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

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