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 gene coding for apolipoprotein AI (apoAl), a lipid binding protein involved in the transport of cholesterol and other lipids in the plasma, is expressed in mammals predominantly in the liver and the intestine.Liver-specific expression is controlled 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 between nucleotides -222 and -110 upstream of the apoAl gene transcription start site (+1). Previous studies in our laboratory have shown that ARP-1, a member of the nuclear receptor superfamily whose ligand is unknown (orphan receptor), binds to site A and represses transcription of the apoAI gene in liver cells. In a more recent series of experiments, we found that site A is a retinoic acid (RA) response element that responds preferentially to the recently identified RA-responsive receptor RXRea over the previously characterized RA receptors RARae and RARI3. In this study we investigated the combined effects of ARP-1 and RXRa on apoAl gene expression in liver cells. Transient transfection assays showed that site A is necessary and sufficient for RXRa-mediated transactivation of the apoAl gene basal promoter in human hepatoma HepG2 cells in the presence of RA and that this transactivation is abolished by increasing amounts of cotransfected ARP-1. Electrophoretic mobility shift assays and subsequent Scatchard analysis of the data revealed that ARP-1 and RXRoa bind to site A with similar affinities. These assays also revealed that ARP-1 and RXRai bind to site A as heterodimers with an affinity approximately 10 times greater than that of either ARP-1 or RXRa alone. Further transfection assays in HepG2 cells, using as a reporter a construct containing the apoAl gene basal promoter and its upstream regulatory elements (including site A) in their natural context, revealed that RXRai has very little effect on the levels of expression regardless of the presence or absence of RA. However, while ARP-1 alone or ARP-1 and RXRa together dramatically repress expression in the absence of RA, the repression by ARP-1 and RXRai together, but not ARP-1 alone, is almost completely alleviated in the presence of RA. These results indicate that transcriptional repression by ARP-1 sensitizes apoAl gene responsiveness to RXRa and RA and suggest that the magnitude of this responsiveness is regulated by the intracellular ratio of ARP-1 to RXRa. These observations raise the possibility that transcriptional repression is a general mechanism for switching gene transcription between alternative transcription activation pathways.A large body of epidemiologic, genetic, pharmacologic, and biochemical evidence suggests that high-density lipoprotein (HDL) levels in plasma play an important role in regulation of cellular cholesterol homeostasis and atherosclerosis progression and regression. Although changes in plasma HDL levels have been associated with a diverse number of dietary, hormonal, and str...

Many laboratory strains ofBaciUus subtilis contain 9 rather than 10 rRNA operons due to deletions occurring within the rrnj-rrnW or rrnl-rrnH-rrnG gene cluster. These operons are members of two sets of closely spaced clusters located in the cysA-arol region. Analysis of rescued DNA from integrants with insertions into rrnG and rrnH indicated that these tandemly arranged operons allowed frequent deletions of an rrn operon equivalent. These events may arise spontaneously by intrachromosomal recombination or by simultaneous double crossovers with a multimeric integrative plasmid.The endospore-forming bacterium Bacillus subtilis has 10 rRNA gene sets, of which 9 are clustered between the origin and 75 degrees on the genomic map (1, 8) ( Fig. 1). In a separate study, we demonstrated by integrative mapping, Southern analysis, and transductional crosses two wellseparated sets of closely spaced rrn clusters in the region between cysA14 and aroI906 (Fig. 1). The first two, rrnJ and rrnW, are assigned to BclI homologs of 4.9 and 5.8 kilobases (kb), respectively, are located between cysAJ4 and rpsE2. The second set, rrnl, rrnH and rrnG, of which the latter two are assigned to BclI homologs of 5.5 and 4.8 kb, respectively, are located between attSPO2 and glpT6 (E. D. Jarvis, R. L. Widom, G. LaFauci, Y. Setoguchi, I. R. Richter, and R. Rudner, manuscript in preparation) (Fig. 1). Southern analysis showed that in certain common laboratory strains of B. subtilis, the number of BclI rrn homologs is 9 instead of 10, revealing the loss of either the 4.8-or the 5.8-kb fragments assigned to rrnG and rrnW, respectively. Our earlier studies with strains of the first group (6) and similar analyses in another laboratory (10) revealed the loss of a ribosomal homolog in other restriction enzyme digests of genomic DNA. During transformation with integrable plasmids containing rDNA, several strains were created which resembled the naturally occurring deletion strains with respect to their BclI and EcoRI Southern patterns.We report that rescue of DNA from the chromosomal region of rrn deletions indicates that the naturally occurring deletions arose by homologous recombination within the closely spaced operons rrnJ-rrnW and rrnI-rrnH-rrnG. Restriction analyses of rescued DNA from the region of the deletion provide additional evidence for the existence of rrnI, the third operon in the second cluster, whose location was predicted by the heteroduplex mapping of Chow and Davidson (2) but so far has had no plasmid insertions. MATERIALS AND METHODSBacterial strains and plasmids. Some of the B. subtilis strains and plasmids (pGR102 and pWR103) used in this study were described previously (8). Integrable plasmid pGR110, which contains a 0.5-kb PstI-SmaI 16S fragment,