Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
PLoS One. 2010 Sep 20;5(9):e12847. doi: 10.1371/journal.pone.0012847.
Tumor suppressor gene (TSG) RASSF1A and candidate TSG BLU are two tandem head-to-tail genes located at 3p21.3. We hypothesized that there may be a concordance on their gene expression and promoter methylation status. If not, then there may be an insulator located between RASSF1A and BLU genes that provides a barrier activity.
METHODOLOGY/PRINCIPAL FINDINGS: We first identified potential transcriptionally important CpG sites using the methylation-specific oligonucleotide array in relation to mRNA expression of RASSF1A and BLU genes in primary lung tumors. We demonstrated that E2F1 bound to the potential transcriptionally important CpG sites in RASSF1A gene of a normal lung cell line expressing RASSF1A transcripts, whereas loss of E2F1 binding to RASSF1A in A549 cancer cell line was the result of DNA methylation. Both RASSF1A and BLU genes had their own potential transcriptionally important CpG regions. However, there was no correlation of methylation status between RASSF1A and BLU. Using gel shift assay and chromatin immunoprecipitation-PCR (ChIP-PCR), we found that CCCTC-binding factor (CTCF) bound to insulator sequences located between these two genes. Bisulfite sequencing and ChIP-PCR revealed distinct methylation and chromatin boundaries separated by the CTCF binding domains in normal cells, whereas such distinct epigenetic domains were not observed in cancer cells. Note that demethylation reagent and histone deacetylase inhibitor treatments led to CTCF binding and recovery of barrier effect for RASSF1A and BLU genes in cancer cells.
CONCLUSIONS/SIGNIFICANCE: Our study dissects the potential transcriptionally important CpG sites for RASSF1A and BLU genes at the sequence level and demonstrates that CTCF binding to the insulator of BLU gene provides a barrier activity within separate epigenetic domains of the juxtaposed BLU and RASSF1A loci in the 3p21.3 gene cluster region.
肿瘤抑制基因（TSG）RASSF1A 和候选 TSG BLU 是位于 3p21.3 的两个串联头尾基因。我们假设它们的基因表达和启动子甲基化状态可能存在一致性。如果没有，那么在 RASSF1A 和 BLU 基因之间可能存在一个绝缘子，提供屏障活性。
方法/主要发现：我们首先使用甲基化特异性寡核苷酸阵列，确定了与原发性肺肿瘤中 RASSF1A 和 BLU 基因的 mRNA 表达相关的潜在转录重要的 CpG 位点。我们证明，E2F1 结合到正常肺细胞系中 RASSF1A 基因的潜在转录重要的 CpG 位点，而 A549 癌细胞系中 E2F1 结合到 RASSF1A 的缺失是 DNA 甲基化的结果。RASSF1A 和 BLU 基因都有自己的潜在转录重要的 CpG 区域。然而，RASSF1A 和 BLU 之间的甲基化状态没有相关性。通过凝胶迁移分析和染色质免疫沉淀-PCR（ChIP-PCR），我们发现 CCCTC 结合因子（CTCF）结合到这两个基因之间的绝缘子序列上。亚硫酸氢盐测序和 ChIP-PCR 显示，在正常细胞中，CTCF 结合的绝缘子序列分隔了这两个基因的不同甲基化和染色质边界，而在癌细胞中则没有观察到这种明显的表观遗传边界。值得注意的是，去甲基化试剂和组蛋白去乙酰化酶抑制剂处理导致 RASSF1A 和 BLU 基因在癌细胞中结合 CTCF，并恢复了屏障效应。
结论/意义：我们的研究在序列水平上剖析了 RASSF1A 和 BLU 基因的潜在转录重要的 CpG 位点，并证明了 CTCF 结合 BLU 基因的绝缘子在 3p21.3 基因簇区域相邻的 BLU 和 RASSF1A 基因座的独立表观遗传域内提供了屏障活性。
