REGULATORY SEQUENCES FOR THE TRANSCRIPTION OF THE LAMININ B2 GENE IN ASTROCYTES

Astrocytes synthesize only the B2 chain of laminin and that this chain is sufficient to stimulate neurite outgrowth. In this study, we have examined laminin B1 and B2 promoter constructs in various cell types i n order to understand the transcriptional regulation of laminin B2 gen e in astrocytes. Comparison of nuclear factor binding by Southwestern analysis with the highly active B2 promoter fragment revealed differen t patterns of nuclear factor binding. In HepG2 cells, two proteins of 105 and 98 kDa were identified while, in primary astrocytes, human U25 1 and rat C6 glioma cells, a greater number of nuclear proteins rangin g from 43 to 212 kDa were detected. The laminin B1 promoter construct was inactive in transient transfection experiments in astrocytes yet a ctive in the HepG2 hepatoma cells which synthesize both the B1 and B2 chains. In contrast, the laminin B2 promoter construct was active in b oth astrocytes and HepG2 cells. These results are consistent with the lack of laminin B1 mRNA expression in astrocytes and suggest that the differential regulation of the laminin B1 and B2 gene is controlled at the transcriptional level. Delineation of the 5'-flanking regions res ponsible for basal levels of B2 laminin promoter activity revealed a s ilencer-like segment between -830 and -224 which reduced promoter acti vity. Deletion analysis further revealed that B2 laminin promoter poss esses a highly active short promoter (-94 to +106) and basal transcrip tional activity resides within -61 to +106. DNase 1 footprinting, gel- shift competition assays and site-directed mutagenesis of a highly act ive short promoter revealed that this region contained binding sites f or cell-type nuclear factors. The shortest construct containing only r esidues -21 to +106 was inactive in HepG2 and U251 glioma cells. In pr imary astrocytes, however, this construct showed a high level of trans criptional activity. Deletion of 47 bp (+59 to +106) in 5'-UTR complet ely blocked promoter activity in astrocytes confirming that this downs tream region is important for transcriptional activity in primary astr ocytes. Together, these results suggest that astrocytes may utilize mu tually exclusive transcription factors and regulatory sequences, in ad dition to common factors in the control of the laminin B2 promoter.