The goal is to explore the role of epigenetic modification in DNA in aberrant silencing or re-expression of genes in melanoma that can serve as markers for early detection, assessment, and prognosis. Epigenetic alteration in chromatin contributes to differential expression of imprinted, X-inactivated, cell-specific, and cancer genes. It involves methylation/demethylation of cytosine at cytosine guanine pair (CpG) rich islands in promoter regions and postranscriptional modification (methylation/acetylation) of histones. The proposal is based on information derived from our global differential gene expression studies comparing normal human melanocytes to melanoma cells. We will focus on Rab33A as a model system, because this gene is expressed specifically in melanocytes and its transcripts are uniformly downregulated in advance melanoma cells. Rab33A is located on the X-chromosome and our studies will determine if aberrant silencing in melanomas recapitulates the cell-type specific inactivation process. We have preliminary data that support the notion that Rab33A expression inversely correlates with DNA methylation of specific CpG dinucleotides in the promoter region.
In Aim 1 we will identify patterns of DNA methylation associated with Rab33 A expression/silencing in normal melanocytes, non-melanocytic normal skin cells and melanoma cells. We will assess CpG methylation in Rab33A CpG rich promoter region and Rab33A expression employing the bisulfite converted genomic DNA PCR and RT-PCR, respectively.
In Aim 2 we will evaluate the utility of Rab33A suppression and DNA methylation as markers for malignant transformation and progression in a cohort of melanocytic lesions. Toward this end, antibodies to Rab33A will be raised and used to probe tissue microarrays composed of benign, premalignant and malignant melanocytic lesions. These studies will address the clinical and biological implications of Rab33a suppression and promoter methylation as well as a basic biological question, i.e., the molecular basis for epigenetic cell- and cancer specific gene inactivation on the X-chromosome. They will serve as the basis for similar evaluations of other genes whose aberrant expression/suppression in melanoma is likely to be regulated by epigenetic changes in chromatin. Altogether, the results are likely to generate a novel marker(s) that can be used to assess propensity for malignant transformation, tumor progression and/or sensitivity to chemotherapeutic drugs that target chromatin conformation.
| Cheng, Elaine; Trombetta, Sergio E; Kovacs, Daniela et al. (2006) Rab33A: characterization, expression, and suppression by epigenetic modification. J Invest Dermatol 126:2257-71 |
