Evidence That Silencing of the HPRT Promoter by DNA Methylation Is Mediated by Critical CpG Sites

Abstract

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 ...

Molecular Mechanisms of Gene Silencing Mediated by DNA Methylation

“…Moreover, we were able to change the specific methylated sites, holding the number constant by annealing different amplification products to ssDNA. With this strategy, we verified that the results we obtained do not depend on modification of key sites (11). Figure 3 shows that all regionally methylated templates, with the exception of the construct containing a single methylated CpG, inhibit transcription.…”

“…Using regionally methylated plasmids, parameters such as position, length, or density of methylated cytosines have been reported as crucial for the efficiency of repression (23,24,28). Other authors suggested that transcriptional inhibition relies on methylation at specific critical CpG sites (11,21,56). It is possible that the patch-modified templates used in these experiments, characterized by the modification of CpG-rich prokaryotic vector DNAs, and the analyses performed on different promoters in different cell lines have caused these contradictory results.…”

Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter

“…Conversely, one allele of the HPRT gene also must undergo the reverse remodeling of nucleosomal architecture in the promoter region, from the active nucleosomal organization to an inactive organization, during the process of X chromosome inactivation in female embryogenesis. Little is known about the mechanisms or factors that mediate these changes in nucleosomal organization in vivo, but repression of the HPRT promoter (and, presumably, maintenance of a repressive chromatin conformation) appears to involve methylation of specific critical CpG sites in the promoter region (6).…”

SWI/SNF Chromatin-Remodeling Factors Induce Changes in DNA Methylation to Promote Transcriptional Activation

“…PCR analysis of methylated DNA. Genomic DNA from cells and tissues was extracted, precipitated, and suspended in TE (16). For each sample, 250 ng of genomic DNA were incubated with HpaII, MspI, or HhaI (New England Biolabs) in the recommended buffer or with buffer alone in a total volume of 20 AL for 2 hours at 37jC.…”