An Sp1/Sp3 Binding Polymorphism Confers Methylation Protection

Hundreds of genes show aberrant DNA hypermethylation in cancer, yet little is known about the causes of this hypermethylation. We identified RIL as a frequent methylation target in cancer. In search for factors that influence RIL hypermethylation, we found a 12-bp polymorphic sequence around its transcription start site that creates a long allele. Pyrosequencing of homozygous tumors revealed a 2.1-fold higher methylation for the short alleles (P<0.001). Bisulfite sequencing of cancers heterozygous for RIL showed that the short alleles are 3.1-fold more methylated than the long (P<0.001). The comparison of expression levels between unmethylated long and short EBV-transformed cell lines showed no difference in expression in vivo. Electrophorectic mobility shift assay showed that the inserted region of the long allele binds Sp1 and Sp3 transcription factors, a binding that is absent in the short allele. Transient transfection of RIL allele-specific transgenes showed no effects of the additional Sp1 site on transcription early on. However, stable transfection of methylation-seeded constructs showed gradually decreasing transcription levels from the short allele with eventual spreading of de novo methylation. In contrast, the long allele showed stable levels of expression over time as measured by luciferase and ∼2–3-fold lower levels of methylation by bisulfite sequencing (P<0.001), suggesting that the polymorphic Sp1 site protects against time-dependent silencing. Our finding demonstrates that, in some genes, hypermethylation in cancer is dictated by protein-DNA interactions at the promoters and provides a novel mechanism by which genetic polymorphisms can influence an epigenetic state.

[1]  Shuji Ogino,et al.  MGMT germline polymorphism is associated with somatic MGMT promoter methylation and gene silencing in colorectal cancer. , 2007, Carcinogenesis.

[2]  E. Estey,et al.  RIL, a LIM gene on 5q31, is silenced by methylation in cancer and sensitizes cancer cells to apoptosis. , 2007, Cancer research.

[3]  S. Leung,et al.  Heritable germline epimutation of MSH2 in a family with hereditary nonpolyposis colorectal cancer , 2006, Nature Genetics.

[4]  J. Issa.  CpG island methylator phenotype in cancer , 2004, Nature Reviews Cancer.

[5]  Victor X Jin,et al.  Loss of Estrogen Receptor Signaling Triggers Epigenetic Silencing of Downstream Targets in Breast Cancer , 2004, Cancer Research.

[6]  S. Clark,et al.  Transcriptional Gene Silencing Promotes DNA Hypermethylation through a Sequential Change in Chromatin Modifications in Cancer Cells , 2004, Cancer Research.

[7]  K A Baggerly,et al.  Sensitive and quantitative universal Pyrosequencing methylation analysis of CpG sites. , 2003, BioTechniques.

[8]  Ulrich K Laemmli,et al.  Structural and dynamic functions establish chromatin domains. , 2003, Molecular cell.

[9]  Susan J Clark,et al.  DNA methylation and gene silencing in cancer: which is the guilty party? , 2002, Oncogene.

[10]  M. Turker.  Gene silencing in mammalian cells and the spread of DNA methylation , 2002, Oncogene.

[11]  S. Clark,et al.  Hypermethylation trigger of the glutathione-S-transferase gene (GSTP1) in prostate cancer cells , 2002, Oncogene.

[12]  S. Baylin,et al.  Identification of differentially methylated sequences in colorectal cancer by methylated CpG island amplification. , 1999, Cancer research.

[13]  J. Simpson,et al.  The primary function of a redundant Sp1 binding site in the mouse aprt gene promoter is to block epigenetic gene inactivation. , 1998, Nucleic acids research.

[14]  M. Markelov,et al.  The human RIL gene: mapping to human chromosome 5q31.1, genomic organization and alternative transcripts. , 1998, Gene.

[15]  V. Sukhatme,et al.  Sp1 Is a Critical Regulator of the Wilms' tumor-1 Gene* , 1997, The Journal of Biological Chemistry.

[16]  A. Aguzzi,et al.  Expression of ril, a novel LIM domain gene, is down-regulated in Hras-transformed cells and restored in phenotypic revertants. , 1995, Oncogene.

[17]  A. Bird,et al.  Sp1 sites in the mouse aprt gene promoter are required to prevent methylation of the CpG island. , 1994, Genes & development.

[18]  Z. Siegfried,et al.  Spl elements protect a CpG island from de novo methylation , 1994, Nature.

[19]  S. Clark,et al.  High sensitivity mapping of methylated cytosines. , 1994, Nucleic acids research.