Duplicated downstream enhancers control expression of the human apolipoprotein E gene in macrophages and adipose tissue

Two distal enhancers that specify apolipoprotein (apo) E gene expression in isolated macrophages and adipose tissue were identified in transgenic mice that were generated with constructs of the human apoE/C-I/C-I'/C-IV/C-II gene cluster. One of these enhancers, multienhancer 1, consists of a 620-nucleotide sequence located 3.3 kilobases (kb) downstream of the apoE gene. The second enhancer, multienhancer 2, is a 619-nucleotide sequence located 15.9 kb downstream of the apoE gene and 5.9 kb downstream of the apoC-I gene. The two enhancers are 95% identical in sequence, and they are likely to have arisen as a consequence of the gene duplication event that yielded the apoC-I gene and the apoC-I' pseudogene. Both enhancer sequences appear to have equivalent activity in directing apoE gene expression in peritoneal macrophages and in adipocytes, suggesting that their activity in specific cell types may be determined by common regulatory elements.     

Two copies of the human apolipoprotein C-I gene are linked closely to the apolipoprotein E gene

The gene for human apolipoprotein (apo) C-I was selected from human genomic cosmid and lambda libraries. Restriction endonuclease analysis showed that the gene for apoC-I is located 5.5 kilobases downstream of the gene for apoE. A copy of the apoC-I gene, apoC-I', is located 7.5 kilobases downstream of the apoC-I gene. Both genes contain four exons and three introns; the apoC-I gene is 4653 base pairs long, the apoC-I' gene 4387 base pairs. In each gene, the first intron is located 20 nucleotides upstream from the translation start signal; the second intron, within the codon of Gly-7 of the signal peptide region; and the third intron, within the codon for Arg39 of the mature plasma protein coding region. The upstream apoC-I gene encodes the known apoC-I plasma protein and differs from the downstream apoC-I' gene in about 9% of the exon nucleotide positions. The most important difference between the exons results in a change in the codon for Gln-2 of the signal peptide region, which introduces a translation stop signal in the downstream gene. Major sequence differences are found in the second and third introns of the apoC-I and apoC-I' genes, which contain 9 and 7.5 copies, respectively, of Alu family sequences. The apoC-I gene is expressed primarily in the liver, and it is activated when monocytes differentiate into macrophages. In contrast, no mRNA product of the apoC-I' gene can be detected in any tissue, suggesting that it may be a pseudogene. The similar structures and the proximity of the apoE and apoC-I genes suggest that they are derived from a common ancestor. Furthermore, they may be considered to be constituents of a family of seven apolipoprotein genes (apoE, -C-I, -C-II, -C-III, -A-I, -A-II, and -A-IV) that have a common evolutionary origin.     

Meiotic expression of human ornithine transcarbamylase in the testes of transgenic mice.

In an attempt to use mouse metallothionein-I (mMT-I) regulatory sequences to direct expression of human ornithine transcarbamylase in the liver of transgenic animals, fusion genes joining either 1.6 kilobases or 185 base pairs of the mMT-I regulatory region to the human ornithine transcarbamylase protein-coding sequence were used to produce transgenic mice. In mice carrying the fusion gene with 1.6 kilobases of the mMT-I 5'-flanking sequences, transgene expression was observed in a wide range of tissues, but, unexpectedly, expression in liver was never observed. Surprisingly, in mice carrying the fusion gene regulated by only 185 base pairs of the mMT-I 5'-flanking sequences, the transgene was expressed exclusively in male germ cells during the tetraploid, pachytene stage of meiosis.     

Human apolipoprotein C-I expression in mice impairs learning and memory functions

The H2 allele of APOC1, giving rise to increased gene expression of apolipoprotein C-I (apoC-I), is in genetic disequilibrium with the APOE4 allele and may provide a major risk factor for Alzheimer's disease (AD). We found that apoC-I protein is present in astrocytes and endothelial cells within hippocampal regions in both human control and AD brains. Interestingly, apoC-I colocalized with beta-amyloid (Abeta) in plaques in AD brains, and in vitro experiments revealed that aggregation of Abeta was delayed in the presence of apoC-I. Moreover, apoC-I was found to exacerbate the soluble Abeta oligomer-induced neuronal death. To establish a potential role for apoC-I in cognitive functions, we used human (h) APOC1(+/0) transgenic mice that express APOC1 mRNA throughout their brains and apoC-I protein in astrocytes and endothelial cells. The hAPOC1(+/0) mice displayed impaired hippocampal-dependent learning and memory functions compared with their wild-type littermates, as judged from their performance in the object recognition task (P = 0.012) and in the Morris water maze task (P = 0.010). ApoC-I may affect learning as a result of its inhibitory properties toward apoE-dependent lipid metabolism. However, no differences in brain mRNA or protein levels of endogenous apoE were detected between transgenic and wild-type mice. In conclusion, human apoC-I expression impairs cognitive functions in mice independent of apoE expression, which supports the potential of a modulatory role for apoC-I during the development of AD.     

Identification of cis sequences controlling efficient position-independent tissue-specific expression of human major histocompatibility complex class I genes in transgenic mice.

We previously reported that genomic major histocompatibility complex class I human leukocyte antigen (HLA)-B7 gene constructs with as little as 0.66 kb of 5'- and 2.0 kb of 3'-flanking DNA were expressed efficiently and appropriately in transgenic mice. To identify and characterize the relevant cis-acting regulatory elements in more detail, we have generated and analyzed a series of transgenic mice carrying native HLA-B7 genes with further 5' truncations or intronic deletions and hybrid constructs linking the 5'-flanking region of B7 to a reporter gene. We were unable to detect a specific requirement for sequence information within introns 2 to 7 for either appropriate constitutive or inducible class I expression in adult animals. The results revealed the presence of cis-acting regulatory sequences between -0.075 kb and -0.66 kb involved in driving efficient copy number-dependent constitutive and gamma interferon-enhanced tissue-specific expression. The region from -0.11 to -0.66 kb is also sufficient to prevent integration site-specific "position effects," because in its absence HLA-B7 expression is frequently detected at significant levels at inappropriate sites. Conserved sequence elements homologous to the H-2 class I regulatory element, or enhancer A, and the interferon response sequence are located between about -151 and -228 bp of the B7 gene. Our results also indicate the existence of sequences downstream of -0.11 kb which can influence the pattern of tissue-specific expression of the HLA-B7 gene and the ability of this gene to respond to gamma interferon.     

Tissue-specific and insulin-dependent expression of a pancreatic amylase gene in transgenic mice.

The regulatory properties of mouse pancreatic amylase genes include exclusive expression in the acinar cells of the pancreas and dependence on insulin and glucocorticoids for maximal expression. We have characterized a murine pancreatic amylase gene, Amy-2.2y, whose promoter sequence is 30% divergent from those of previously sequenced amylase genes. To localize sequences required for tissue-specific and hormone-dependent activation, we established two lines of transgenic mice. The first line contained a single copy of the complete Amy-2.2y gene as well as 9 kilobases of 5'-flanking sequence and 5 kilobases of 3'-flanking sequence. The second line carried a minigene which included 208 base pairs of 5'-flanking sequence and 300 base pairs of 3'-flanking sequence. In both lines the transgene was expressed at high levels exclusively in the pancreas. Both constructs were dependent on insulin and induced by dexamethasone. Thus, the transferred genes contained the sequences required for tissue-specific and hormonally regulated expression.     

Differential regulation of the rat phosphoenolpyruvate carboxykinase gene expression in several tissues of transgenic mice.

The selective expression of a unique copy gene in several mammalian tissues has been approached by studying the regulatory sequences needed to control expression of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene in transgenic mice. A transgene containing the entire PEPCK gene, including 2.2 kb of the 5'-flanking region and 0.5 kb of the 3'-flanking region, exhibits tissue-specific expression in the liver, kidney, and adipose tissue, as well as the hormonal and developmental regulation inherent to endogenous gene expression. Deletions of the 5'-flanking region of the gene have shown the need for sequences downstream of position -540 of the PEPCK gene for expression in the liver and sequences downstream of position -362 for expression in the kidney. Additional sequences upstream of position -540 (up to -2200) are required for expression in adipose tissue. In addition, the region containing the glucocorticoid-responsive elements of the gene used by the kidney was identified. This same sequence was found to be needed specifically for developmental regulation of gene expression in the kidney and, together with upstream sequences, in the intestine. The apparently distinct sequence requirements in the various tissues indicate that the tissues use different mechanisms for expression of the same gene.     

Expression and regulation of the human GLUT4/muscle-fat facilitative glucose transporter gene in transgenic mice.

To study the molecular basis of tissue-specific expression of the GLUT4/muscle-fat facilitative glucose transporter gene, we generated lines of transgenic mice carrying 2.4 kilobases of the 5'-flanking region of the human GLUT4 gene fused to a chloramphenicol acetyltransferase (CAT) reporter gene (hGLUT4[2.4]-CAT). This reporter gene construct was specifically expressed in tissues that normally express GLUT4 mRNA, which include both brown and white adipose tissues as well as cardiac, skeletal, and smooth muscle. In contrast, CAT reporter activity was not detected in brain or liver, two tissues that do not express the GLUT4 gene. In addition, the relative levels of CAT mRNA driven by the human GLUT4 promoter in various tissues of these transgenic animals mirrored those of the endogenous mouse GLUT4 mRNA. Since previous studies have observed alterations in GLUT4 mRNA levels induced by fasting and refeeding (Sivitz, W. I., DeSautel, S. L., Kayano, T., Bell, G. I., and Pessin, J. E. (1989) Nature 340, 72-74), the regulated expression the hGLUT4[2.4]-CAT transgene was also assessed in these animals. Fasting was observed to decrease CAT activity in white adipose tissue which was super-induced upon refeeding. These alterations in CAT expression occurred in parallel to the changes in endogenous mouse GLUT4 mRNA levels. Although CAT expression in skeletal muscle and brown adipose tissue was unaffected, the endogenous mouse GLUT4 mRNA was also refractory to the effects of fasting/refeeding in these tissues. These data demonstrate that 2.4 kilobases of the 5'-flanking region of the human GLUT4 gene contain all the necessary sequence elements to confer tissue-specific expression and at least some of the sequence elements controlling the hormonal/metabolic regulation of this gene.     

Disruption of TR4 orphan nuclear receptor reduces the expression of liver apolipoprotein E/C-I/C-II gene cluster

Apolipoprotein E (apoE) is synthesized in many tissues, and the liver is the primary site from which apoE redistributes cholesterol and other lipids to peripheral tissues. Here we demonstrate that the TR4 orphan nuclear receptor (TR4) can induce apoE expression in HepG2 cells. This TR4-mediated regulation of apoE gene expression was further confirmed in vivo using TR4 knockout mice. Both serum apoE protein and liver apoE mRNA levels were significantly reduced in TR4 knockout mice. Gel shift and luciferase reporter gene assays further demonstrated that TR4 can induce apoE gene expression via a TR4 response element located in the hepatic control region that is 15 kb downstream of the apoE gene. Furthermore our in vivo data from TR4 knockout mice prove that TR4 can also regulate apolipoprotein C-I and C-II gene expression via the TR4 response element within the hepatic control region. Together our data show that loss of TR4 down-regulates expression of the apoE/C-I/C-II gene cluster in liver cells, demonstrating important roles of TR4 in the modulation of lipoprotein metabolism.     

Overexpression of apoC-I in apoE-null mice: severe hypertriglyceridemia due to inhibition of hepatic lipase

Apolipoprotein C-I (apoC-I) has been proposed to act primarily via interference with apoE-mediated lipoprotein uptake. To define actions of apoC-I that are independent of apoE, we crossed a moderately overexpressing human apoC-I transgenic, which possesses a minimal phenotype in the WT background, with the apoE-null mouse. Surprisingly, apoE-null/C-I mice showed much more severe hyperlipidemia than apoE-null littermates in both the fasting and non-fasting states, with an almost doubling of cholesterol, primarily in IDL+LDL, and a marked increase in triglycerides; 3-fold in females to 260 +/- 80 mg/dl and 14-fold in males to 1409 +/- 594 mg/dl. HDL lipids were not significantly altered but HDL were apoC-I-enriched and apoA-II-depleted. Production rates of VLDL triglyceride were unchanged as was the clearance of post-lipolysis remnant particles. Plasma post-heparin hepatic lipase and lipoprotein lipase levels were undiminished as was the in vitro hydrolysis of apoC-I transgenic VLDL. However, HDL from apoC-I transgenic mice had a marked inhibitory effect on hepatic lipase activity, as did purified apoC-I. LPL activity was minimally affected. Atherosclerosis assay revealed significantly increased atherosclerosis in apoE-null/C-I mice assessed via the en face assay. Inhibition of hepatic lipase may be an important mechanism of the decrease in lipoprotein clearance mediated by apoC-I.     

Analysis of the human alpha globin upstream regulatory element (HS-40) in transgenic mice.

We have analysed the effect of a 1.4 kb segment of DNA containing the upstream alpha globin regulatory element (HS-40) on human alpha globin gene expression in fetal mice and lines of transgenic mice. High levels of tissue-specific, human alpha mRNA expression were seen in all transgenic animals and in this sense expression was position independent. However, the level of human alpha mRNA expression per integrated gene copy decreased during development and was inversely related to copy number. The limitation in expression with increasing gene copy number was shown to be in cis since homozygotes for the transgene produced twice as much human alpha mRNA as hemizygotes. In many respects HS -40 appears similar to single elements within the previously described beta globin locus control region and in cross breeding experiments we have shown that HS -40 behaves in a similar manner to such elements in transgenic mice.     

Accumulation of human apolipoprotein E in the plasma of transgenic mice

Three separate lines of transgenic mice were created with integrated copies of an 11.1-kilobase pair human DNA fragment containing the apolipoprotein (apo) E gene. The endogenous mouse apoE gene is primarily expressed in the liver with varying levels of expression in other tissues. However, in all three transgenic lines high levels of human apoE mRNA were detected only in the kidney, with lower levels found in the liver and other tissues; despite this profile of human apoE mRNA, human apoE was found in the plasma of the transgenic mice at levels comparable to those found in human plasma. All of the human apoE in the plasma of the transgenic mice was associated with lipoproteins. These results suggest that the domain responsible for the high level of apoE expression in liver lies outside of the microinjected DNA fragment and that an ectopic site of expression of an introduced gene may be permissive for the accumulation of its protein in plasma.     

Identification and expression of cosmids with an allelic variant of class I alcohol dehydrogenase in transgenic mice

The mouse Adh1 gene exhibits tissue-specific regulation, is developmentally regulated, and is androgen regulated in kidney and adrenal tissue. To study this complex regulation phenotype a transgenic mouse approach has been used to investigate regulatory regions of the gene necessary for proper tissue expression and hormonal control. Transgenic mice have been produced with an Adh1 minigene as a reporter behind either 2.5- or 10 kb of 5'-flanking sequence [1]. Complete androgen regulation in kidney requires a region between -2.5 and -10 kb. A sequence extending to -10 kb does not confer liver expression in this minigene construct. B6.S mice express an electrophoretically variant protein resulting from a known nucleotide substitution resulting in a restriction endonuclease length polymorphism. Transgenic mice harboring B6.S cosmids can be studied for expression analysis at both protein and mRNA levels, identification of transgenic founders and inheritance studies are greatly facilitated by a PCR-restriction endonuclease cleavage approach, the entire mouse gene is used as a reporter, and the formation of heterodimeric enzyme molecules can be used to infer expression of the transgene in the proper cell types within a given tissue. Expression of a B6.S cosmid containing the entire Adh1 gene and 6 kb of 5'- and 21 kb of 3'-flanking region occurs in transgenic mice in a copy number dependent manner in a number of tissues, but expression in liver does not occur. The ability to analyze expression at the protein and mRNA levels has been confirmed using this system. Future directions will involve the use of large BAC clones modified by RARE cleavage to identify the liver specific elements necessary for expression.     

Ostrich (Struthio camelus) eggshell matrix contains two different C-type lectin-like proteins. Isolation, amino acid sequence, and posttranslational modifications

Comparative analysis of the human and mouse genomic sequences downstream of the apolipoprotein E gene (APOE) revealed a highly conserved element with previously undefined function. In reporter gene transfection studies, this element which is located approximately 42 kb distal to APOE was found to have silencer activity in a subset of cell lines examined. Analysis of transgenic mice containing a fusion construct linking this distal 631 bp conserved element to a reporter gene comprised of the human APOE gene with its proximal promoter resulted in robust brain expression of the transgenic human apoE mRNA in three independent transgenic lines, supporting the identification of a novel brain controlling region (BCR). Further studies using immunohistochemistry revealed widespread human apoE localization throughout the brains of the BCR-apoE transgenic mice with prominent expression in the cortex and diencephalon. In addition, double-label immunofluorescence performed on brain sections and cultures of primary cortical cells localized human apoE protein to cortical neurons and microglia. These studies demonstrate that comparative sequence analysis is a successful strategy to predict candidate regulatory regions in vivo, although they do not imply that this element controls apoE expression physiologically.