GENETIC-ANALYSIS OF A TRANSCRIPTIONAL ACTIVATION PATHWAY BY USING HEPATOMA-CELL VARIANTS

A hierarchy of liver-enriched transcription factors plays an important role in activating expression of many hepatic genes. In particular, h epatocyte nuclear factor 4 (HNF-4) is a major activator of the gene en coding HNF-1, and HNF-1 itself activates expression of more than 20 li ver genes. To dissect this activation pathway genetically, we prepared somatic cell variants that were deficient in expression of the liver- specific alpha 1-antitrypsin (alpha 1AT) gene, which requires both HNF -1 and HNF-4 for high-level gene activity. This was accomplished in tw o steps. First, hepatoma transfectants that stably expressed two selec table markers under alpha 1AT promoter control were prepared; second, variant sublines that could no longer express either transgene were is olated by direct selection. In this report, we demonstrate that the va riants contain defects in the HNF-4/HNF-1 activation pathway. These de fects functioned in trans, as expression of many liver genes was affec ted, but the variant phenotypes were recessive to wild type in somatic cell hybrids. Three different variant classes could be discriminated by their phenotypic responses to ectopic expression of either HNF-4 or HNF-1. Two variant clones appeared specifically deficient in HNF-4 ex pression, as transfection with an HNF-4 expression cassette fully rest ored their hepatic phenotypes. Another line activated HNF-1 in respons e to forced HNF-4 expression, but activation of downstream genes faile d to occur. One clone was unresponsive to either HNF-1 or HNF-4. Using the variants, we demonstrate further that the chromosomal genes encod ing alpha 1AT, aldolase B, and alpha-fibrinogen display strict require ments for HNF-1 activation in vivo, while other liver genes were unaff ected by the presence or absence of HNF-1 or HNF-4. We also provide ev idence for the existence of an autoregulatory loop in which HNF-1 regu lates its own expression through activation of HNF-4.