Aberrant Silencing of the CpG Island-Containing Human O⁶-Methylguanine DNA Methyltransferase Gene Is Associated with the Loss of Nucleosome-Like Positioning

Abstract

Tumor-associated aberrant silencing of CpG island-containing genes has been correlated with increased cytosine methylation, a "closed" chromatin structure, and exclusion of transcription factor binding in the CpG island/promoter regions of affected genes. Given the lack of understanding of what constitutes a closed chromatin structure in CpG islands, however, it has been difficult to assess the relationship among cytosine methylation, chromatin structure, and inappropriate gene silencing. In this study, nuclease accessibility analysis was used to more clearly define the chromatin structure in the CpG island of the human O 6 -methylguanine DNA methyltransferase (MGMT) gene. Chromatin structure was then related to in vivo DNA-protein interactions and cytosine methylation status of the MGMT CpG island in human glioma cells varying in MGMT expression. The results of these studies indicated that the "open" chromatin structure associated with the MGMT CpG island in MGMT ؉ cells consisted of an approximately 250-bp transcription factor-binding, nuclease-accessible, nucleosome-free region of DNA, whose formation was associated with at least four flanking, precisely positioned nucleosome-like structures. In MGMT ؊ cells, this precise nucleosomal array was lost and was replaced by randomly positioned nucleosomes (i.e., the closed chromatin structure), regardless of whether methylation of the CpG island was spread over the entire island or limited to regions outside the transcription factor binding region. These results suggest that CpG islands facilitate the expression of housekeeping genes by facilitating nucleosomal positioning and that the conditions that alter the formation of this array (such as perhaps methylation) may indirectly affect CpG island-containing gene expression.

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

“…The strict translational positioning of nucleosomes over the HPRT promoter may act to preferentially expose specific transcription factor binding sites and the multiple transcription initiation sites to facilitate transcriptional activation. This hypothesis is consistent with several studies that indicate that the promoters and enhancers of expressed genes are assembled into translationally positioned nucleosomal arrays in which the nucleosomes over the transcription initiation site are absent or modified (2,33,46,51). In contrast, the randomly positioned nucleosomes on the inactive promoter may serve to obstruct access to critical cis-acting elements by their cognate transcriptional activators and interfere with transcription initiation since transcriptional initiation (but not elongation) is strongly inhibited by nucleosomal assembly (28).…”

Novel Mechanism whereby Nuclear Factor κB Mediates DNA Damage Repair through Regulation of O6-Methylguanine-DNA-Methyltransferase

“…A recent study analyzed time to tumor progression in relation to MGMT promoter methylation in patients with glioblastoma moltiforme and found significantly improved results in patients with methylated MGMT promoter (7)(8)(9). It is conceivable that methylated regions of the MGMT promoter are located in closed nucleosome structures (32), impeding transcription factor access to the promoter (33,34). DNA methylation may affect transcription through this mechanism.…”

Activation of human O6-methylguanine-DNA methyltransferase gene by glucocorticoid hormone

“…The molecular basis for extinction of the MGMT gene is not known. It was recently suggested that altered chromatin organization and nucleosome positioning and aberrant silencing in the CpG island of the promoter region, surrounding of the MGMT gene is linked to Mex 7 phenotype (Patel et al, 1997;Watts et al, 1997). However, the GRE sequences are located outside these CpG island regions and it appears that GREs in the MGMT promoter are involved in activation rather than repression.…”

“…Thus elucidation of the molecular mechanisms controlling MGMT expression may lead to development of drugs that speci®cally inhibit or enhance MGMT expression, and is of signi®cant clinical importance. Earlier studies showed that MGMT expression can be enhanced by a variety of genotoxic agents in rodent and human cell lines (Fritz et al, 1991) and more recently studies have implicated altered chromatin organization and nucleosome positioning and aberrant silencing in the CpG island in the promoter region in regulating the expression of the gene (Patel et al, 1997;Watts et al, 1997). However, the cis-elements present in the promoter or the transacting factors responsible for the variable, cell-speci®c expression of MGMT are not known.…”