For methylation and histone deacetylation, respectively, and they have been widely used for investigating epigenetic alteration of many tumor suppressor genes. These inhibitors usually cause global changes in gene expression by remodeling chromatin via directly converting methylated DNA to unmethylated DNA or unacetylated histones to the acetylated state, thereby allowing easy access of the transcription machinery to gene promoters. However, some inhibitors might be doing more, and their anti-cancer properties could be much more complicated. For instance, many non-histone cellular proteins such as transcription factors are also substrates of HDAC, and their transcriptional activities could be affected by the HDAC inhibitor TSA as well. Most tumor suppressor genes are epigenetically silenced by either DNA methylation and/or histone EX 527 HDAC inhibitor deacetylation in their promoters. To our knowledge, there is no report showing that the expression of such genes can be differentially regulated by inhibitors of methylation or histone deacetylation in a cancerspecific fashion without having epigenetic modifications in the promoter. The regulation of MIG-6 by these inhibitors, as we show here, unveils a novel mechanism by which a tumor suppressor gene can be epigenetically silenced in an indirect and tissuespecific manner. Our luciferase reporter assay results indicated that the regulation of MIG-6 expression in melanoma and in lung cancer was most likely mediated by different factors. We have identified a minimal TSA response NVP-BEZ235 element in exon 1 of MIG-6 proximal to its promoter, while location of the 5aza-dC response element is still uncertain. We speculate that the TSA response element in the MIG-6 gene is most likely regulated by a factor whose expression is affected by histone deacetylation in its promoter or whose protein activity is 
directly regulated by acetylation/deacetylation. This factor would be activated in lung cancer cells upon TSA treatment, but not in melanoma cells. Within the minimal TSAresponse element that we identified in MIG-6 gene exon 1, there are putative DNA binding sequences for the transcription factor activator protein-2, which has five family members and binds to the consensus sequence 59GCCNNNGGC-39. When the putative TFAP2 binding sites were mutated, we observed a significant drop in TSAresponsiveness, indicating that those sequences are crucial for TSA-mediated regulation. It will be interesting to see if TFAP2 or other factor binds to those sequences and regulates MIG-6 gene expression. As for 5-aza-dC, its response element is likely outside the tested 1.383-kb MIG-6 promoter regulatory region; that is, it is either directly affected by methylation in its DNA sequences or is indirectly mediated by another transcriptional regulator whose promoter is modified by methylation in melanoma cells. Extensive studies will be required to determine what those factors are and how they control MIG-6 expression. Cancer-type regulation of gene expression by inhibitors of methylation and histone deacetylation is not unique to MIG-6. Other genes such as EGR1 are also differentially regulated in lung cancer and melanoma cells by those inhibitors. It remains to be determined whether-like the MIG-6 promoter-the EGR1 promoter is neither hypermethylated nor affected by histone deacetylation in those cells. If these characteristics are the same in the two promoters, it will be interesting to see if they are regulated by same factor or via different mechanisms. We report here that MIG-6 expression is differentially regulated by inhibitors of methylation and histone deacetylation in lung cancer and melanoma cells without physical epigenetic alterations in its promoter.