Identification of the 5′ regulatory elements of avian lipoprotein lipase gene: Synergistic effect of multiple factors

Abstract

The organization of cis-acting regulatory elements of the chicken lipoprotein lipase gene was investigated in 5.4 kb of 5′ flanking sequences. Various lengths of 5′ flanking sequence were linked to the bacterial chloramphenicol acyltransferase (CAT) gene and transfected into primary cultures of chicken adipocytes by DEAE-dextran transfection method. Negative elements are present between −1947 and −139 of the 5′ flanking sequence. Removal of these sequences revealed the presence of positive elements located within 138 bp upstream of the major transcription start site. Sequence analysis showed that the region from the major transcription start site to −138 contains an inverted GC box (ACCACGCCCC), a CCAAT element and two direct repeats of the octamer motif, ATTTGCAT. DNase I footprinting assays using a probe extending from −175 to +191, identified three sites protected by nuclear factors. Site I (−126 to −123), a C-rich sequence, GCCC, was identified only on the coding strand. Site II covered the sequence from −95 to −68 and includes the GC box. Site III, from −53 to −26, contained two octamer repeats. Site I is the 5′ portion of a 10 bp sequence (CCCTCCCCCC; −126/−116) which is perfectly conserved in the avian and the human promoter. Single or multiple copies of a 37 bp DNA fragment (−138/−102) containing the 10 bp conserved sequence were cloned into LPLCAT-51, upstream or downstream of the major transcription start site and in both orientations; transfection and CAT activity assays with these constructs indicate that the −138/−102 fragment has an enhancer like activity. Additional 5′ and internal deletions of LPLCAT-138 suggest that the factors binding to the C-rich element, the GC box and the two octamer repeats have a synergistic effect on promoter activity.