Functional activity of hepatocyte nuclear factor-1 is specifically decreased in amino acid-limited hepatoma cells

Limitation of cultured rat hepatoma cells for an essential amino acid resul ts in a specific decrease in expression of several genes that are preferent ially expressed in the liver, including the serum albumin and transthyretin genes. In the work presented here, we examined whether the coordinate repr ession of these genes is caused by decreased activity of one or more of the liver-enriched transcription factors, hepatocyte nuclear factor-1 (HNF-1), HNF-3, HNF-4 or C/EBP. To address this question, HepG2 human hepatoma cell s were transiently transfected with luciferase reporter constructs containi ng multiple copies of individual transcription factor binding sites. Limita tion for an essential amino acid resulted in specific repression of a const ruct in which luciferase expression was directed by HNF-1. A single HNF-1 b inding site located adjacent to the TATA box plays a major role in transcri ption directed by the serum albumin promoter in transient transfection assa ys. Amino acid limitation of cells transfected with an albumin promoter/luc iferase reporter construct resulted in specific repression of promoter acti vity. In addition, bacterial methylation or site-directed mutagenesis of th e HNF-1 binding site in the albumin proximal promoter region eliminated the regulation of an albumin promoter-luciferase reporter construct under cond itions of amino acid limitation. These results demonstrated that the HNF-1 binding site played a major role in regulation of the albumin promoter by a mino acid availability. Deletion analysis of the albumin promoter confirmed regulation through the HNF-1 binding site and also identified a second ami no acid regulatory element in the upstream region of the albumin promoter, which has been shown previously to contain a functional binding site for HN F-3. The repression of albumin promoter and HNF-1 reporter constructs in am ino acid-limited cells occurred without a change in the DNA binding activit y of HNF-1. Moreover, HNF-3 DNA binding activity was also not decreased in amino acid-limited cells. These results suggest that the regulation of tran scription by amino acids occurs at the level of transcriptional activation by HNF-1 and HNF-3, rather than by alteration of the DNA binding activity o f either factor. (C) 1999 Elsevier Science B.V. All rights reserved.