An association study of 43 SNPs in 16 candidate genes with atorvastatin response.

Variation in individual response to statin therapy has been widely studied for a potential genetic component. Multiple genes have been identified as potential modulators of statin response, but few study findings have replicated. To further examine these associations, 2735 individuals on statin therapy, half on atorvastatin and the other half divided among fluvastatin, lovastatin, pravastatin and simvastatin were genotyped for 43 SNPs in 16 genes that have been implicated in statin response. Associations with low-density lipoprotein cholesterol (LDL-C) lowering, total cholesterol lowering, HDL-C elevation and triglyceride lowering were examined. The only significant associations with LDL-C lowering were found with apoE2 in which carriers of the rare allele who took atorvastatin lowered their LDL-C by 3.5% more than those homozygous for the common allele and with rs2032582 (S893A in ABCB1) in which the two groups of homozygotes differed by 3% in LDL-C lowering. These genetic effects were smaller than those observed with the demographic variables of age and gender. The magnitude of all the differences found is sufficiently small that genetic data from these genes should not influence clinical decisions on statin administration.

Cholesteryl ester transfer protein promoter single-nucleotide polymorphisms in Sp1-binding sites affect transcription and are associated with high-density lipoprotein cholesterol.

Genetic variation in the human cholesteryl ester transfer protein (CETP) promoter has been shown to be associated with high-density lipoprotein cholesterol (HDL-C) levels and cardiovascular disease. Some of this variation occurs in Sp1/Sp3 binding sites in the proximal promoter. We find that both the known promoter polymorphism at -629 and the previously uncharacterized polymorphism at -38 are associated with HDL-C levels in vivo and affect transcription in vitro. While the -629 polymorphism is common in all ethnic groups, the -38 polymorphism is found at significant levels (6.4%) only among African Americans. Those homozygous for the less common -38A allele have higher HDL-C levels than those with the more frequent -38G allele. This association was found in a population of African Americans at risk of cardiovascular disease and then replicated in a different population chosen from among patients with extremes of HDL-C. When studied in vitro, the most transcriptionally active allele (-629C/-38G) yields 51% more reporter protein than the least active allele (-629A/-38A) in HepG2 cells. These transcriptional effects reflect the projected impact of increased CETP expression on HDL-C phenotypes seen in vivo.

Mutation of a protease-sensitive region in firefly luciferase alters light emission properties.

Luciferase (EC 1.13.12.7) from the North American firefly, Photinus pyralis, is widely used as a reporter enzyme in cell biology. One of its distinctive properties is a pronounced susceptibility to proteolytic degradation that causes luciferase to have a very short intracellular half-life. To define the structural basis for this behavior and possibly facilitate the design of more stable forms of luciferase, limited proteolysis studies were undertaken using trypsin and chymotrypsin to identify regions of the protein whose accessible and flexible character rendered them especially sensitive to cleavage. Regions of amino acid sequence 206-220 and 329-341 were found to be sensitive, and because the region around 206-220 had high homology with other luciferases, CoA ligases, and peptidyl synthetases, this region was selected for mutagenesis experiments intended to determine which of its amino acids were essential for activity. Surprisingly, many highly conserved residues including Ser198, Ser201, Thr202, and Gly203 could be mutated with little effect on the luminescent activity of P. pyralis luciferase. One mutation, however, S198T, caused several alterations in enzymatic properties including shifting the pH optimum from 8.1 to 8.7, lowering the Km for Mg-ATP by a factor of 4 and increasing the half-time for light emission decay by a factor of up to 150. While the S198T luciferase was less active than wild type, activity could be restored by the introduction of the additional L194F and N197Y mutations. In addition to indicating the involvement of this region in ATP binding, these results provide a new form of the enzyme that affords a more versatile reporter system.

Transcriptional modulators affect in vivo protein binding to the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase promoters.

Treatment of HepG2 cells with known effectors of low density lipoprotein receptor (LDLR) gene expression altered the in vivo pattern of protein-DNA interactions in the promoter. The observed changes are consistent with proteins binding in vivo to the sterol regulatory element (SRE), to Sp1-like sites, as well as to other regions. Protein bound to the SRE in all conditions, but the nature of the dimethyl sulfate reactivity changed depending on the physiological state of the cell. Hypermethylation within the SRE of the low density lipoprotein receptor promoter was observed when cells were treated with cholesterol synthesis inhibitors, insulin, or phorbol 12-myristate 13-acetate, suggesting that the SRE regulates this promoter through sterol-independent as well as sterol-dependent mechanisms. No significant changes were observed in binding to the Sp1-like sites, suggesting that differential binding to these sites does not play a role in altered transcription levels. Analysis of the 3-hydroxy-3-methylglutaryl coenzyme A reductase promoter also revealed protections that varied in a cell type-specific manner. Binding to the 3-hydroxy-3-methylglutaryl coenzyme A reductase SRE and putative nuclear factor 1 sites could be observed but varied little in different physiological conditions.

Protein binding to the low density lipoprotein receptor promoter in vivo is differentially affected by gene activation in primary human cells.

Protein-DNA interactions within a region of the LDL receptor promoter involved in sterol-mediated feedback repression of transcription were examined using in vivo genomic footprinting with dimethylsulfate (DMS). A broad region of protein-DNA contacts spanning from repeat 1 to beyond the transcription start sites was observed in primary cultures of human skin fibroblasts and hepatocytes. Hypermethylation of guanine -59 within the sterol regulatory element-1 (SRE-1, repeat 2) occurred within a 4.0 h incubation of fibroblasts with media containing lipoprotein-deficient serum (LPDS) and cholesterol synthesis inhibitors. Methylation of this residue was reduced to control levels within 2.0 h after the addition of a mixture of 25-hydroxycholesterol and mevalonic acid. The time-dependent changes in DMS-reactivity of guanine -59 induced by the cholesterol synthesis inhibitors or oxysterols were paralleled by alterations in LDL receptor mRNA. In contrast to the results with fibroblasts, neither cholesterol synthesis inhibitors nor oxysterols produced consistent effects on the DMS-reactivity of guanine -59 in hepatocytes despite induction or repression of LDL receptor mRNA in these cells. Interestingly, no other changes in the protection pattern over repeats 1, 2, and 3 were apparent in either fibroblasts or hepatocytes. These results demonstrate that hypermethylation of guainine -59 within the SRE-1 is positively associated with activation of LDL receptor gene transcription in skin fibroblasts. Furthermore, the absence of demonstrable changes in DMS-reactivity of other purines within this region suggests that the LDL receptor promoter is poised to activate transcription with only minimal changes of protein binding to the proximal promoter in vivo.

Cholesterol 7 alpha hydroxylase promoter separated from cyclophilin pseudogene by Alu sequence.

The promoter for human cholesterol 7-alpha hydroxylase has been cloned and sequenced. In the regions previously described, our sequence agrees well with one report but not with another. At position -469, we find a widespread MaeII polymorphism. At -2636, there is an Alu sequence flanked by runs of adenines. Upstream of the Alu sequence, there is a cyclophilin pseudogene oriented in the opposite direction.

Modulation of firefly luciferase stability and impact on studies of gene regulation.

Two of the reporter enzymes most commonly used in studies of eukaryotic gene expression are chloramphenicol acetyl-transferase (CAT) and firefly luciferase (Luc). CAT has a half-life of about 50 h in mammalian cells, making it useful for transient transfection assays but less suitable for assays with stable cell lines. Luc has a half-life of only 3 h in mammalian cells, making it much more responsive in stable cell lines. Luc instability arises from its sensitivity to proteolysis both in vivo and in vitro. Compounds that resemble its natural substrate, luciferin, act as effective competitive inhibitors in vitro. When these compounds (e.g., phenylbenzothiazole) are added to either prokaryotic or eukaryotic cells, more than tenfold increases in Luc activity can be observed. This increased activity results from a lower rate of degradation of the enzyme in vivo and can be mimicked in vitro as phenylbenzothiazole protects Luc from trypsin digestion while it has no effect on the rate of digestion of alkaline phosphatase.