Introduction: 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.
Materials & 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.