CpG islands frequently contain gene promoters or exons and are usually unmethylated in normal cells. Methylation of CpG islands is associated with delayed replication, condensed chromatin and inhibition of transcription initiation. The investigation of aberrant CpG-island methylation in human cancer has primarily taken a candidate gene approach, and has focused on less than 15 of the estimated 45,000 CpG islands in the genome. Here we report a global analysis of the methylation status of 1,184 unselected CpG islands in each of 98 primary human tumours using restriction landmark genomic scanning (RLGS). We estimate that an average of 600 CpG islands (range of 0 to 4,500) of the 45,000 in the genome were aberrantly methylated in the tumours, including early stage tumours. We identified patterns of CpG-island methylation that were shared within each tumour type, together with patterns and targets that displayed distinct tumour-type specificity. The expression of many of these genes was reactivated by experimental demethylation in cultured tumour cells. Thus, the methylation of particular subsets of CpG islands may have consequences for specific tumour types.

The current paper demonstrates that cholesterol and its hydroxylated derivative, 25-hydroxycholesterol (25-HC), inhibit cholesterol synthesis by two different mechanisms, both involving the proteins that control sterol regulatory element-binding proteins (SREBPs), membrane-bound transcription factors that activate genes encoding enzymes of lipid synthesis. Using methyl-␤-cyclodextrin as a delivery vehicle, we show that cholesterol enters cultured Chinese hamster ovary cells and elicits a conformational change in SREBP cleavage-activating protein (SCAP), as revealed by the appearance of a new fragment in tryptic digests. This change causes SCAP to bind to Insigs, which are endoplasmic reticulum retention proteins that abrogate movement of the SCAP⅐SREBP complex to the Golgi apparatus where SREBPs are normally processed to their active forms. Direct binding of cholesterol to SCAP in intact cells was demonstrated by showing that a photoactivated derivative of cholesterol cross-links to the membrane domain of SCAP. The inhibitory actions of cholesterol do not require the isooctyl side chain or the ⌬5-double bond of cholesterol, but they do require the 3␤-hydroxyl group. 25-HC is more potent than cholesterol in eliciting SCAP binding to Insigs, but 25-HC does not cause a detectable conformational change in SCAP. Moreover, a photoactivated derivative of 25-HC does not cross-link to SCAP. These data imply that cholesterol interacts with SCAP directly by inducing it to bind to Insigs, whereas 25-HC works indirectly through a putative 25-HC sensor protein that elicits SCAP-Insig binding.Nearly 30 years ago, during early studies of feedback inhibition of cholesterol synthesis in cultured cells, it was noted that oxygenated sterols such as 25-hydroxycholesterol were more than 50-fold more potent than cholesterol in reducing the activity of 3-hydroxy-3-methylglutaryl-CoA reductase, the rate-controlling enzyme in cholesterol biosynthesis (1-4). These experiments were conducted by dissolving sterols in ethanol and adding them to protein-containing aqueous culture media in which cholesterol forms an amorphous suspension and thus has poor access to the interior of the cell. When cholesterol was delivered to cells in low density lipoprotein (LDL), 1 a physiologic carrier that enters cells through LDL receptors, the ability of cholesterol to suppress 3-hydroxy-3-methylglutaryl-CoA reductase was enhanced (5). Later, when methods were devised to reconstitute LDL with sterol esters, it was observed that 25-hydroxycholesterol was only about 5-fold more potent than cholesterol when both sterol esters were reconstituted into LDL and delivered through LDL receptors (6). The question of whether cholesterol itself is a regulator or whether it must be converted to an oxygenated metabolite, like 25-hydroxycholesterol, remained unresolved (7). In view of this ambiguity, studies of feedback regulation in our laboratory have generally used a mixture of cholesterol and 25-hydroxycholesterol in a 10:1 molar ratio added in ethanol.In recent years...

Sterol accumulation in membranes blocks the exit of SCAP from the ER, preventing SREBP cleavage and reducing cholesterol synthesis. Sterols act through SCAP's sterol-sensing domain by an obscure mechanism. Here, we show that addition of cholesterol to ER membranes in vitro causes a conformational change in SCAP, detected by the unmasking of closely spaced trypsin cleavage sites. Two mutant forms of SCAP (Y298C and D443N) that are refractory to sterol regulation in vivo are also refractory to sterol-induced conformational change in vitro. 25-hydroxycholesterol, a potent regulator of SCAP in vivo, fails to change SCAP's conformation in vitro, suggesting that oxysterols act in intact cells by translocating cholesterol from plasma membrane to ER. These studies demonstrate an in vitro effect of cholesterol on the sterol regulatory machinery.

Proteasomes mediate the regulated degradation of Insig-1, a membrane protein of the endoplasmic reticulum (ER) that plays a crucial role in lipid metabolism. We showed previously that sterols inhibit this degradation by blocking ubiquitination of Insig-1. Here we show that unsaturated fatty acids stabilize Insig-1 without affecting its ubiquitination. Instead unsaturated fatty acids inhibit extraction of ubiquitinated Insig-1 from membranes, a process known to be mediated by valosin-containing protein and necessary for ER-associated degradation. Valosin-containing protein is recruited to Insig-1 through the action of another protein, Ubxd8. Unsaturated fatty acids block the binding between Ubxd8 and Insig-1, thereby abrogating the membrane extraction of Insig-1. Unsaturated fatty acidmediated stabilization of Insig-1 enhances the ability of sterols to inhibit proteolytic activation of SREBP-1, which activates transcription of genes involved in fatty acid synthesis. The current study provides a molecular mechanism for regulation of proteasome-mediated ER protein degradation at a postubiquitination step.

Insig-1 is an intrinsic protein of the endoplasmic reticulum (ER) that regulates the proteolytic processing of membrane-bound sterol regulatory element-binding proteins (SREBPs), transcription factors that activate the synthesis of cholesterol and fatty acids in mammalian cells. When cellular levels of sterols rise, Insig-1 binds to the membranous sterol-sensing domain of SREBP cleavage-activating protein (SCAP), retaining the SCAP/SREBP complex in the ER and preventing it from moving to the Golgi for proteolytic processing. Under conditions of sterol excess, Insig-1 also binds to the ER enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, facilitating its ubiquitination and proteasomal degradation. Here, we use protease protection, glycosylation site mapping, and cysteine derivitization to define the topology of the 277-amino acid human Insig-1. The data indicate that short segments at the N and C termini of Insig-1 face the cytosol. Most of the protein is buried within the membrane, forming six transmembrane segments separated by five short luminal and cytosolic loops that range from ϳ5 to 16 amino acids. The membranous nature of Insig-1 is consistent with its sterol-dependent binding to hydrophobic sterolsensing domains in SCAP and HMG CoA reductase.