Extinction of alpha 1-antitrypsin gene expression in somatic cell hybrids: evidence for multiple controls

Expression of the liver-specific alpha 1-antitrypsin (alpha 1AT) gene is extinguished in hepatoma/fibroblast hybrids. To define the mechanism of extinction, we identified DNA sequences involved in this process by transiently transfecting mutant alpha 1AT promoters into parental and hybrid cells. The wild-type alpha 1AT promoter (-554 to +44 bp) was highly expressed in rat hepatoma cells, but activity was 100-fold less in fibroblasts or cell hybrids. Mutations in this region failed to activate alpha 1AT expression in nonhepatic cells, but mutations in the binding site for liver factor B1 (LF-B1) reduced hepatic-specific expression greater than 100-fold. Furthermore, the hybrid cells failed to express LF-B1-binding activity and mRNA. This suggested that alpha 1AT extinction in hybrids might be an indirect, lack-of-activation phenotype mediated primarily through repression of LF-B1. To test this possibility, we stably transfected an LF-B1 expression cassette into parental and hybrid cells and monitored expression of transfected and endogenous alpha 1AT genes. Surprisingly, although constitutive LF-B1 expression could activate alpha 1AT-CAT transgenes in these cells, it neither prevented nor reversed extinction of the chromosomal alpha 1AT genes. We conclude that although extinction of the LF-B1 trans-activator accompanies alpha 1AT extinction in cell hybrids, it does not play a causal role in this process.