DNA methylation is an epigenetic mark critical for regulating the chromatin structure and gene transcription. DNA methylation is mainly catalyzed by three DNA methyltransferases (Dnmts) encoded by Dnmt1, Dnmt3α and Dnmt3b. Dnmt1, the maintenance DNA methyltransferase and a major Dnmt in adult cells, plays an important role in cancer progression associated with epigenetic silencing of tumor suppressor genes. Previous work has shown that the mouse Dnmt1 promoter is independently activated by Sp1/Sp3 (25
) and E2F (26
), and the human DNMT1 by signal transducer and activator of transcription-3 (STAT3) (28
) transcription factors. Dnmt1 expression can also be modulated by Rb (41
), AUF1 (42
), BRCA1 (43
) and small non-coding microRNAs miR-152 (44
). In addition to its expression regulation, Dnmt1 protein stability is controlled by SET7 (46
) and LSD1 (47
), and its enzymatic activity is mediated by G9α (48
), EZH2 (49
), PML-RAR (50
) and hNaa10p (51
). A recent study demonstrated that p53 is a negative regulator of the Dnmt1 promoter activity (52
). However, the mechanism of Dnmt1 overexpression in cancers remains largely unknown. The results in this study identified a zinc-mediated activation of Dnmt1 that is modulated by MTF-1 and SHP crosstalk.
An important finding of this study is our establishment of a regulatory link between zinc/MTF-1 and Dnmt1 in HCC. Although there is no report investigating the specific function of MTF-1 in the development of liver cancer, a recent study showed that MTF-1 protein levels were significantly elevated in breast, lung and cervical carcinomas (53
), suggesting a role for MTF-1 in human tumor development and growth. MTF-1 was also proposed as a candidate lymphoma susceptibility gene (54
), and loss of MTF-1 resulted in delayed tumor growth associated with increased matrix collagen deposition and reductions in vasculature density (55
). Both tissue hypoxia and oxidative stress are well documented to be common features of most solid tumors (56
). MTF-1 has been associated with hypoxic-induced placenta growth factor (PIGF) expression, an angiogenic factor expressed in many tumors (57
). Up-regulation of MTF-1 and PIGF occurred in human intrahepatic cholangiocarcinoma due to loss of liver–intestine cadherin, which contributed to tumor differentiation and vascular invasion, and thus poor prognosis (58
Interestingly, we found that the induction of Dnmt1 by MTF-1 requires the presence of zinc. Zinc not only increases MTF-1 expression, as seen by other studies (59
), but more importantly, it also enhances the recruitment of MTF-1 to the Dnmt1 promoter and causes transcriptionally active configuration of the local chromatin. It should be noted that, in the 14 HCC specimens that we analyzed (), the expression of MTF-1 was not significantly altered, and no strong positive correlation between MTF-1 and DNMT1 mRNA was observed (data not shown). This suggests that the disrupted intracellular zinc homeostasis, but not merely changes of MTF-1 mRNA, may be critical in promoting MTF-1-mediated activation of Dnmt1. Although it remains to be determined how zinc metabolism is altered during HCC growth, MTF-1 may play a role by activating Dnmt1 under aberrant metal conditions. Up-regulated Dnmt1 may further silence other tumor suppressors and stimulate HCC progression. It would be interesting to determine in future studies whether hypoxia or oxidative stress contribute to Dnmt1 expression regulation by MTF-1. In this regard, MTF-1 could be a potential therapeutic target that offers the opportunity to manipulate metal or redox homeostasis in tumor cells.
One intriguing observation is the cross-inhibition between SHP and MTF-1. SHP directly represses MTF-1 expression at the transcriptional level. Conversely, induction of MTF-1 by zinc inhibits SHP expression by binding to the SHP promoter and repressing the basal, as well as LRH-1-induced SHP promoter activity. Surprisingly, the basal level of SHP is decreased in MTF-1-deficient MEFs, which is induced by zinc. On the other hand, the induction of MTF-1 target gene MTI, as well as Dnmt1, is observed in MTF-1−/− cells, which is repressed by zinc. The changes in SHP may be responsible for the alterations of Dnmt1. It is postulated that MTF-1 may play a predominant role to control a zinc-dependent activation of Dnmt1 through inhibition of SHP. In the absence of MTF-1, zinc may turn on other zinc responsive genes that function as SHP activators, resulting in the elevation of SHP and reduction of Dnmt1.
Recently we showed that SHP also inhibits Dnmt1 promoter transactivation by ERRγ in several cancer cells (29
). The effect of SHP is through a direct protein–protein interaction with ERRγ to convert the local chromatin structure of the Dnmt1 promoter from a transcriptionally active mode to an inactive mode. Because SHP does not interact with the MTF-1 protein directly, SHP appears to repress Dnmt1 through decreasing MTF-1 expression. The induction of MTF-1 by zinc may activate Dnmt1 by repressing SHP, which represents a feed-forward inhibitory mechanism between SHP and MTF-1 that controls Dnmt1 expression. Thus, SHP modulates the expression of Dnmt1 by at least two distinct mechanisms ().
Figure 7. Schematic showing SHP inhibition of the Dnmt1 promoter through two distinct mechanisms. Our recent study showed that SHP inhibits ERRγ transactivation of the Dnmt1 promoter. The present study identified a zinc-mediated induction of Dnmt1 which (more ...)
In conclusion, we identified a second pathway through which SHP represses Dnmt1. SHP inhibition of Dnmt1 may affect global DNA methylation and alter methylation levels of tumor suppressors. Targeting SHP may prove a useful approach to demethylate and reactivate the silenced tumor suppressors to slow the progression of HCC.