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Trafficking in Cholesterol: Investigating the Human ABCA1 Gene
By Barbra Rodriguez

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Researchers have identified two key regulatory sequences for a human gene that plays a role in transporting cholesterol and is associated with Tangier disease, a lipid disorder involving the accumulation of cholesterol in certain tissues. As part of a larger investigation of how disease genes are regulated, the researchers are testing the function of the human promoter sequences in mice.

The gene, ATP binding cassette transporter 1 (ABCA1), is among more than 240 being studied at the Lawrence Berkeley National Laboratory in Berkeley, California. The researchers are comparing the sequences of genes across multiple species, including human, mouse, chimp, dog, rabbit, and opossum. The genes in the study have previously been linked to heart disease, stroke and other cardiovascular diseases.

"We're not interested in discovering new genes involved in cardiovascular biology," says Edward M. Rubin, senior scientist at the Berkeley Laboratory. Rather, his research group is interested in using "cross-species comparisons to identify functionally relevant regions that cardiovascular biologists can now target in their genes of interest."

The Berkeley sequencing effort is one of 11 projects funded by the US National Heart, Lung, and Blood Institute (NHLBI) last year to use genomic approaches in biological research. In other projects, researchers are using knockout mice to clarify the role of disease-related genes and microarray analyses to study global gene expression patterns.

The gene structure and expression pattern of hABCA1. View full

"The overall goal of the NHLBI project is to help move the products of the genome program out of databases to empower cardiovascular biologists," says Rubin.

The ABCA1 gene encodes a protein that pumps cholesterol from different types of cells. Macrophages, for example, are cells of the immune system that may use the transporter to help keep arteries functional and unclogged. The transporter allows the cells to unload cholesterol into high-density lipoprotein vesicles (HDL), which then travel to the liver.

The researchers identified two stretches of genomic sequence that were highly conserved in human and mouse ABCA1, in addition to 29 other potential regulatory sites for the transporter. The results were published in the April issue of Genomics.

One DNA sequence, located within the gene itself, was especially well conserved, with 88 percent sequence homology. "It was too good a homology to have just occurred [randomly]," says Jan-Fang Cheng, who led the study.

The researchers determined that this and a second potential regulatory region were well conserved in ABCA1 from rat, dog, rabbit, cow, and pig. Further analyses revealed more than a dozen potential binding sites for regulatory elements within the internal sequence. The researchers also found that the second sequence—located outside the gene—contained the main ABCA1 promoter.

Expression of hABCA1 in mice mimics that of the endogenous mABCA1. View full

Cheng notes that the comparative sequencing effort allowed his group to peg the external sequence as the promoter before more labor-intensive, mutational studies by others came to this conclusion. "That shows the power of comparative data," he says.

The comparative research also revealed that other sites for regulatory elements are likely to exist outside the gene. "That probably implies that there's a more complicated regulatory mechanism [for ABCA1] than we thought," Cheng says, noting that this could confound efforts to develop drugs targeting the gene.

The researchers have since found evidence of this intricate regulation in studies of transgenic mice that express one of two forms of ABCA1. Mice with the full-length transporter gene expressed the protein in many tissues. However, animals accidentally given a shortened version of the gene expressed high levels of the protein only in the liver, suggesting the use of an alternate promoter.

"The transgenics, sort of by mistake, led us to something that had physiological relevance," says Rubin, who led the transgenic research project. The findings were published in May in The Journal of Biological Chemistry.

The liver form of the transporter could serve a unique role by directing cholesterol to the gall bladder on its way to the intestines. To test this hypothesis, the researchers are developing mice that express only the shortened human ABCA1 gene and assessing how the animals handle excess dietary cholesterol.

The findings imply that mammals economize by using the same cardiovascular-related genes for different functions, says Cheng. "If you find one gene with different promoters that express differently in different tissues, that could imply they play slightly different functional roles," he explains.

Model for the role of PPARg and LXR in macrophage cholesterol efflux. View larger

An additional control element for ABCA1 was identified in January. Two studies linked the expression of ABCA1 to a known regulator of lipid metabolism. Peroxisome proliferator-activated receptor gamma (PPAR-g) stimulates ABCA1 function by activating another genetic element called liver X receptor alpha (LXR-a), the studies showed. Bart Staels, of the Institut Pasteur, in Lille, France, and colleagues published one study in Nature Medicine.

In the second study, Peter Tontonoz, of the University of California, Los Angeles, and colleagues found that a more severe form of atherosclerosis develops in mice that are prone to the disease and lack PPAR-g expression in their macrophages.

"That's the first direct link between PPAR-g actions, specifically in macrophages, and a reduced risk of atherosclerosis," says Tontonoz, a Howard Hughes Medical Institute investigator at UCLA. His group determined that mature macrophages express more ABCA1 and export more cholesterol when exposed to the factors that activate PPAR-g or LXR-a. The study was published in the journal Molecular Cell.

The results have raised hope among researchers that PPAR-g may be a key regulatory switch for activating ABCA1 function. Drugs that activate PPAR-g, called thiozolidinediones, are used to treat diabetes, and could be tested for reducing atherosclerosis.

"It's likely that there are multiple good effects that can come from PPAR-g-targeted therapy," says Tontonoz. "These ligands are known to have beneficial effects on the overall lipid profile."

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Cavelier, L.B. et al. Regulation and activity of the human ABCA1 gene in transgenic mice. J Biol Chem 276, 18046-18051 (May 2001).
Qiu, Y. et al. Human and mouse ABCA1 comparative sequencing and transgenesis studies revealing novel regulatory sequences. Genomics 73, 66-76 (April 2001).
Chawla, A. et al. A PPARg-LXR-ABCA1 pathway in macrophages is involved in cholesterol efflux and atherogenesis. Mol Cell 7, 161-171 (January 2001).
Chinetti, G. et al. PPAR-a and PPAR-g activators induce cholesterol removal from human macrophage foam cells through stimulation of the ABCA1 pathway. Nat Med 7, 53-58 (January 2001).

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