Abstract 2863: DNA methylation dictates transcription factor binding and gene activation in brain tumor

Abstract

Abstract Dysregulation of DNA methylation is a hallmark in various types of cancers including malignant brain tumors. Although genome-wide methylation profiles (i.e., methylome) in brain tumors are increasingly becoming available, 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 krüppel-like factor 4 (KLF4), preferentially bind to mCpG-containing motifs and thereby transactivate gene expression. The cancer driver KLF4 is up-regulated in high-grade gliomas. We found that in addition to binding to conventional unmethylated DNA motifs, KLF4 bound to mCpG-containing motifs in a sequence-specific manner and thereby transactivated gene expression. We further demonstrated that the dual-binding activities of KLF4 were through distinct domains. Based on the crystal structure of KLF4, we generated a KLF4 mutant (R458A) that no longer bound to methylated DNA motif but retained KLF4 binding to unmethylated motif. This KLF4R458A mutant allowed us to uncouple the molecular and biological effects of KLF4-binding to methylated and unmethylated motifs in biological relevant systems such as glioblastoma (GBM) cells. 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, compared with KLF4-mut expressing cells, KLF4-wt 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 by scratch and transwell assays. 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 were up-regulated by KLF4-wt but not KLF4-mut. Furthermore, for one of these genes, the small GTPase RhoC, ChIP-PCR demonstrated that KLF4 bound to its promoter region via a mCpG-dependent mechanism. Our separate large scale ChIP-sequencing revealed that KLF4 bound to ∼2733 DNA fragments through a methylation-dependent manner in brain tumor cells. These data reveal the novel mechanism by which DNA methylation at CpG sites regulates cancer cell phenotype through transcription factor-dependent gene transactivation. These findings are paradigm shifting and substantially impact our understanding on how CpG methylation influences tumor cell behavior such as migration and invasion. Citation Format: Shuli Xia, Qifeng Song, Jun Wan, Yijing Su, Yaxue Zheng, Hongjun Song, Jiang Qian, John Laterra, Heng Zhu. DNA methylation dictates transcription factor binding and gene activation in brain tumor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2863. doi:10.1158/1538-7445.AM2015-2863