Structural And Functional Characterization Of Human Hepatic Stimulator Substance (hHSS) Gene Promoter

Human hepatic stimulator substance (hHSS) is a newly identified growth - promoting factor in liver. HSS is capable to stimulate the hepatic regeneration in hepaectomized rats and promotes the growth of hepatic tumor cells. To understand and elucidate the regulation of hHSS on hepatocyte growth at genetic level, the 4890bp of 5' - flanking region of hHSS gene have been isolated and sequenced. Based on comparison of the 5' - flanking sequences with GenBank, hHSS gene was localized on human chromosome 16. Transcriptional start site was identified by reverse transcriptase - polymerase chain reaction ( RT -PCR) and 5' rapid amplification cDNA end (5'RACE). The initiation nucleotide was defined as "C", which located 254bp upstream of the reported sequence of exon I. No TATA box or GAATT are found in this region but a GC rich segment (>70% ) of more than 400bp immediately upstream of exon I . Deletion mapping was applied to i-dentify the promoter region and cis - acting elements 5' of hHSS gene. Eight serial deletions produced by PCR were inserted upstream of the reporter gene (LacZ) to form recombinant plasmids, which were then transiently transfected into HepG2 and 313 cells. The results revealed that the regions -577 to -441 in HepG2 cell line, -876 to -577 and -1865 to -1075 in 3T3 cell line, and -2872 to-1865 in both cell lines possessed positive regulatory function for hHSS expression; i. e. , increased gene expression. There were putative cis - acting ele-ments, including activator protein - 1 (AP1), activator protein - 2 (AP2) , activator protein-4 (AP4) , hepatocyte nuclear factor - 3 (HNF3) and nuclear factor - 1 ( NF1) which have an important role in activating growth in liver, directly or indirectly. Two negative elements were found at nucleotide position - 441 to - 246 in both cell types and -1075 to - 876 in 3T3 cell line. Deletion of them resulted in an increase of β-galactosidase activity. The results of electropho-resis mobility shift assay (EMSA) confirmed the promoter activity of 5'-flanking region of hHSS gene. In addition, it was found that in these regions, there were many cis - acting elements related to the transcriptional regulation including SP1 binding sites responsible for the transcriptional promotion and some unknown regulatory suppression elements. The promoter of hHSS might be located in the region of -2872 to +104 of hHSS gene.