Extinction of a^-antitrypsin gene expression in somatic cell hybrids: evidence for multiple controls

Expression of the liver-specific a,-antitrypsin (a,AT) 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 a^AT promoters into parental and hybrid cells. The wild-type WjAT 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 a^AT expression in nonhepatic cells, but mutations in the binding site for liver factor Bl (LF-Bl) reduced hepatic-specific expression > 100-fold. Furthermore, the hybrid cells failed to express LF-Bl-binding activity and mRNA. This suggested that a^AT extinction in hybrids might be an indirect, lack-of-activation phenotype mediated primarily through repression of LF-Bl. To test this possibility, we stably transfected an LF-Bl expression cassette into parental and hybrid cells and monitored expression of transfected and endogenous a^AT genes. Surprisingly, although constitutive LF-Bl expression could activate ajAT-CAT transgenes in these cells, it neither prevented nor reversed extinction of the chromosomal a,AT genes. We conclude that although extinction of the LF-Bl trans-activator accompanies a^AT extinction in cell hybrids, it does not play a causal role in this process.