To determine the cis-and trans-regulatory elements which control the expression of the apolipoprotein (apo) A-I gene, several DNA-protein binding assays, namely, gel mobility shift, exonuclease 111 protection, and exonuclease 111 footprinting assays, were employed to identify these elements. It is demonstrated that nuclear proteins of Hep G2 cells bind to five regions of DNA sequences between 252 and 149 base pairs upstream from the transcription initiation site of the rat apo A-I gene. Using South-Western blot analysis, it is determined that DNA-binding protein has a molecular mass of approximately 90 kDa. It is also shown that the DNA-binding protein was present in Hep G2 cells and rat livers but absent in rabbit livers. The results suggest that the lack of expression of the apo A-I gene in rabbit livers is due to the absence of this DNA-binding protein.Apolipoprotein (apo) A-I is an important plasma protein.It is a major constituent of plasma high-density lipoproteins and a cofactor of plasma lecithin -cholesterol acyltransferase [l] which catalyzes the formation of most of the cholesteryl esters in plasma. It is thought that apo A-I, together with lecithin -cholesterol acyltransferase, facilitates the removal of cholesterol from peripheral tissues and transports it to the liver for catabolism [2]. Patients with a defective apo A-I gene show low levels of plasma high-density lipoprotein and apo A-I and accelerated atherosclerosis [3]. Several epidemiological studies have established that plasma levels of high-density lipoprotein and apo A-I are negatively correlated with the incidence of coronary heart diseases [4 -61. Hence it is beneficial to raise plasma levels of both high-density lipoproteinThe expression of apo A-I can be induced at transcriptional levels by several pharmacological agents: phenobarbital, estrogen, insulin and dexamethasone [7 -91. Usually, the expression of eukaryotic genes is regulated by the 5'-flanking promoter and enhancer elements of the genes. These elements interact with and bind to specific trans-acting nuclear proteins [lo -121. Thus, the identification of the trans-acting elements which bind to and interact with the enhancer elements of the apo A-S gene becomes crucial for designing therapeutic agents which intervene in the expression of this gene. The nucleotide sequences of the rat and human apo A-I genes have been reported [13, 141. Recently, we have identified a 45-base-pair nucleotide element, located between 235 and 190 nucleotides upstream from the transcription initiation site of the rat apo A-I gene, which is essential for the expression of this gene in Hep G2 cells and that this element exerts its function with an enhancerlike activity [l 51. In the current research we studied the binding of nuclear proteins from Hep G2 cells and rat livers to the enhancer element of the rat apo A-I gene; our data suggest and apo A-S. that the lack of expression of the apo A-I gene in rabbit livers is due to decreased levels of this binding protein in rabbit livers. MATERIALS AND ...