4506

Many genes in the human genome are associated with CpG-dense DNA known as CpG “islands” at their 5’ ends. Aberrant methylation of CpG sites within such islands is associated with inappropriate gene silencing and has been implicated in the inactivation of tumor suppressor genes in human cancers. However, the mechanism responsible for eliciting these changes has yet to be elucidated. Our lab has identified a CpG island associated gene called TMS1 (Target of methylation-induced silencing-1) that is subject to epigenetic silencing in 40% of primary breast tumors and other cancers. The CpG island of TMS1 in normal somatic cells contains three DNaseI hypersensitive sites (HS) with two forming at the boundary between unmethylated and densely methylated flanking DNA. Aberrant methylation of TMS1 CpG island in cancer cells is accompanied by the loss of CpG island-specific DNaseI HS sites and gene silencing, indicating that local alterations in chromatin structure play an important role in methylation-dependent silencing. We identified a methylation-sensitive complex that bound to HS2 located in the first intron of TMS1. Using DNA affinity chromatography we identified one component of the complex as GABPα, a heterotetrameric ets family transcription factor. Interestingly, the interaction of GABP with its recognition sequence has been shown to be blocked by DNA methylation. It is our hypothesis that the GABP complex plays a role in maintaining the unmethylated, open chromatin structure across the CpG island domain of TMS1 in normal cells, either through direct effects on TMS1 transcription or by recruiting factors that mark the CpG island domain with a “permissive” histone code. To this end, we find that the GABP complex binds to a 55bp fragment of HS2 containing tandem ets transcription factor binding sites separated by 29bp. Methylation of a CpG in either ets site blocks GABP complex binding in vitro. Preliminary studies using chromatin immunoprecipitation show that the binding of GABP to the endogenous TMS1 locus is also sensitive to the methylation status of the locus in that HS2 is occupied by GABPα in cells that are unmethylated and express TMS1, but devoid of GABP binding in cells in which TMS1 is methylated and silent. We further examined the impact of HS2/GABP on TMS1 promoter activity through the use of luciferase assays. Inclusion of the HS2 element in the sense or anti-sense orientation resulted in a 2-3 fold increase in transcriptional activity from the TMS1 promoter in a normal fibroblast cell line and a transformed breast cancer cell line. Initial siRNA experiments indicate that transient knockdown of GABPα leads to a corresponding decrease in TMS1 expression. Taken together, these data support a role for the GABP complex and HS2 in the regulation of TMS1 expression. Spurious methylation of the GABP binding sites might be one mechanism contributing to the silencing of TMS1 in human cancer cells. Supported by NIH grants GM078787 and CA077337.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA