sequences of the filensin gene, namely the tsp and the minimal filensin promoter.
Masaki
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S
Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi 480-0392, Japan.
Kamachi
Y
Y
Quinlan
R A
RA
Yonezawa
S
S
Kondoh
H
H
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002642:Chick Embryo; D004247:DNA / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D017930:Genes, Reporter; D007381:Intermediate Filament Proteins / Q000235:genetics*; D007908:Lens, Crystalline / Q000166:cytology / Q000378:metabolism; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000187:drug effects; D015183:Restriction Mapping; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000378:metabolism; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism
D017931:DNA Primers; D004268:DNA-Binding Proteins; D005136:Eye Proteins; D007381:Intermediate Filament Proteins; D016329:Sp1 Transcription Factor; D050656:Transcription Factor AP-2; D014157:Transcription Factors; C079098:filensin; D016222:Fibroblast Growth Factor 2; D004247:DNA; D008156:Luciferases