In mammalian cells, CpG methylation in a promoter is a primary epigenetic mechanism for silencing genes, and is involved in the control of cellular function and homeostasis. It is therefore reasonable to predict that aberrant methylation of CpG regions within a promoter could manifest in a pathology associated with some chronic diseases. Recent studies have shown that TLR2 promoter hypomethylation is associated with increased expression of TLR2 in CF bronchial epithelial cells. This is consistent with CF airway pathology that typically shows increased proinflammatory responses to TLR2 bacterial ligands [17
]. The present study shows that a specific CpG region of the TLR2 promoter, adjacent to an SP1 binding site, is significantly demethylated, and that this promoter region is both necessary and sufficient to maintain basal TLR2 promoter activity in human epithelial cells. These data suggest that there are CFSMPs (CF-specific methylation patterns) within TLR2 promoter, that might be used as markers to facilitate the discovery of anti-inflammatory CF drugs, as has been the case with anti-tumor drug discovery [20
The studies presented here also sought to clarify the molecular mechanisms underlying demethylation-dependent enhancement of TLR2 gene expression in CF epithelial cells. SP1, but not SP3, appears to play a critical role in maintaining basal TLR2 promoter activation in human epithelial cells. Furthermore, SP1 appears to be involved in regulating DNA demethylation-dependent TLR2 transcriptional activity and expression. It is important to note that the introduction of tri-methylation into the promoter region adjacent to SP1 binding site did not abrogate SP1 binding, and suggests that SP1 binding is not rate limiting in regulating TLR2 mRNA expression. Binding of some additional unknown "X" factors to the tri-methylated probe, regardless of the source of nuclear extract, was indicated in the EMSA assay (Fig. , lanes 10 and 12, denoted by a sharp). This "X" factor band was not observed in the non- or mono-methylated probes (Fig. , lanes 2, 4, 6 and 8), suggesting that the increased methylation of CpG#18-20 in non-CF epithelial cells enhances recruitment of factors that recognize tri-methylated CpGs and suppress SP1-dependent transcription without affecting the SP1 binding (Fig. , left panel). On the other hand, the decreased methylation of CpG#18-20, often observed in CF epithelial cells, might abolish the recruitment and activity of these "X" factors, and allowed SP1-dependent transcription of TLR2 (Fig. , right panel).
Figure 9 A potential model representing the mechanisms underlying hypomethylation-dependent TLR2 promoter activation in human epithelial cells. Enhanced methylation of the CpG#18-20 site within TLR2 promoter observed in non-CF epithelial cells is followed by the (more ...)
The modulation of SP1 binding to its target sequences by DNA methylation is controversial. In the promoters of p21Cip1
, 11-hydroxysteroid dehydrogenase type 2, and GSTP1 (glutathione S-transferase p1), SP1 binding was found to be diminished by DNA methylation [22
]. In contrast, SP1 binding to the claudin-4 (CLDN4) promoter was not influenced by DNA methylation [25
]. It was found that the methyl-CpG-binding domain protein 2 (MBD2) was recruited to CpG sites and silenced the CLDN4 gene in ovarian cancer cell lines without interfering with SP1 binding. The present study clearly indicates that SP1 binding to a TLR2 promoter region was not inhibited by DNA methylation. This is similar to what was observed for the CLDN4 promoter [25
]. Recruitment of methyl binding proteins to the SP1 binding site in the TLR2 promoter needs to be verified. MBD2 and/or the methyl-CpG-binding protein 1 (MeCP1) complex, a large protein complex that includes MBD2 [26
], could be candidate "X" factors that interact with the methylated TLR2 promoter.
It is still unclear how the methylation patterns of the TLR2 promoter are determined in non-CF and CF epithelial cell lines. Whether it is recruitment of factors required for transcriptional activation or the recruitment of factors required for silencing requires further study. The A549 and HeLa, cell lines used as non-CF epithelial cells in this study, are not thought to express CFTR mRNA, yet their TLR2 expression was comparable to that of the 16HBE14o- cells. Therefore, it appears that wt CFTR expression may not be the only factor that would determine the TLR2 gene expression in epithelial cells. However, the processing of ΔF508CFTR might affect the transcription of the TLR2 gene through a mechanism that involves the methylation of DNA and the expression of specific transcriptional regulatory factors. Preliminary studies comparing CF and non-CF cells showed no difference in the expressions of DNA methyl transferase genes, DNmt1, DNmt3a, and DNmt3b [27
], or in the DNA demethylases, MBD2 and Gadd45a [27
], (unpublished data). One possible mechanism could involve the recruitment of factors that releases the methylation-induced transcription block by activating demethylation of the promoter in the nucleus. Clearly, further investigation is required to elucidate these mechnaisms.