title: Computational Modeling Approach in Probing the Effects of Cytosine Methylation on the Transcription Factor Binding to DNA.
abstract: BACKGROUND
  Cytosine methylation at CpG dinucleotides is a chief mechanism in epigenetic modification of gene expression patterns. Previous studies demonstrated that increased CpG methylation of Sp1 sites at -268 and -346 of protein kinase C ε promoter repressed the gene expression.
  
  
  METHODS
  The present study investigated the impact of CpG methylation on the Sp1 binding via molecular modeling and electrophoretic mobility shift assay. Each of the Sp1 sites contain two CpGs. Methylation of either CpG lowered the binding affinity of Sp1, whereas methylation of both CpGs produced a greater decrease in the binding affinity. Computation of van der Waals (VDW) energy of Sp1 in complex with the Sp1 sites demonstrated increased VDW values from one to two sites of CpG methylation. Molecular modeling indicated that single CpG methylation caused underwinding of the DNA fragment, with the phosphate groups at C1, C4 and C5 reoriented from their original positions. Methylation of both CpGs pinched the minor groove and increased the helical twist concomitant with a shallow, hydrophobic major groove. Additionally, double methylation eliminated hydrogen bonds on recognition helix residues located at positions -1 and 1, which were essential for interaction with O6/N7 of G-bases. Bonding from linker residues Arg565, Lys595 and Lys596 were also reduced. Methylation of single or both CpGs significantly affected hydrogen bonding from all three Sp1 DNA binding domains, demonstrating that the consequences of cytosine modification extend beyond the neighboring nucleotides.
  
  
  RESULTS
  The results indicate that cytosine methylation causes subtle structural alterations in Sp1 binding sites consequently resulting in inhibition of side chain interactions critical for specific base recognition and reduction of the binding affinity of Sp1.
authors.affiliation: Department of Chemistry and Biochemistry, California State University, San Bernardino, California 92407, United States.
authors.forename: John
authors.initials: J
authors.lastname: Tenayuca
authors.affiliation: Department of Chemistry and Biochemistry, California State University, San Bernardino, California 92407, United States.
authors.forename: Kimberley
authors.initials: K
authors.lastname: Cousins
authors.affiliation: Department of Chemistry & Biochemistry, California State University, San Bernardino, CA 92407, China.
authors.forename: Shumei
authors.initials: S
authors.lastname: Yang
authors.affiliation: Center for Perinatal Biology, Loma Linda University School of Medicine, Loma Linda, California 92350, United States.
authors.forename: Lubo
authors.initials: L
authors.lastname: Zhang
mesh_terms: D001665:Binding Sites; D003596:Cytosine / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D019175:DNA Methylation; D024202:Electrophoretic Mobility Shift Assay; D006860:Hydrogen Bonding; D008958:Models, Molecular; D011485:Protein Binding; D016329:Sp1 Transcription Factor / Q000737:chemistry / Q000378:metabolism*
chemical_list: D016329:Sp1 Transcription Factor; D003596:Cytosine; D004247:DNA
keywords: DNA methylation; Molecular modeling; Protein-DNA recognition; Sp1; Zinc finger