Stacy Taylor

Two Hepatic Enhancers, HCR.1 and HCR.2, Coordinate the Liver Expression of the Entire Human Apolipoprotein E/C-I/C-IV/C-II Gene Cluster

We show that the liver-specific expression of all four genes in the human apolipoprotein (apo) E/C-I/C-IV/C-II gene cluster in transgenic mice is determined by the coordinate action of two distinct hepatic control regions (HCR). These enhancers are positioned 15 kilobases (kb) (HCR.1) and 26 kb (HCR.2) downstream of the apoE gene. To investigate the action of each HCR, transgenic mice were generated with a 70-kb human genomic fragment that contained the complete apoE gene cluster or with this fragment modified by the specific deletion of HCR.1, HCR.2, or both HCR domains. Hepatic expression of all four apolipoprotein genes was observed in transgenic mice in which either HCR.1 or HCR.2 was deleted, but no transgene expression was found in the liver in the absence of both HCR domains. The overall patterns of transgene expression suggested that HCR.2 was the dominant element for apoC-IV and apoC-II expression and that HCR.1 was dominant for the apoE/C-I expression. No liver-specific transcriptional activity was identified for the proximal promoter of any gene in the cluster; all liver-specific activity was associated with HCR.1 and HCR.2. Thus, the HCRs of the apoE gene cluster constitute unique regulatory domains for determining the requirements for apolipoprotein gene expression in the liver.

Structure of the Hepatic Control Region of the Human Apolipoprotein E/C-I Gene Locus

The specificity of expression in the liver of the human apolipoprotein (apo) E/C-I gene locus is determined by a hepatic control region (HCR) that is located 15 kilobases downstream of the apoE gene. DNase I footprint studies of this sequence using nuclear extracts identified a region of the HCR that is enriched in nuclear protein-binding sites. Nuclease analysis of chromatin revealed liver-specific DNase I-hypersensitive sites that were associated with this region, and additional liver-specific nuclease-sensitive sites associated with the apoE gene were identified. The HCR domain has a limited binding affinity for the nuclear scaffold. The specific domain required for liver expression was tested by ligating subfragments of the HCR to the apoE gene and examining their activity in transgenic mice. A segment of 319 nucleotides that contained several potential regulatory sequences was required for full activity of liver-specific transcription with shorter segments yielding much lower levels of expression in the liver. All constructs that contained a fully active HCR were expressed in approximately a copy-dependent manner, suggesting that transgene expression was independent of integration position. Taken together, the properties of the HCR are consistent with its function as a locus control region for the liver-specific expression of the apoE gene.

Overexpression of Human Apolipoprotein B-100 in Transgenic Rabbits Results in Increased Levels of LDL and Decreased Levels of HDL

In this study, and 80-kb human genomic DNA fragment spanning the human apoB gene was used to generate transgenic New Zealand White rabbits that expressed human apoB-100. The concentration of human apoB in the plasma of the transgenic rabbits ranged between 5 and 100 mg/dL. The transgenic rabbits had nearly threefold elevations in the plasma levels of triglycerides and cholesterol compared with nontransgenic controls. Nearly all the cholesterol and human apoB in the plasma was in the LDL fraction. Pronounced triglyceride enrichment of the LDL fraction was a striking feature of human apoB overexpression in the transgenic rabbits, in which the LDL fraction contained more than 75% of the plasma triglycerides. The triglyceride-enriched LDL particles were smaller and more dense than the native rabbit LDL and contained markedly increased amounts of apoE and apoC-III. In the nontransgenic control animals most of the triglycerides were in the VLDL, and most of the apoE and apoC-III were in the VLDL and HDL fractions. In addition to increased LDL levels, overexpression of human apoB in rabbits resulted in lower plasma levels of HDL cholesterol and apoA-I. In our prior studies on transgenic mice expressing human apoB, we documented triglyceride-rich LDL and reduced levels of HDL cholesterol. These prior findings in mice, together with the present findings in transgenic rabbits, suggest that triglyceride-rich LDL and lowered levels of HDL cholesterol may be hallmark features of apoB overexpression.

Apolipoprotein B gene expression in a series of human apolipoprotein B transgenic mice generated with recA-assisted restriction endonuclease cleavage-modified bacterial artificial chromosomes. An intestine-specific enhancer element is located between 54 and 62 kilobases 5' to the structural gene.

Prior studies have established that the expression of the human apolipoprotein B (apoB) gene in the intestine is dependent on DNA sequences located a great distance from the structural gene. To identify the location of those sequences, we usedrecA-assisted restriction endonuclease (RARE) cleavage to truncate the 5′- or 3′-flanking sequences from a 145-kilobase (kb) bacterial artificial chromosome spanning the entire human apoB gene. Seven RARE cleavage– modified bacterial artificial chromosomes with different lengths of flanking sequences were used to generate transgenic mice. An analysis of those mice revealed that as little as 1.5 kb of 3′ sequences or 5 kb of 5′ sequences were sufficient to confer apoB expression in the liver. In contrast, apoB gene expression in the intestine required DNA sequences 54–62 kb 5′ to the structural gene. Those sequences retained their ability to direct apoB expression in the intestine when they were moved closer to the gene. These studies demonstrate that the intestinal expression of the apoB gene is dependent on DNA sequences located an extraordinary distance from the structural gene and that the RARE cleavage/transgenic expression strategy is a powerful approach for analyzing distant gene-regulatory sequences.

Taok2 controls behavioral response to ethanol in mice

Despite recent advances in the understanding of ethanols biological action, many of the molecular targets of ethanol and mechanisms behind ethanols effect on behavior remain poorly understood. In an effort to identify novel genes, the products of which regulate behavioral responses to ethanol, we recently identified a mutation in the dtao gene that confers resistance to the locomotor stimulating effect of ethanol in Drosophila. dtao encodes a member of the Ste20 family of serine/threonine kinases implicated in MAP kinase signaling pathways. In this study, we report that conditional ablation of the mouse dtao homolog, Taok2, constitutively and specifically in the nervous system, results in strain-specific and overlapping alterations in ethanol-dependent behaviors. These data suggest a functional conservation of dtao and Taok2 in mediating ethanols biological action and identify Taok2 as a putative candidate gene for ethanol use disorders in humans.

Transgenic mice expressing human apob100 and apob48

Transgenic mice that express human apolipoprotein (apo)B have been developed by microinjecting fertilized mouse oocytes with an 80 kb genomic DNA fragment that encompasses the entire human APOB gene. In the transgenic mice expressing the largest amounts of human apoB, the concentration of human apoB100 in the plasma is nearly as high as the levels observed in normolipidemic humans (50 mg/dl). Virtually all of the human apoB100 in the transgenic plasma is located in the LDL fraction, resulting in substantially increased levels of LDL cholesterol. These human apoB-transgenic mice should be useful animal models for understanding various aspects of lipoprotein metabolism and for further delineating the role of LDL in atherogenesis.