IDENTIFICATION OF A CIS-ACTING NEGATIVE DNA ELEMENT WHICH MODULATES HUMAN HEPATIC TRIGLYCERIDE LIPASE GENE-EXPRESSION

The promoter fragment -1550/+129 of the human hepatic triglyceride lip ase (HTGL) gene drives the expression of the CAT gene in HepG2 cells, albeit at very low levels. Transient transfections in HepG2 and HeLa c ells of 5' deletion constructs indicated that the regulatory elements that control this expression are located in the proximal region of the gene. DNase I footprint analysis with DNA fragments spanning the regi on -483 to +129 and rat liver nuclear extracts identified eight protec ted regions, four upstream of the transcription initiation site (A, -2 8 to -75; B, -96 to -106; C, -1 18 to -158; D, -185 to -255) and four in the first exon of the gene (E1, -5 to +20; E2, +36 to +55; E3, +58 to +83; E4, +86 to +107). DNA binding and foot printing analysis demon strated that the region-75 to -43 within footprint A binds to the live r-specific transcription factor HNF1. The region +28 to +129 contains a functional negative regulatory element (NRE) since deletion of this region results in a 17-fold increase in CAT activity. The NRE can act independent of orientation and position and repress transcription driv en by heterologous promoters. DNA binding assays using native and frac tionated liver nuclear extracts identified two transcription factors t hat bind to element E2 and also to element E3. A dinucleotide mutation in element E2 which causes derepression of the HTGL gene by 10-fold a lso abolishes the binding of these two activities. Transfection experi ments showed that deletion of the NRE allows expression of reporter co nstructs in HeLa cells, indicating that the NRE may play a determinant role for the expression of HTGL gene in hepatic cells.