A methylation center within intron 1 of RIL predisposes to silencing, and is opposed by a polymorphic Sp1/Sp3 site

摘要: 2843 Aberrant promoter methylation is a common feature of cancer, yet the underlying mechanisms it are not well understood. We have previously reported that RIL gene one most frequently methylated genes in different cancer types and candidate tumor suppressor by functional assays. In search for causes abnormal RIL, we designed seven pyrosequencing assays around transcription start site (TSS) as exon 1 intron 1. Here show group 19 normal colon samples, region (-306 to +168) shows low degrees (average density, 25.9± 1.9%) strong correlation with age (R=0.62, P=0.006 measured sum Z-scores), whereas (+370 +384 has very high degree tissues 85.35± 0.8%) no (R=-0.05, P=0.844). These data suggested contains putative center RIL. To test this, generated reporter constructs (-589 +516) without (-590 +20) (the center). Transient transfections 3T3 cells showed 3.8 4.0 fold lower levels expression containing center, suggesting presence repressor sequence within Stable transfection resulted sustained expression, intron-containing gradual decline starting from day 37 after transfection, consistent time-dependednt silencing. polymorphic Sp1/Sp3 TSS area associated vivo . Interestingly, among constructs, Sp1+ transgenes were more resistant time-dependent silencing relative Sp1- transgenes, they substantially higher (4.1-fold on 25 23.8-fold 51). Similar obtained RKO cell line. Our results suggest model whereby predisposes its silencing, naturally-occuring insertional acts barrier, preventing cis-acting spreading heterochromatin.