Aberrant hypermethylation of gene promoters is a major mechanism associated with inactivation of tumor suppressor genes (TSGs) in cancer, We have previously shown that the methyl-CpG targeted transcriptional activation (MeTA) that allows re-expression of TSGs in human cancer cells is accomplished by combining a methyl-CpG binding domain (MBD) with a NF kappa B transcriptional activation domain (AD), accompanied by histone H3K9/K14 acetylation, Herein we demonstrate that p300 histone acetyltransferase (HAT), one of the NF kappa B (AD)-associated coactivators, reactivates epigenetically silenced MLH1 in 293T cells. Interestingly, the HAT domain of p300 is not essential for the reactivation of MLH1; instead, the C-terminal transactivation domain (C-TAD) but not the N-terminal one (N-TAD) reactivates MLH1. Furthermore, all ten of the cancer-related genes analyzed in three types of cancer cells were reactivated by the effect of p300 linked to MBD. These results demonstrate that it is possible to reactivate epigenetically silenced TSGs in human cancer cells by direct targeting of a transcriptional coactivator at highly methylated promoters. All rights reserved (C) 2009 Elsevier Inc. All rights reserved.