Identification of a silencing element in the chicken lipoprotein lipase gene promoter: characterization of the silencer-binding protein and delineation of its target nucleotide sequence

Abstract

Lipoprotein lipase (LPL) hydrolyzes triglycerides in chylomicrons and very low density lipoproteins (VLDL) and plays a central role in lipid metabolism. It is regulated tissue-specifically. By deletion analysis, a negative regulatory element was identified in the chicken LPL gene promoter at base pairs (bp) −263 to −241. This sequence contained two palindromic halves with a three nucleotide spacer. Either half was sufficient for full inhibitory function. A protein complex bound specifically to this element and a high correlation was found between binding of the complex and inhibition of transcription. Its molecular mass, evaluated by native gel electrophoresis and Ferguson plot analysis, was 120 kDa. UV cross-linking followed by SDS–PAGE revealed two protein subunits of 48 kDa and 44 kDa, respectively. This inhibitory protein complex may contribute to the tissue-specific regulation of LPL gene transcription. It was much more abundant in liver than in adipose tissue and heart. Our data showed that this negative element inhibited transcription even when placed at an upstream location (−666), but failed to function in the herpes simplex virus thymidine kinase promoter, indicating that it acted in conjunction with other element(s) in the chicken LPL gene to inhibit transcription.