TRANSCRIPTION OF THE HUMAN HEPATIC LIPASE GENE IS MODULATED BY MULTIPLE NEGATIVE ELEMENTS IN HEPG2 CELLS

The expression of the hepatic lipase (HL) gene is highly tissue specif ic. In order to identify cis-acting elements which regulate the expres sion of this gene in the liver, multiple deletion mutants of the 5'-fl anking region of the HL gene fused to the human growth hormone gene we re transfected in HepG2 cells, which normally produce HL. Transient ex pression assays indicated the presence of negative (at nucleotides (nt ) -1576/-1342 and -623/-407) and positive (at nt -1862/-1576 and -50/- 9) regulatory elements. Transfection of HeLa cells, which do not produ ce HL, with the same deletion constructs resulted in a similar pattern of promoter activities. However, additional negative (nt -138/-50) an d positive (nt -407/-138) elements were found. DNase I footprint analy sis of the proximal and distal HL promoter sequences with HepG2 and He La cell nuclear extracts identified seven protected regions: A, nt -15 40/-1527; B, -1505/-1473; C, -1467/-1460; D, -592/-577; E, -565/-545; F, -234/-220; and G, -70/-48. Sites A, B, C, D and E were located with in regions containing negative regulatory elements. In order to determ ine which nuclear factor interacts with the negative elements, sites B , D and E were mutated and the effects of mutation on competition in a gel retardation assay and on promoter activity were studied. When the binding motif for AP1 in sites B, D and E was mutated, the specific D NA-protein complexes were not competed with the mutant oligonucleotide s and promoter activity increased twofold. The magnitude of the increa se is less than expected from the deletion analysis, and simultaneous mutations did not cause further increase in promoter activity, which s uggests that other sites are involved in this negative modulation. The se results suggest that the transcription of the HL gene in HepG2 cell s is negatively modulated by multiple cis-acting negative elements and AP1-like nuclear factor may play some role in this modulation.