Tenayuca John, Cousins Kimberley, Yang Shumei, Zhang Lubo
Department of Chemistry and Biochemistry, California State University, San Bernardino, California 92407, United States.
Department of Chemistry & Biochemistry, California State University, San Bernardino, CA 92407, China.
Curr Top Med Chem. 2017;17(15):1778-1787. doi: 10.2174/1568026617666161116142031.
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.
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.
CpG二核苷酸处的胞嘧啶甲基化是基因表达模式表观遗传修饰的主要机制。先前的研究表明，蛋白激酶Cε启动子-268和-346处Sp1位点的CpG甲基化增加会抑制基因表达。
本研究通过分子建模和电泳迁移率变动分析研究了CpG甲基化对Sp1结合的影响。每个Sp1位点包含两个CpG。任一CpG的甲基化都会降低Sp1的结合亲和力，而两个CpG均甲基化则会使结合亲和力有更大程度的降低。计算Sp1与Sp1位点复合物的范德华（VDW）能量表明，从一个到两个CpG甲基化位点，VDW值增加。分子建模表明，单个CpG甲基化会导致DNA片段解旋，C1、C4和C5处的磷酸基团从其原始位置重新定向。两个CpG均甲基化会挤压小沟并增加螺旋扭曲，同时伴随浅的、疏水的大沟。此外，双重甲基化消除了位于-1和1位置的识别螺旋残基上的氢键，这些氢键对于与G碱基的O6/N7相互作用至关重要。连接残基Arg565、Lys595和Lys596的键合也减少。单个或两个CpG甲基化均显著影响来自所有三个Sp1 DNA结合结构域的氢键，表明胞嘧啶修饰的后果超出了相邻核苷酸。
结果表明，胞嘧啶甲基化会导致Sp1结合位点发生细微的结构改变，从而导致对特定碱基识别至关重要的侧链相互作用受到抑制，并降低Sp1的结合亲和力。
