Altered expression of C/EBP family members results in decreased adipogenesis with aging.

Fat mass, adipocyte size and metabolic responsiveness, and preadipocyte differentiation decrease between middle and old age. We show that expression of CCAAT/enhancer binding protein (C/EBP)-alpha, a key regulator of adipogenesis and fat cell function, declined substantially with aging in differentiating preadipocytes cultured under identical conditions from rats of various ages. Overexpression of C/EBP alpha in preadipocytes cultured from old rats restored capacity to differentiate into fat cells, indicating that downstream differentiation-dependent genes maintain responsiveness to regulators of adipogenesis. C/EBP alpha-expression also decreased with age in fat tissue from three different depots and in isolated fat cells. The overall level of C/EBP beta, which modulates C/EBP alpha-expression, did not change with age, but the truncated, dominant-negative C/EBP beta-liver inhibitory protein (LIP) isoform increased in cultured preadipocytes and isolated fat cells. Overexpression of C/EBP beta-LIP in preadipocytes from young rats impaired adipogenesis. C/EBP delta, which acts with full-length C/EBP beta to enhance adipogenesis, decreased with age. Thus processes intrinsic to adipose cells involving changes in C/EBP family members contribute to impaired adipogenesis and altered fat tissue function with aging. These effects are potentially reversible.

The activation function-1 of hepatocyte nuclear factor-4 is an acidic activator that mediates interactions through bulky hydrophobic residues.

The hepatocyte nuclear factor-4 (HNF-4) contains two transcription activation domains. One domain, activation function-1 (AF-1), consists of the extreme N-terminal 24 amino acids and functions as a constitutive autonomous activator of transcription. This short transactivator belongs to the class of acidic activators, and it is predicted to adopt an amphipathic alpha-helical structure. Transcriptional analysis of sequential point mutations of the negatively charged residues (Asp and Glu) revealed a stepwise decrease in activity, while mutation of all acidic residues resulted in complete loss of transcriptional activity. Mutations of aromatic and hydrophobic amino acids surrounding the negatively charged residues had a much more profound effect than mutations of acidic amino acids, since even a single mutation of these residues resulted in a dramatic decrease in transactivation, thus demonstrating the importance of hydrophobic residues in AF-1 activity. Like other acidic activators, the AF-1 of HNF-4 binds the transcription factor IIB and the TATA-binding protein directly in vitro. In addition, the cAMP-response-element-binding-protein, a transcriptional adapter involved in the transactivation of a plethora of transcription factors, interacts with the AF-1 of HNF-4 and co-operates in the process of transactivation by HNF-4. The different protein targets of AF-1 suggest that the AF-1 of HNF-4 may be involved in recruiting both general transcription factors and chromatin remodelling proteins during activation of gene expression.

The N-terminal 17% of apoB binds tightly and irreversibly to emulsions modeling nascent very low density lipoproteins.

The N-terminal 17% of apolipoprotein B (apoB-17) readily associates with dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles (MLV) to form large (240-A diameter) discoidal particles. Because apoB is normally secreted with triacylglycerol (TAG)-rich lipoproteins, we studied the binding of apoB-17 to triolein-rich emulsions modeling nascent TAG-rich very low density-like lipoproteins. Emulsions with the following composition (by weight) were prepared: 85--89% triolein, 1.1--1.4% cholesterol, and 10--14% phosphatidylcholines (PC) including either egg yolk (EY)-, dimyristoyl (DM)-, or dipalmitoyl (DP)-PC representing (at 25 degrees C), respectively, a fluid surface, a surface at transition, and a mainly solid surface. The respective sizes were 1,260 +/- 500, 1,070 +/- 450, and 830 +/- 300 A mean diameter. The emulsions were incubated with conditioned medium containing apoB-17, and then reisolated by ultracentrifugation. Analysis of the emulsion-bound proteins by gel electrophoresis showed that all three emulsions bound primarily apoB-17. The DPPC emulsions bound more apoB-17 than EYPC or DMPC emulsions. Immunoaffinity-purified apoB-17 exhibited saturable, high affinity binding to EYPC and DPPC emulsions. The respective K(d) values were 32 +/- 23 and 85 +/- 27 nM and capacities (N) were 10 and 58 molecules of apoB-17 per particle. When apoB-17 bound to emulsions was incubated with DMPC MLV at 26 degrees C for 18 h, it remained bound to the emulsions, indicating that once bound to these emulsions it is unable to exchange off and solubilize DMPC into discs. In contrast, apoE-3 bound to emulsions dissociated from the emulsions when incubated with DMPC MLV and formed discs.Thus, apoB-17 binds strongly and irreversibly to emulsions modeling nascent lipoproteins. It therefore may play an important role in the stabilization of nascent VLDL and chylomicrons.- Herscovitz, H., A. Derksen, M. T. Walsh, C. J. McKnight, D. L. Gantz, M. Hadzopoulou-Cladaras, V. Zannis, C. Curry, and D. M. Small. The N-terminal 17% of apoB binds tightly and irreversibly to emulsions modeling nascent very low density lipoproteins. J. Lipid Res. 2001. 42: 51;-59.

The orphan nuclear receptor SHP inhibits hepatocyte nuclear factor 4 and retinoid X receptor transactivation: two mechanisms for repression.

The orphan nuclear hormone receptor SHP interacts with a number of other nuclear hormone receptors and inhibits their transcriptional activity. Several mechanisms have been suggested to account for this inhibition. Here we show that SHP inhibits transactivation by the orphan receptor hepatocyte nuclear factor 4 (HNF-4) and the retinoid X receptor (RXR) by at least two mechanisms. SHP interacts with the same HNF-4 surface recognized by transcriptional coactivators and competes with them for binding in vivo. The minimal SHP sequences previously found to be required for interaction with other receptors are sufficient for interaction with HNF-4, although deletion results indicate that additional C-terminal sequences are necessary for full binding and coactivator competition. These additional sequences include those associated with direct transcriptional repressor activity of SHP. SHP also competes with coactivators for binding to ligand-activated RXR, and based on the ligand-dependent interaction with other nuclear receptors, it is likely that coactivator competition is a general feature of SHP-mediated repression. The minimal receptor interaction domain of SHP is sufficient for full interaction with RXR, as previously described. This domain is also sufficient for full coactivator competition. Functionally, however, full inhibition of RXR transactivation requires the presence of the C-terminal repressor domain, with only weak inhibition associated with this receptor interaction domain. Overall, these results suggest that SHP represses nuclear hormone receptor-mediated transactivation via two separate steps: first by competition with coactivators and then by direct effects of its transcriptional repressor function.

Cysteine-rich regions of pig gastric mucin contain von willebrand factor and cystine knot domains at the carboxyl terminal(1).

In order to sequence the cysteine-rich regions of pig gastric mucin (PGM), we used our previously identified pig gastric mucin clone PGM-2A to screen a pig stomach cDNA library and perform rapid amplification of cDNA ends to obtain two cysteine-rich clones, PGM-2X and PGM-Z13. PGM-2X has 1071 base pairs (bp) encoding 357 amino acids containing five serine-threonine-rich 16 amino acid tandem repeats, downstream from a cysteine-rich region similar to human and mouse MUC5AC. PGM-Z13 encodes the complete 3'-terminus of PGM and is composed of 3336 bp with a 2964 bp open reading frame encoding 988 amino acids with four serine-threonine-rich tandem repeats upstream from a cysteine-rich region similar to the carboxyl terminal regions of human and rat MUC5AC and human MUC5B. This region is homologous to von Willebrand factor C and D domains involved in acid induced polymerization, and to the carboxyl terminal cystine-knot domain of various mucins, TGF-beta, vWF and norrin, which is involved in dimerization. These newly sequenced cysteine-rich regions of pig gastric mucin may be critical for its gelation and for its observed increased viscosity induced by low pH.

CREB-binding protein is a transcriptional coactivator for hepatocyte nuclear factor-4 and enhances apolipoprotein gene expression.

Hepatocyte nuclear factor-4 (HNF-4) is a liver-enriched transcription factor that is crucial in the regulation of a large number of genes involved in glucose, cholesterol, and fatty acid metabolism and in determining the hepatic phenotype. We have previously shown that HNF-4 contains transcription activation functions at the N terminus (AF-1) and the C terminus (AF-2) which work synergistically to confer full HNF-4 activity. Here, we show that HNF-4 recruits the CREB-binding protein (CBP) coactivator on promoters of genes that contain functional HNF-4 sites. HNF-4 interacts with the N-terminal region of CBP (amino acids 1-771) and the C-terminal region of CBP (amino acids 1812-2441). The two activating functions of HNF-4, AF-1 and AF-2, interact with the N terminus and the N and C terminus of CBP, respectively. In addition, we show that in contrast to the other nuclear hormone receptors the interaction between HNF-4 and CBP is ligand-independent. Recruitment of CBP by HNF-4 results in an enhancement of the transcriptional activity of the latter. CBP does not activate gene expression in the absence of HNF-4, and dominant negative forms of HNF-4 prevent transcriptional activation by CBP, suggesting that the mere recruitment of CBP by HNF-4 is not sufficient for enhancement of gene expression. These findings demonstrate that CBP acts as a transcriptional coactivator for HNF-4 and provide new insights into the regulatory function of HNF-4.

Binding specificity and modulation of the human ApoCIII promoter activity by heterodimers of ligand-dependent nuclear receptors.

Human apolipoprotein CIII (apoCIII) is a major determinant of plasma triglyceride metabolism. The regulatory elements that control both hepatic and intestinal transcription of the human apoCIII gene are localized between nucleotides -792 and -25 of the apoCIII promoter. Elements important for apoCIII promoter activity are three hormone response elements (HREs) and three SP1-binding sites. Orphan members of the nuclear hormone receptor superfamily can bind the HREs and strongly enhance or repress apoCIII promoter activity. In the present study we have investigated the ability of ligand-dependent nuclear hormone receptors to bind and modulate the human apoCIII promoter activity. Experiments using DNA binding and competition assays showed that the proximal element B (-87/-72) binds strongly, in addition to HNF-4, ARP-1, EAR-2, and EAR-3, heterodimers of RXRalpha with RARalpha, and less efficiently, homodimers of RARalpha and heterodimers of RXRalpha with T3Rbeta or PPARalpha. Element G (-669/-648), which was shown previously to bind ARP-1 and EAR-3 but not HNF-4, binds strongly heterodimers of RXRalpha with either RARalpha or T3Rbeta. Finally element I4 (-732/-712), which was shown to bind HNF-4, also binds strongly ARP-1 and EAR-3, as well as RXRalpha/RARalpha heterodimers and less efficiently, RXRalpha/T3Rbeta heterodimers. Methylation interference experiments have identified the protein-DNA interactions between different nuclear receptors and the respective HREs on the apoCIII promoter. RXRalpha/RARalpha heterodimers and HNF-4 homodimers bind to DR-1 motifs on elements B and I4, respectively. RXRalpha/T3Rbeta heterodimers and ARP-1 bind to DR-5 and DR-0 motifs respectively on element G. Cotransfection experiments in HepG2 cells showed that RXRalpha or a combination of RXRalpha and RARalpha increased the apoCIII promoter activity approximately 2-fold in the presence of the ligands 9-cis or all-trans RA. In contrast, a combination of RXRalpha and T3Rbeta transactivated the apoCIII promoter 1.5-fold in the presence of 9-cis RA but it repressed the apoCIII promoter activity in the presence of T3. Mutations in the HREs of elements B, G, or I4 or in the SP1-binding site of element H, which abolished the binding of nuclear hormone receptors or SP1 to their cognate site, reduced the promoter strength and exhibited different responses to the ligand-dependent nuclear receptors. The findings suggest that modulation of the apoCIII promoter activity by orphan and ligand-dependent nuclear receptors involves complex interactions among nuclear receptors, SP1 and possibly other factors bound to the enhancer and the proximal promoter region.

Transactivation of the ApoCIII promoter by ATF-2 and repression by members of the Jun family.

It was shown previously that cytokines such as tumor necrosis factor-alpha that stimulate signal transduction pathways involving transcription factors ATF-2 and Jun repress apoCIII promoter activity in HepG2 cells. In the present study, DNase I footprinting analysis established that ATF-2 protected three regions in the apoCIII promoter. One region (-747/-726) present in the apoCIII enhancer is within the previously identified footprint I and has overlapping boundaries with the binding sites of Sp1 (-764/-742) and HNF-4 (-736/-714). The other two regions represent new footprints and have been designated D/E (-219/-199) and B/C (-102/-75). The B/C region overlaps with the previously identified footprint B which contains an HNF-4 binding site (-87/-63). Cotransfection experiments in HepG2 cells showed that ATF-2 transactivated the -890/+24 apoCIII promoter 1.6-fold. In addition, mutations in the proximal D/E (-219/-199) and distal I (-747/-726) ATF-2-binding sites reduced the apoCIII promoter strength to 33 and 9% of control, respectively, indicating that ATF-2 is a positive regulator of apoCIII gene transcription. Cotransfections with ATF-2 and HNF-4 expression plasmids resulted in additive transactivation of the apoCIII promoter. Furthermore, apoCIII promoter constructs bearing mutations in the D/E and I ATF-2 binding sites were efficiently transactivated by HNF-4, suggesting that these two factors contribute independently to the apoCIII promoter strength. Members of the Jun family (c-Jun, JunB, and JunD) caused a dose-dependent inhibition of the -890/+24 apoCIII promoter activity. A synthetic promoter containing the apoCIII enhancer in front of the minimal AdML promoter was also repressed by Jun. In contrast, apoCIII promoter segments lacking the enhancer region were transactivated by Jun. The findings suggest that homodimers of Jun or heterodimers of Jun with other AP-1 subunits could be responsible for the observed repression by interfering with the function(s) of the apoCIII enhancer. Repression by Jun could be reversed in the presence of ATF-2 and HNF-4, suggesting that ATF2 and possibly Jun/ATF-2 heterodimers exert a positive effect on apoCIII gene transcription, as opposed to Jun homodimers or heterodimers with other AP-1 members. These findings suggest a role for members of the Jun family and ATF-2 that participate in signal transduction pathways in basal or induced apoCIII promoter activity in cells of hepatic origin.

Functional domains of the nuclear receptor hepatocyte nuclear factor 4.

The hepatocyte nuclear factor 4 (HNF-4) is a member of the nuclear receptor superfamily and participates in the regulation of several genes involved in diverse metabolic pathways and developmental processes. To date, the functional domains of this nuclear receptor have not been identified, and it is not known whether its transcriptional activity is regulated by a ligand or other signals. In this report, we show that HNF-4 contains two transactivation domains, designated AF-1 and AF-2, which activate transcription in a cell type-independent manner. AF-1 consists of the extreme N-terminal 24 amino acids and functions as a constitutive autonomous activator of transcription. This short transactivator belongs to the class of acidic activators, and it is predicted to adopt an amphipathic alpha-helical structure. In contrast, the AF-2 transactivator is complex, spanning the 128-366 region of HNF-4, and it cannot be further dissected without impairing activity. The 360-366 region of HNF-4 contains a motif that is highly conserved among transcriptionally active nuclear receptors, and it is essential for AF-2 activity, but it is not necessary for dimerization and DNA binding of HNF-4. Thus, HNF-4 deletion mutants lacking the 361-465 region bind efficiently to DNA as homo- and heterodimers and behave as dominant negative mutants. Remarkably, the full transactivation potential of AF-2 is inhibited by the region spanning residues 371-465 (region F). The inhibitory effect of region F on the HNF-4 AF-2 activity is a unique feature among members of the nuclear receptor superfamily, and we propose that it defines a distinct regulatory mechanism of transcriptional activation by HNF-4.

Distal apolipoprotein C-III regulatory elements F to J act as a general modular enhancer for proximal promoters that contain hormone response elements. Synergism between hepatic nuclear factor-4 molecules bound to the proximal promoter and distal enhancer sites.

Transient transfection assays have shown that the distal apoC-III promoter segments that contain the regulatory elements F to J enhance the strength of the tandemly linked proximal apoA-I promoter 5- to 13-fold in hepatic (HepG2) cells. Activation in intestinal (CaCo-2) cells to levels comparable to those obtained in HepG2 cells requires a larger apoA-I promoter sequence that extends to nucleotide -1500 as well as the presence of hepatic nuclear factor-4 (HNF-4). The distal apoC-III regulatory elements can also enhance 4- to 8-fold the strength of the heterologous apoB promoter in HepG2 and CaCo-2 cells. Finally, these elements in the presence of HNF-4 enhance 14.5- to 18.5-fold the strength of the minimal adenovirus major late promoter linked to two copies of the hormone response element (HRE) AID of apoA-I in both HepG2 and CaCo-2 cells. In vitro mutagenesis of the promoter/enhancer cluster established that the enhancer activity is lost by a mutation in the HRE present in the 3' end of the regulatory element I (-736 to -714) and is reduced significantly by point mutations or deletions in one or more of the regulatory elements F to J of the apoC-III enhancer. The enhancer activity also requires the HREs of the proximal apoA-I promoter. The apoC-III enhancer can also restore the activity of the proximal apoA-I and apoB promoters that have been inactivated by mutations in CCAAT/enhancers binding protein binding sites, indicating that C/EBP may not participate in the synergistic activation of the promoter/enhancer cluster. The findings suggest that the regulatory elements F to J of the apoC-III promoter act as a general modular enhancer that can potentiate the strength of proximal promoters that contain HREs. Such potentiation in the HepG2 cells can be accounted for by synergistic interactions between HNF-4 or other nuclear hormone receptors bound to the proximal and distal HREs and SP1 or other factors bound to the apoC-III enhancer. Additional factors may be required for optimal activity in CaCo-2 cells as well as for the function of this region as an intestinal enhancer.

Isolation and characterization of cDNA clones encoding pig gastric mucin.

Polyclonal antibodies raised to deglycosylated pig gastric mucin were used to screen a cDNA library constructed with pig stomach mucosal mRNA. Immunocytochemistry indicated that the antibody recognizes intracellular and secreted mucin in surface mucous cells of pig gastric epithelium. A total of 70 clones producing proteins immunoreactive to this antibody were identified, two of which (PGM-2A,9B) were fully sequenced from both ends. Clone PGM-9B hybridized to a polydisperse mRNA (3-9 kb) from pig stomach, but not liver, intestine or spleen, nor to mRNA from human, mouse, rabbit or rat stomach. Sequence analysis indicated that PGM-9B encodes 33 tandem repeats of a 16-amino-acid consensus sequence rich in serine (46%) and threonine (17%). Using the restriction enzyme MwoI, which has a single target site in the repeat, it was demonstrated that PGM-9B consists entirely of this tandem repeat. Southern-blot analysis indicated that the repeat region is contained in a 20 kb HindIII-EcoRI fragment, and BamHI digestion suggested that most of the repeats are contained in a 10 kb fragment. In situ hybridization with an antisense probe to PGM-9B showed an intense signal in the entire gastric gland. Clone PGM-2A also contains the same repeat sequence as 9B, but, in addition, has a 64-amino-acid-long non-repeat region at its 5' end. Interestingly the non-repeat region of PGM-2A has five cysteine residues, the arrangement of which is identical with that reported for human intestinal mucin gene MUC2.

Identification of a cis-acting negative DNA element which modulates human hepatic triglyceride lipase gene expression.

The promoter fragment -1550/+129 of the human hepatic triglyceride lipase (HTGL) gene drives the expression of the CAT gene in HepG2 cells, albeit at very low levels. Transient transfections in HepG2 and HeLa cells of 5' deletion constructs indicated that the regulatory elements that control this expression are located in the proximal region of the gene. DNase I footprint analysis with DNA fragments spanning the region -483 to +129 and rat liver nuclear extracts identified eight protected regions, four upstream of the transcription initiation site (A, -28 to -75; B, -96 to -106; C, -118 to -158; D, -185 to -255) and four in the first exon of the gene (E1, -5 to +20; E2, +36 to +55; E3, +58 to +83; E4, +86 to +107). DNA binding and footprinting analysis demonstrated that the region -75 to -43 within footprint A binds to the liver-specific transcription factor HNF1. The region +28 to +129 contains a functional negative regulatory element (NRE) since deletion of this region results in a 17-fold increase in CAT activity. The NRE can act independent of orientation and position and repress transcription driven by heterologous promoters. DNA binding assays using native and fractionated liver nuclear extracts identified two transcription factors that bind to element E2 and also to element E3. A dinucleotide mutation in element E2 which causes derepression of the HTGL gene by 10-fold also abolishes the binding of these two activities. Transfection experiments showed that deletion of the NRE allows expression of reporter constructs in HeLa cells, indicating that the NRE may play a determinant role for the expression of HTGL gene in hepatic cells.

Transcriptional regulation of human apolipoprotein genes ApoB, ApoCIII, and ApoAII by members of the steroid hormone receptor superfamily HNF-4, ARP-1, EAR-2, and EAR-3.

Apolipoproteins B, CIII, and AII are synthesized primarily in the liver and intestine and play an important role in lipid and cholesterol metabolism. It was previously shown that the cis-acting elements (BA1 (-79 to -63), CIIIB (-87 to -63), and AIIJ (-740 to -719) present in the regulatory regions of the human apoB, apoCIII, and apoAII genes, respectively, are recognized by common transcription factors present in hepatic nuclear extracts. This report shows that four members of the steroid receptor superfamily, ARP-1, EAR-2, EAR-3, and HNF-4, bind specifically to the regulatory elements BA1, CIIIB, and AIIJ. Dissociation constant measurements showed that ARP-1, EAR-2, and HNF-4 bind to elements BA1 and CIIIB with similar affinities (Kd 1-3 nM). Cotransfection experiments in HepG2 cells revealed that ARP-1, EAR-2, and EAR-3 repressed the BA1, CIIIB, and AIIJ element-dependent transcription of the reporter gene constructs and the transcription driven by homopolymeric promoters containing either five BA1 or two CIIIB elements. In contrast, HNF-4 activated transcription of reporter genes containing the elements BA1, CIIIB, and AIIJ and reversed the ARP-1-mediated repression of the apoB and apoCIII genes. These results suggested that the opposing transcription effects observed between HNF-4 and ARP-1 may be due to competition for binding to the same regulatory element. Mutations which affected the binding of HNF-4 to elements BA1 and CIIIB affected its ability to activate transcription of the apoB and apoCIII reporter genes, respectively. Transcriptional activation by HNF-4 depended on the presence of elements II (-112 to -94) and III (-86 to -62) of the apoB and H (-705 to -690), I (-766 to -726), and J (-792 to -779) of the apoCIII promoters, indicating that transcriptional activation of apoB and apoCIII genes by HNF-4 requires the synergistic interaction of factors binding to these elements. The finding that HNF-4, ARP-1, EAR-2 and EAR-3 can regulate the expression of the apoB, apoCIII, and apoAII genes suggest that these nuclear hormone receptors may be an important part of the signal transduction pathways modulating lipid metabolism and cholesterol homeostasis.

Expression, secretion, and lipid-binding characterization of the N-terminal 17% of apolipoprotein B.

The N-terminal 17% of human apolipoprotein B (apoB-17) was expressed in murine C127 cells following transfection with a bovine papilloma virus-based expression vector. A permanent cell line overexpressing the expected 89-kDa protein was selected and characterized. Pulse-chase experiments showed that the depletion of intracellular apoB-17 follows an apparent first-order kinetics with t1/2 = 51 min. Under conditions of continuous labeling, greater than 60% of the total synthesized apoB-17 was secreted in a soluble form, approximately 98% lipid-poor and approximately 2% lipid-bound. Inclusion of 1.2 mM oleate resulted in 5- and 2.5-fold increases in the amount of labeled apoB-17 in the p less than 1.063 g/ml and 1.063 less than p less than 1.21 g/ml fractions, respectively, which was coordinated with increased secretion of radiolabeled core lipids, triacylglycerols, and cholesteryl esters. Thus under conditions in which lipid pools are enriched a greater fraction of apoB-17 may be secreted on lipoprotein-like particles. The lipid-poor apoB-17 present in p greater than 1.21 g/ml readily associates with exogenously added dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles to form discoidal particles. Discs formed with DMPC/apoB-17, 7:1 (wt/wt), are 239 +/- 43 A in diameter and 61 +/- 4 A thick and contain approximately 2 molecules of apoB-17 and 2250 molecules of DMPC per disc. Based on volume calculations we conclude that apoB-17 forms an annulus about one bilayer high and 10 A thick surrounding the DMPC disc. Circular dichroic spectra of apoB-17 on DMPC discs showed apoB-17 to contain 39% alpha-helix, 36% beta-sheet, 9% beta-turn, and 16% random coil. To be consistent with this model greater than 70% of apoB-17 on DMPC discs must bind to lipid. These data suggest that the N-terminal 17% of apoB-100 can bind lipid and may contribute to some extent to the stabilization of triglyceride-rich lipoproteins.

Characterization of the promoter elements required for hepatic and intestinal transcription of the human apoB gene: definition of the DNA-binding site of a tissue-specific transcriptional factor.

The promoter elements important for intestinal and hepatic transcription of the human apoB gene have been localized downstream of nucleotide -150. Footprinting analysis using hepatic nuclear extracts identified four protected regions, -124 to -100, -97 to -93, -86 to -33, and +33 to +52. Gel electrophoretic mobility shift assays showed that multiple factors interact with the apoB sequence -86 to -33, while the region -88 to -61 binds a single nuclear factor. Methylation interference analysis and nucleotide substitution mutagenesis identified the binding site of the factor between residues -78 and -68. Binding competition experiments indicate that this factor recognizes the regulatory elements of other liver-specific genes.

Promoter elements and factors required for hepatic and intestinal transcription of the human ApoCIII gene.

We have mapped the promoter elements required for hepatic and intestinal transcription of the human apoCIII gene by deletion, nucleotide substitution, and DNase I footprinting analyses of the promoter region (nucleotides -1411 to +24). Deletion of the region -1020 to -871 increased 2-fold intestinal transcription without affecting hepatic transcription. Deletion of the region -890 to -686 decreased hepatic and intestinal transcription 34- and 13-fold respectively. Internal deletions of the -686 to -553 region increased intestinal transcription 2-fold and decreased hepatic transcription 9-fold. Finally, internal deletions in the region -408 to -163 decreased hepatic transcription 2- to 4-fold without affecting the intestinal transcription. Footprinting analysis using rat liver nuclear extracts identified 10 protected regions as follows: A, -32 to -25; B, -87 to -72; C, -138 to -119; D, -160 to -142; E, -414 to -403; F, -611 to -592; G, -669 to -648; H, -705 to -690; I, -766 to -726; and J, -792 to -779. The findings indicate that the region -890 to -686 is recognized by nuclear factors which promote both intestinal and hepatic transcription, whereas the region -686 to -553 is recognized by factors which promote only hepatic transcription. DNA binding and methylation interference assays indicated that the region -86 to -74 is recognized by two mutually exclusive nuclear factors with overlapping domains. One factor (CIIIB1) is unique to apoCIII and the other (CIIIB2) recognizes the regulatory elements of other apolipoprotein promoters. Binding of factor CIIIB2 is associated with normal transcription. In contrast, binding of factor CIIIB1 is associated with reduced transcription indicating that this factor may act as a modulator of transcription.

Expression of ApoE gene in Chinese hamster cells with a reversible defect in O-glycosylation. Glycosylation is not required for apoE secretion.

The effects of O-glycosylation on the synthesis and secretion of apolipoprotein E (apoE, a glycoprotein with O- but not N-linked sugars) were studied with a UDP-galactose/UDP-N-acetylgalactosamine 4-epimerase-deficient cell mutant (ldlD cells) which expresses a reversible defect in protein O-glycosylation. Under normal culture conditions the mutant ldlD cells cannot add N-acetylgalactosamine (GalNAc) to proteins. GalNAc is the first sugar of mucin-type O-linked oligosaccharides attached to the protein. This O-glycosylation defect is rapidly corrected when GalNAc is added to the culture medium. These cells also require external sources of galactose for the addition of this sugar to O-linked and other oligosaccharides. A bovine papilloma virus-based expression vector for human apoE and the human metallothionein 1A gene were transfected into ldlD cells, and apoE-expressing cell clones resistant to CdCl2 were selected and used in the present studies. The structure and secretion of apoE in these cells were examined by immunoprecipitation and one- and two-dimensional gel electrophoresis and autoradiography. The synthesis, rate, and extent of secretion of apoE were unaffected by O-glycosylation (GalNAc-independent). In the presence of both galactose and GalNAc, multiple apoE isoforms were synthesized in ldlD cells as a result of variation in the extent of sialylation. ApoE sialylation was dependent on the addition of galactose as well as GalNAc to the extracellular medium, suggesting that addition of galactose to the nascent oligosaccharide chains was required for the addition of sialic acid.

The rev (trs/art) protein of human immunodeficiency virus type 1 affects viral mRNA and protein expression via a cis-acting sequence in the env region.

The study of expression of several human immunodeficiency virus type 1 proviral mutants in human cells in the presence or absence of rev (trs/art) protein reveals that rev increases the levels of unspliced and env mRNA and the accumulated structural viral proteins. rev protein produced from appropriate expression vectors fully complements the rev-defective mutants. rev requires the presence of a specific cis-acting sequence for its function. This rev-responsive element sequence has been localized within a 520 base-pair fragment in the env region of human immunodeficiency virus type 1. gag and env expression is coordinately regulated by rev. Two independent cis-acting elements localized in the gag and env regions are responsible for the low levels of gag and env mRNA in the absence of rev. These elements are different than the rev-responsive element and act independent of each other.

rev protein of human immunodeficiency virus type 1 affects the stability and transport of the viral mRNA.

rev (trs/art) is an essential human immunodeficiency virus type 1 (HIV-1) regulatory protein. rev increases the levels of the gag- and env-producing mRNAs via a cis-acting element in the env region of HIV-1, named rev-responsive element. Our results show that rev increases the stability of the unspliced viral mRNA, while it does not affect the stability of the multiply spliced viral mRNAs that do not contain the rev-responsive element. The study of mutated proviral constructs producing mRNA that cannot be spliced revealed that the effect of rev on stability is independent of splicing. Our experiments also indicate that rev promotes the transport of the viral mRNA containing the rev-responsive element from the nucleus to the cytoplasm. The proposed functions of rev are consistent with its nuclear localization as shown by immunofluorescence. The selective effects of rev on the levels of the viral mRNA suggest a model for feedback regulation by rev leading to a steady state of viral expression.