Human MGMT protects cells against lethality and mutagenicity of intrinsic and environmental DNA damage by removing adducts from O(6)-guanine and thus is a critical determinant of response to anticancer chloroethylating (e.g. BCNU), and methylating (e.g. temozolomide) drugs, as well as their potential carcinogenicity. Because normal tissues and tumors exhibit a wide spectrum of MGMT expression, while the gene is completely silenced in a subset of tumor, we wish to understand the mechanisms for regulation of MGMT. Having earlier characterized the 5' promoter region of the gene, demonstrating a tight correlation between methylation of """"""""hotspots"""""""" within the 5' -CpG island and MGMT silencing, we now hypothesize (Aim 1) that a methylated CpG-binding protein (e.g. MeCP2) binds the methylated region, followed by recruitment of histone deacetylase (HDAC), whose activity leads to local chromatin compaction and exclusion of transcription factors. We shall test this model by determining the acetylation status of histones H3 and H4 located at the MGMT promoter, in expressing versus silenced cells. We shall examine the effect on histone acetylation and MGMT expression, of inhibiting HDAC with Trichostatin A, or inhibiting methylation with 5 -azadeoxycytidine. Based on our observation that the MGMT 5'enhancer binding protein (MEBP) is excluded from the nucleus of MGMT silenced cells, we now propose to address the hypothesis that MGMT expression requires the MEBP to be localized to the nucleus. We shall initially isolate, identify and characterize the protein (Aim 2) enabling us to generate antibodies with which to monitor MEBP intracellular localization and determine correlation with MGMT expression levels. 3, to isolate the cDNA for MEBP will enable us to generate probes with which to explore MEBP function by modulating its expression, initially in reporter gene studies. Ultimately, armed with the molecular tools for monitoring and modulating promoter methylation and histone-acetylation, as well as MEBP expression and localization, we shall be able to explore the interrelationships between all these parameters. The long-term goal is to test the hypothesis, that MEBP enhancer binding is a primary event in MGMT expression, while methylation and deacetylation at the promoter represent secondary events that stabilize the silenced gene state. If nuclear localization of MEBP is indeed a primary event, regulation of its localization would become a primary focus for future studies. Elucidation of these factors involved in regulation of MGMT expression may provide new markers for susceptibility to environmental carcinogens or of potential resistance to cancer chemotherapy, and may suggest new interventions in these processes.
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