Apolipoprotein CIII (apoCIII), a lipid-binding protein involved in the transport of triglycerides and cholesterol in the plasma, is synthesized primarily in the liver and the intestine. A cis-acting regulatory element, C3P, located at -90 to -66 upstream from the apoCIII gene transcriptional start site (+1), is necessary for maximal expression of the apoCIII gene in human hepatoma (HepG2) and intestinal carcinoma (Caco2) cells.This report shows that three members of the steroid receptor superfamily of transcription factors, hepatocyte nuclear factor 4 (HNF-4), apolipoprotein Al regulatory protein 1 (ARP-1), and Ear3/COUP-TF, act at the C3P site. HNF-4 activates apoCIII gene expression in HepG2 and Caco2 cells, while ARP-1 and Ear3/COUP-TF repress its expression in the same cells. HNF-4 activation is abolished by increasing amounts of ARP-1 or Ear3/COUP-TF, and repression by ARP-1 or Ear3/COUP-TF is alleviated by increasing amounts of HNF-4. HNF-4 and ARP-1 bind with similar affinities to the C3P site, suggesting that their opposing transcriptional effects may be mediated by direct competition for DNA binding. HNF-4 and ARP-1 mRNAs are present within the same cells in the liver and intestine, and protein extracts from hepatic tissue, HepG2, and Caco2 cells contain significantly more HNF-4 than ARP-1 or Ear3/COUP-TF binding activities. These findings suggest that the transcription of the apoCIII gene in vivo is dependent, at least in part, upon the intracellular balance of these positive and negative regulatory factors.Apolipoprotein CIII (apoCIII) is a major protein constituent of the triglyceride-rich lipoproteins, very low density lipoproteins, and chylomicrons, and it appears to play an important role in their metabolism by inhibiting the hydrolysis of triglycerides by lipoprotein lipase (4,12,56) and inhibiting the removal of chylomicrons and triglyceride-rich lipoproteins by hepatocytes (44,53,59). ApoCIII plasma levels are often elevated in hypertriglyceridemic individuals (5, 45). Furthermore, overexpression of apoCIII in transgenic mice results in profound hypertriglyceridemia (21). However, the mechanism whereby apoCIII influences triglyceride metabolism has not been clearly defined.

Rats and mice have two, equally expressed, nonallelic genes encoding preproinsulin (genes I and II). Cytological hybridization with metaphase chromosomes indicated that both genes reside on rat chromosome 1 but are approximately 100,000 kilobases apart. In mice the two genes reside on two different chromosomes.DNA sequence comparisons of the gene-flanking regions in rats and nmice indicated that the preproinsulin gene I has lost one of the two introns present in gene II, is flanked by a long (41-base) direct repeat, and has a remnant of a polydeoxyadenylate acid tract preceding the downstream direct repeat. These structural features indicated that gene I was generated by an RNA-mediated duplication-transposition event involving a transcript of gene II which was initiated upstream from the normal capping site. Sequence divergence analysis indicated that the pair of the original gene and its retroposed, but functional, counterpart (which appeared about 35 million years ago) is maintained by strong negative selection operating primarily on the segments encoding the chains of the mature hormone, whereas the segments encoding the parts of the polypeptide that are eliminated during processing and also the introns and the flanking regions are evolving neutrally.Rats (as well as mice and three fish species) have two insulins instead of one, in contrast to other organisms (13,23). These rat hormones are the products of two nonallelic preproinsulin genes (57) that are almost equally expressed (11). Characterization of the duplicated genes (36), isolated from a rat chromosomal DNA library, indicated a significant structural difference between them. The gene for preproinsulin I has a single 119-base-pair (bp) intron interrupting the segment corresponding to the 5' noncoding region of the mRNA, whereas the other gene, encoding preproinsulin II, contains in addition to this small intron a second 499-bp intron interrupting the segment encoding the C-peptide. Since the structures of the unique chicken (46) and human (2, 62) genes are similar to that of the rat gene II (two introns at corresponding positions) we concluded that the two-intron organization corresponds to that of the common ancestor and that introns can be lost during evolution (46). This conclusion was strengthened by the subsequent determination of the structures of the unique dog (30) and guinea pig (7) preproinsulin genes, which are of the twointron type.Intron loss was subsequently documented in a mouse ot-globin pseudogene (45, 69) and later in other pseudogenes that exhibit clearly the features of processed genes (22; see references 34, 56, and 68 for reviews). These observations raised the possibility that the rat preproinsulin I gene was a * Corresponding author. retroposon (51) that had been generated by an RNAmediated duplication-transposition event but which for some reason remained functional. To examine this possibility we mapped the chromosomal location of the two genes and characterized their flanking regions by DNA sequencing to define the break po...

The gene coding for apolipoprotein Al, a plasma protein involved in the transport of cholesterol and other lipids in the plasma, is expressed predominantly in liver and intestine. Previous work in our laboratory has shown that hepatocyte-specific expression is determined by synergistic interactions between transcription factors bound to three separate sites, sites A (-214 to -192), B (-169 to -146), and C (-134 to -119), within a powerful liver-specific enhancer located in the region -222 to -110 nucleotides upstream of the apolipoprotein AI gene transcription start site (+ 1). In this study, it was found that site A is a highly selective retinoic acid-responsive element (RARE) that responds preferentially to the recently identified retinoic acid receptor RXRa over the previously characterized retinoic acid receptors RARa and RARI. Control experiments indicated that a RARE in the regulatory region of the laminin Bl gene responds preferentially to RARaL and RAR, over RXRa, while a previously described palindromic thyroid hormone-responsive element responds similarly to ail three of these receptors. Gel retardation experiments showed that the activity of these RAREs is concordant with receptor binding. These results indicate that different RAREs may play a fundamental role in defining distinctive retinoic acid cellular response pathways and suggest that retinoic acid response pathways mediated by RXRa play an important role in cholesterol and retinoid transport and metabolism.Apolipoprotein Al (apoAI) is a major protein constituent of plasma high-density lipoproteins and intestinally derived lipoproteins known as chylomicrons. High-density lipoproteins are involved in a large number of diverse intravascular metabolic processes including the process of reverse cholesterol transport, in which cholesterol from extrahepatic tissues is transported to the liver for conversion to bile acids and eventual excretion (for a recent review, see reference 16). This process is thought to play an important role in protection against premature coronary heart disease (16). Chylomicrons, on the other hand, transport dietary lipids including retinol in the form of retinyl esters to the liver for storage and/or secretion as lipoprotein complexes (reviewed in references 2 and 11).Although several recent studies suggest that dietary, hormonal, and other environmental factors regulate apoAl gene expression, the molecular basis of the mechanisms involved remains poorly understood (16). Based on transient transfection assays, it was recently concluded that in cultured human hepatoma (HepG2) cells, nearly all the transcriptional activity of the apoAl gene is determined by a powerful liver-specific enhancer located in the region -222 to -110 nucleotides upstream of the apoAI gene transcription start site (+1) (36). It was also observed that maximal transcriptional activity of this enhancer was dependent on synergistic interactions between transcription factors bound to three * Corresponding author. t Present address: Cardiology Division, Jew...

The gene coding for apolipoprotein Al (apoAl), a plasma protein involved in the transport of cholesterol and other lipids in the plasma, is expressed predominantly in liver and intestine. Previous work in our laboratory has shown that different cis-acting elements in the 5'-flanking region of the human apoAl gene control its expression in human hepatoma (HepG2) and colon carcinoma (Caco-2) cells. Hepatocyte-specific expression is mediated by elements within the -256 to -41 DNA region relative to the apoAl gene transcription start site (+1). In this study it was found that the -222 to -110 apoAI gene region is necessary and sufficient for expression in HepG2 cells. It was also found that this DNA region functions as a powerful hepatocyte-specific transcriptional enhancer. Gel retardation and DNase I protection experiments showed that HepG2 cells contain proteins that bind to specific sites, sites A (-214 to -192), B (-169 to -146), and C (-134 to -119), within this enhancer. Site-directed mutagenesis that prevents binding of these proteins to individual or different combinations of these sites followed by functional analysis of these mutants in HepG2 cells revealed that protein binding to any one of these sites in the absence of binding to the others was not sufficient for expression. Binding to any two of these sites in any combination was sufficient for only low levels of expression. Binding to all three sites was essential for maximal expression. These results indicate that the transcriptional activity of the apoAl gene in liver cells is dependent on synergistic interactions between transcription factors bound to its enhancer.The accumulation and utilization of cholesterol by tissues are dependent on a dynamic balance between the mechanisms that determine the rates of de novo cholesterol synthesis, the rates of synthesis and hydrolysis of stored pools of cholesteryl esters, and the rates of uptake and removal of cholesterol from cells by plasma lipoproteins (reviewed in references 4 and 20). Removed cholesterol binds to a species of high-density lipoprotein (HDL) particles containing primarily apolipoprotein AI (apoAI). After its esterification by lecithin:cholesterol acyltransferase (an enzyme activated by apoAI), cholesterol is transported to the liver, where it is excreted either directly or in the form of bile acids (reviewed in references 3, 20, and 24). The critical role of HDL and apoAI in cholesterol homeostasis, and in particular in preventing deposition of excessive amounts of cholesterol in coronary and other arteries, is exemplified by epidemiological and genetic evidence indicating a strong correlation between decreased HDL and apoAI plasma levels and the development of atherosclerotic heart disease (reviewed in references 3, 39, and 51). Thus, the recent observation that there is a direct correlation between apoAI plasma levels and hepatic apoAI mRNA concentrations (54, 55) suggests that factors controlling expression of the apoAI gene in liver could play an important role in tissue cholesterol a...

The estrogen receptor (ER) belongs to a superfamily of ligand-inducible transcription factors. Functions of these proteins (dimerization, DNA binding, and interaction with other transcription factors) are modulated by binding of their corresponding ligands. It is, however, controversial whether various ER ligands affect the receptor's ability to bind its specific DNA element (ERE).By using real time interaction analysis we have investigated the kinetics of human (h)ER binding to DNA in the absence and presence of 17␤-estradiol, 17␣-ethynyl estradiol, analogs of tamoxifen, raloxifene, and ICI-182,780. We show that ligand binding dramatically influences the kinetics of hER interaction with specific DNA. We have found that binding of estradiol induces the rapid formation of a relatively unstable ER⅐ERE complex, and binding of ICI-182,780 leads to slow formation (k a is approximately 10 times lower) of a stable receptor-DNA complex (k d is almost 2 orders of magnitude lower). Therefore, binding of estradiol accelerates the frequency of receptor-DNA complex formation more than 50-fold, compared with unliganded ER, and more than 1000-fold compared with ER liganded with ICI-182,780. We hypothesize that a correlation exists between the rate of gene transcription and the frequency of receptor-DNA complex formation. We further show that a good correlation exists between the kinetics of hER-ERE interaction induced by a ligand and its biological effect.Steroid hormones are widely distributed small, lipophilic molecules that participate in intracellular communication and control a wide spectrum of developmental and physiological processes. Their effects are mediated by specific intracellular receptors, a family of proteins that are characterized by a high affinity for the corresponding hormones and an ability to discriminate between structurally closely related ligands. These ligand-inducible receptors can modulate transcription of target genes by virtue of their binding to a specific sequence on DNA in target promoters known as hormone response elements. Although distinct proteins, these receptors are members of a large superfamily of steroid hormone receptors and share many common structural and functional features (1-4).Binding of 17␤-estradiol (E 2 ) 1 to estrogen receptor (ER) is followed by a conformational change, leading to dissociation of the receptor from the complex with the heat shock proteins hsp90 and p59 (5, 6), dimerization (7,8), and activation of DNA binding. After DNA binding the activated receptor can interact with basal transcription factors (9). These interactions are thought to stabilize the preinitiation complex at the promoter, allowing RNA polymerase to initiate transcription (10). Recently a number of transcriptional intermediary factors have been identified that can modify estrogen responsiveness, and several of these proteins interact with the ligand binding domain of the ER in a ligand-dependent manner (11-13). It is obvious that the ligand plays a key role in initiating this cascade of events. ...