Abstract
We have identified the regulatory sequences that govern the expression of the human transferrin gene in cultured brain cells and compared them with the data obtained with the neuronal cell line B103. Oligodendrocytes and epithelial choroid plexus cells from rat brain were cultured and used for transient expression experiments. Deletion analysis of 1.8 kilobase pairs of the 5' regulatory sequences revealed a -1530/-1140 positive-acting region in oligodendrocytes. The -164/+1 promoter region was sufficient to confer cell type-specific transcription in oligodendrocytes, epithelial choroid plexus cells, and B103 cells. DNase I footprinting experiments revealed three protected sequences, the proximal regions I and II, and the central region I. Gel retardation and antibody reactivity data allowed us to identify most of the nuclear factors present in oligodendrocytes interacting with the promoter sequences. Chicken ovalbumin upstream promoter transcription factor, a CAAT/enhancer-binding protein, and a cAMP response element-binding protein called CRI-BP interact with the proximal regions I and II and central region I sites, respectively. These data confirm the results obtained with the neuronal cell line and emphasize the importance of the three promoter elements for the transferrin gene-specific expression in the central nervous system compared with only two elements required for liver- and testis-specific expression.
Highlights
From the $Departments of Anatomy andCell Biology, and Psychiatry, Mental Retardation Research Center, University of California, Los Angeles, California 90024-1759,the White #Expression des Ghnes Eucaryotes, Znstitut Pasteur, 28, rue du Docteur R o w, 75724 Paris Cedex 15, and the **Znstitut National de la Recherche Agronomique, Station de Physiologie de la Reproduction, 37380 Nouzilly, France
We have identified the regulatory sequences that gsotuvd-ies concerning transferrin gene expressionin the mammaern the expression of the human transferrin gene ilniancburla-in
We have previously shown by immunocytochemistry in rat elements for the transferrin gene-specific expression inprimary glial cultures that transferrin is an eadrelvyelopmenthe central nervous system compared with only twelo- tal marker for oligodendrocytes (Espinosa de 10s Monteros et ements required for liver- taenstdis-specificexpression. al., 1988)
Summary
Brain-As described by several authors, transfemnis svnthesized hyoligodendroc-ytes (Rloch ct 01.. 1985: Espinosn de Ins dishescontaining Ilulbecco's minimumessentialmedium(LifeTrchnologies, Inc.), supplemented with5%fetal calf serum in a 37 "C water jackrted incubator in 5% CO,. A series of 5' deletion vectors (Fig. A ) was inal., 1974), theyalso have the ability to express the transferrin troduced into neurohlastoma R10.7 cells, oligodendrocytes, and gene, providingan alternative to primary culturoefsCNS neu- choroid plexus epithelial cellsby the calcium phosphate coprerons that can be generated only in limited amounts. Choroid plexus epithelial cells were able to secrete a positive-acting region between-1140 and -872 hp that moduabout eight times more transferrin than oligodendrocytes. In primary cultured epithelial cells of choroid plexus, the positive control pSV2-CAT vector was transcriptionally very active, indicating that thesecells were efficiently transfected. Thepromoter appears located between-164 bp and the cap site(Fig. 20) These results suggest that inall three cell types examined, the transferrin promoter elements reside within the -164/+1 region
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