Oxidized LDL (ox-LDL) plays a critical role in atherogenesis, including apoptosis. As hypercholesterolemia causes epigenetic changes resulting in long-term phenotypic consequences, we hypothesized that repeated and continuous exposure to ox-LDL may alter the pattern of apoptosis in human umbilical vein endothelial cells (HUVECs). We also analyzed global and promoter-specific methylation of apoptosis-related genes. As expected, ox-LDL evoked a dose-dependent increase in apoptosis in the first passage HUVECs that was completely abrogated by lectin-like ox-LDL receptor (LOX-1)-neutralizing antibody. Ox-LDL-induced apoptosis was associated with upregulation of proapoptotic LOX-1, ANXA5, BAX, and CASP3 and inhibition of antiapoptotic BCL2 and cIAP-1 genes accompanied with reciprocal changes in the methylation of promoter regions of these genes. Subsequent passages of cells displayed attenuated apoptotic response to repeat ox-LDL challenge with blunted gene expression and exaggerated methylation of LOX-1, BAX, ANXA5, and CASP3 genes (all P < 0.05 vs. first exposure to ox-LDL). Treatment of cells with LOX-1 antibody before initial ox-LDL treatment prevented both gene-specific promoter methylation and expression changes and reduction of apoptotic response to repeat ox-LDL challenge. Based on these data, we conclude that exposure of HUVECs to ox-LDL induces epigenetic changes leading to resistance to apoptosis in subsequent generations and that this effect may be related to the LOX-1-mediated increase in DNA methylation.

Differential chromatin structure and accessibility, particularly at the promoter, have long been recognized as characteristics that distinguish active from inactive genes. Active genes are in general more accessible to regulatory factors than inactive genes, as indicated by nuclease sensitivity. In addition, the promoters of active genes often exhibit marked DNase I hypersensitivity, especially in the vicinity of transcription factor binding sites (10,20). This hypersensitivity is postulated to be due to changes in the chromatin architecture of the promoter and may represent nucleosomal remodeling or displacement, stretches of single-stranded DNA, torsionally stressed DNA, or other distortions in chromatin structure arising from factor binding (18,20,62).The functional effect of nucleosomes on transcription initiation is thought to be repressive since in vitro assembly of nucleosomal arrays on DNA templates drastically reduces the capacity of these templates to support basal transcription (25,35,36,57). Furthermore, the differential accessibility and transcriptional potential of chromatin structure in active versus inactive promoters are often associated with differential nucleosomal organization (3,5,23,30,46,62). Thus, remodeling the nucleosomal architecture of a promoter is likely to be an integral feature of mechanisms of gene activation and/or silencing, which may involve histone acetylation and chromatinremodeling complexes such as SWI/SNF (15,20,60).The organization of genomic DNA into nucleosomal arrays is defined by both the translational position of the nucleosome relative to the linear nucleotide sequence and the rotational orientation of the DNA helix relative to the surface of the histone octamer. The translational position of nucleosomes on a DNA template (i.e., the linear position of the nucleosome relative to the DNA sequence [46]) has been shown to affect the accessibility of cis-acting elements in the promoter to various transcription factors as well as the basal transcription complex. Whether a transcription factor binding site is incorporated into a nucleosomal core or is in the linker region between nucleosomes can dramatically affect its accessibility to its cognate binding protein(s) in vitro (25,58). However, the extent to which incorporating a transcription factor binding site into a nucleosomal core reduces the accessibility of that site can vary widely among transcription factors (4, 25). The rotational orientation of the DNA helix wound around a nucleosomal core also affects the accessibility of that DNA to transcription factors (25). For instance, the rotational orientation of the TATA box, the glucocorticoid response element, and the thyroid response element within a nucleosome strongly affects the accessibility of these elements to their cognate binding factors (14,24,59). These findings suggest that both the translational position and rotational orientation of cis-acting

The strong correlation between promoter hypermethylation and gene silencing suggests that promoter methylation represses transcription. To identify methylation sites that may be critical for maintaining repression of the human HPRT gene, we treated human/hamster hybrid cells containing an inactive human X chromosome with the DNA demethylating agent 5-azadeoxycytidine (5aCdr), and we then examined the high resolution methylation pattern of the HPRT promoter in single cell-derived lines. Reactivation of HPRT correlated with complete promoter demethylation. In contrast, the 61 5aCdr-treated clones that failed to reactivate HPRT exhibited sporadic promoter demethylation. However, three specific CpG sites remained methylated in all unreactivated clones, suggesting these sites may be critical for maintaining transcriptional silencing of the HPRT gene. Re-treatment of partially demethylated (and unreactivated) clones with a second round of 5aCdr did not increase the frequency of HPRT reactivation. This is consistent with mechanisms of methylation-mediated repression requiring methylation at specific critical sites and argues against models invoking overall levels or a threshold of promoter methylation. Treatment of cells with the histone deacetylase inhibitor, trichostatin A, failed to reactivate HPRT on the inactive X chromosome, even when the promoter was partially demethylated by 5aCdr treatment, suggesting that transcriptional repression by DNA methylation is unlikely to depend upon a trichostatin A-sensitive histone deacetylase.In mammals, DNA methylation at CpG dinucleotides in the 5Ј region of genes is frequently associated with transcriptional silencing (1), particularly in housekeeping genes on the inactive X chromosome. Numerous studies suggest that this association between promoter hypermethylation and transcriptional repression has a functional basis. For instance, individual loci on the inactive human X chromosome in human/ hamster hybrid cell lines may be reactivated using DNA-demethylating agents such as 5-azacytidine (2, 3) and 5-azadeoxycytidine, which inhibit the maintenance methyltransferase and incorporate into newly synthesized DNA (4), resulting in a failure to maintain methylation patterns during growth. Likewise, in vitro methylation of various promoter constructs results in inhibition of transcription in transient expression assays (5-9). However, despite significant evidence that DNA methylation represses transcription, specific mechanisms of this repression are only now becoming apparent.Recent reports suggest that DNA methylation mediates transcriptional repression indirectly, via binding of the methylated DNA-binding protein MeCP2, which in turn recruits histone deacetylases that modify the local chromatin structure (10, 11). However, additional mechanisms may also act to repress transcription by methylation, such as direct inhibition of transcription factor binding to its cognate site in DNA. Indeed, methylation of the binding sites of several transcription factors has been shown to alter ...

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