CBIO-45DNA METHYLATION DICTATES TRANSCRIPTION FACTOR BINDING AND GENE ACTIVATION IN BRAIN TUMOR

Abstract

Dysregulation of DNA methylation is a hallmark of cancers including malignant brain tumors; yet the functional impact of DNA methylation on tumor malignancy remains largely elusive. Classical dogma states that promoter CpG island methylation (mCpG) influences tumor biology by silencing tumor suppressor genes through interfering with transcription factor (TF)-DNA binding. In this study, we challenge this view by showing that many TFs, including kruppel-like factor 4 (KLF4), preferentially bind to mCpG-containing motifs, and have the ability to activate gene expression. KLF4 is a cancer driver gene up-regulated in high-grade gliomas. We found that KLF4 can bind to mCpG-containing motifs in a sequence-specific manner and activate gene expression. We generated a KLF4 mutant (R458A) that no longer bound to methylated DNA motif but retained KLF4 binding to unmethylated motif. We engineered GBM cell lines to express either doxycycline (Dox)-inducible KLF4 wild type (KLF4-wt) or Dox-inducible KLF4R458A mutant (KLF4-mut) transgenes. Neither KLF4-wt nor KLF4-mut had appreciable effect on cell proliferation and cell viability. However, KLF4-wt but not KLF4-mut induced noticeable morphology changes characterized by elongated, spindle-like phenotypes. We further showed that KLF4-wt but not KLF4-mut promoted GBM cell migration and adhesion. This cell response was partially reversed by the DNA methytransferase inhibitor 5-aza-2′-deoxycytidine (5-Aza), confirming by alternate methods a DNA methylation-dependent mechanism. We employed cell-motility q-PCR arrays and found that ∼10 genes involved in the cell motility pathway, including the small GTPase RhoC, were up-regulated by KLF4-wt but not KLF4-mut. ChIP-PCR demonstrated that KLF4 bound to its promoter region of RhoC via a mCpG-dependent mechanism. These data reveal the novel mechanism by which DNA methylation at CpG sites regulates cancer cell phenotype through transcription factor-dependent gene transactivation. Our studies will be paradigm shifting and will substantially impact our understanding on how CpG methylation influences tumor cell behavior such as migration and invasion.