Abstract
Epigenetic silencing involving the aberrant DNA methylation of promoter-associated CpG islands is one mechanism leading to the inactivation of tumor suppressor genes in human cancers. However, the molecular mechanisms underlying this event remains poorly understood. TMS1/ASC is a novel proapoptotic signaling factor that is subject to epigenetic silencing in human breast and other cancers. The TMS1 promoter is embedded within a CpG island that is unmethylated in normal cells and is spanned by three DNase I-hypersensitive sites (HS). Silencing of TMS1 in cancer cells is accompanied by local alterations in histone modification, remodeling of the HS, and hypermethylation of DNA. In this study, we probed the functional significance of the CpG island-specific HS. We identified a methylation-sensitive complex that bound a 55-bp intronic element corresponding to HS2. Affinity chromatography and mass spectrometry identified a component of this complex to be the GA-binding protein (GABP) alpha. Supershift analysis indicated that the GABPalpha binding partner, GABPbeta1, was also present in the complex. The HS2 element conferred a 3-fold enhancement in TMS1 promoter activity, which was dependent on both intact tandem ets binding sites and the presence of GABPalpha/beta1 in trans. GABPalpha was selectively enriched at HS2 in human cells, and its occupancy was inversely correlated with CpG island methylation. Down-regulation of GABPalpha led to a concomitant decrease in TMS1 expression. These data indicate that the intronic HS2 element acts in cis to maintain transcriptional competency at the TMS1 locus and that this activity is mediated by the ets transcription factor, GABPalpha.
Highlights
Methylation of DNA in the human genome is tightly controlled during development by the action of DNA methyltransferases
HS1 and HS3 demarcate the boundaries between the unmethylated CpG island and heavily methylated flanking DNA, whereas HS2 resides in the center of the CpG island, near the exon 1/intron 1 interface (Fig. 1A)
We hypothesized that there may be DNA-binding proteins defined by the hypersensitive sites (HS) sites that protect the CpG island from methylation in normal cells, and that loss of function at these sites might contribute to aberrant methylation, changes in chromatin structure, and gene silencing
Summary
Preparation of Nuclear Extracts—Cells were washed in 1ϫ phosphate-buffered saline and resuspended in 20 packed cell volumes of ice-cold 0.67ϫ phosphate-buffered saline, swollen on ice 10 min, pelleted, and resuspended in 5 packed cell volumes of ice-cold hypotonic buffer A (10 mM HEPES, pH 7.9, 1 mM MgCl2, 10 mM NaCl, 0.2 mM phenylmethylsulfonyl fluoride, 0.5 mM DTT, 0.7 g/ml pepstatin, 0.5 g/ml leupeptin). Binding reactions (20 l) were performed at room temperature for 30 min and contained nuclear extract (5–10 g) plus 1.5 g of poly(dI-dC) and ϳ20 fmol of end-labeled double-stranded DNA (ϳ50,000 cpm) in 1ϫ Binding Buffer (50 mM NaCl, 20 mM HEPES, pH 7.9, 12% glycerol, 0.2 mM EDTA, 1 mM DTT). Nuclear extract (207 mg) was prepared as described above and applied to a 50-ml SP-Sepharose Fast Flow (17-0729-01, Amersham Biosciences) cation exchange column in 1ϫ binding buffer (20 mM HEPES, pH 7.9, 20% glycerol, 50 mM NaCl, 0.2 mM EDTA, 1 mM DTT, 0.5 g/ml leupeptin, 0.5 g/ml pepstatin). For experiments incorporating GABP␣/ siRNA, MCF7 cells (5 ϫ 104) were seeded in 24-well plates and transfected the following day with 200 ng of reporter plasmids and 40 ng of pRL-TK control plus 10 pmol each of GABP␣ and GABP siRNA (Invitrogen StealthTM Select, supplemental Table S1) or 20 pmol of scrambled siRNA using 1.5 l of Lipofectamine 2000. At 24 h postinfection, cells were placed under puromycin selection (0.5 g/ml) and harvested 5 days later for immunoblot analysis
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