An Sp1Sp3 Binding Polymorphism Confers Methylation Protection 英文参考文献.docVIP

AnSp1/Sp3BindingPolymorphismConfersMethylation Protection YanisA.Boumber1,2,YutakaKondo1¤a,XuqiChen1¤b,LanlanShen1,YiGuo1,CarmenTellez1,MarcosR.H. Este′cio1,SairaAhmed1,Jean-PierreJ.Issa1,2* 1DepartmentofLeukemia,M.D.AndersonCancerCenter,UniversityofTexas,Houston,Texas,UnitedStatesofAmerica,2PrograminCancerBiology,GraduateSchoolof BiomedicalSciences,UniversityofTexas,Houston,Texas,UnitedStatesofAmerica Abstract 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).ThecomparisonofexpressionlevelsbetweenunmethylatedlongandshortEBV-transformedcelllinesshowed nodifferenceinexpressioninvivo.Electrophorecticmobilityshiftassayshowedthattheinsertedregionofthelongallele binds Sp1 and Sp3 transcription factors, a binding that is absent in the short allele. Transient transfection of RIL allele- specifictransgenesshowednoeffectsoftheadditionalSp1siteontranscriptionearlyon.However,stabletransfectionof methylation-seeded constructs showed gradually decreasing transcription levels from the short allele with eventual spreadingofdenovomethylation.Incontrast,thelongalleleshowedstablelevelsofexpressionovertimeasmeasuredby luciferaseand,2–3-foldlowerlevelsofmethylationbybisulfitesequencing(P,0.001),suggestingthatthepolymorphic 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 p