Abstract 637 Gastroenterology and Nutrition Platform, Monday, 5/3

Background: The orphan homeobox gene, Hex, has a limited domain of expression in the developing mouse which includes the liver and biliary tree, and which persists in adult rodents in the liver ( Ped. Res. 251A, 1998). We have shown that Hex can trans-activate a liver-specific gene, ntcp, and that nuclear levels of Hex binding to the ntcp promoter are suppressed in the acute phase ( Hepatology 28:505A,1998). The aims of this study were to clone and characterize the factors regulating the mouse Hex promoter. Methods: A ≈7kb nucleotide region of mouse genomic DNA was subcloned from a single lambda phage clone obtained by screening a mouse genomic DNA library. Analysis of automated sequencing of the promoter region was performed by computer generated searches. The transcription start site was determined by primer extension. Cell line-specific Hex RNA and protein expression were determined by Northern and Western blots, respectively. 5′-truncated promoter fragments were generated by PCR and restriction digestion, cloned into a luciferase reporter vector, and transiently transfected into liver-derived HepG2 and fibroblast (COS) cells. Results: Hex RNA and protein expression in HepG2 cells were confirmed by Northern and Western blots, respectively. Comparison of a series of 5′ deletion Hex promoter constructs (from ≈-7kb, ≈-3.5kb, -583bp, -233bp, and -103bp all ending at nt +22) in Hep G2 cells indicated that the minimal region required for basal activity resided between -233/+22. Sequence analysis revealed an extensive GC-rich region with multiple Sp1 binding elements. Compared to vector (pGL3Basic) expression, these inserts increased activity by 2x, 3x, 15x, 15x, and 5x respectively, suggesting the presence of negative-acting elements between nt -583 and ≈-3.5kb. Interestingly, the -233/+22 and -103/+22 constructs were active in both the sense and reverse orientations. Although the -583/+22 construct was also active in the Hex null COS cell line, Hex protein could not be detected by Western blot analysis, suggestive of either a cell line-specific upstream transcriptional silencer or post-transcriptional regulation of Hex. Conclusions: Basal activity of the Hex promoter in HepG2 cells is derived largely from the -233/-103 region, which can function in a bidirectional manner and contains putative Sp1 response elements. Additional activity is derived from the -103/+22 region, which contains a putative HNF3 response element. An upstream silencer region has also been identified. The Hex minimal promoter construct was also active in COS cells, yet there was no detectable Hex protein in these cells. Taken together, these data demonstrate that Hex exhibits both transcriptional and post-transcriptional mechanisms of cell line-specific regulation. Further studies are ongoing to characterize the transcription factors which regulate Hex promoter activity, and the presumed cofactors which contribute to activation of target genes by Hex.