Tumor suppressor genes RASSF1A and BLU are two tandem head-to-tail genes located at 3p21.3.Therefore, we hypothesized that there may be a regional effect on their gene expression and promoter methylation status.If not, then there may be an insulator between RASSF1A and BLU genes.We first identified transcriptional important CpG sites using the methylation-specific oligonucleotide microarray in relation to the mRNA expression of RASSF1A and BLU genes in 32 primary lung tumors.We demonstrated that the E2F1 bound to the transcription important CpG sites in RASSF 1A gene and activated RASSF 1A promoter, and this transcriptional regulation was impaired when the targeted CpGs were hypermethylated.Both RASSF1A and BLU genes had their own transcriptional important CpG regions, and methylation of these regions correlated with low mRNA/protein expressions.However, there was no correlation between RASSF1A and BLU protein expressions.Using chromatin immunoprecipitation-PCR, we found that CCCTC-binding factor (CTCF) bound to insulator sequences located between these two genes and may exhibit barrier activity between RASSF1A and BLU promoters.Our study dissects the transcriptional important CpG sites for both RASSF1A and BLU genes and demonstrates that CTCF protein may protect the RASSF1A and BLU genes against the encroachment ofncighboring silencing or activating signals.