CpG methylation in cis-regulatory elements is generally believed to repress gene expression by disrupting transcription factor (TF)-DNA interactions directly or indirectly via the recruitment of proteins containing methyl-CpG-binding domain (MBD), which are largely sequence independent. However, this dogma is challenged by several recent studies including a recent publication by us. In this study, many TFs, including krüppel-like factor 4 (KLF4), were found to preferentially bind to mCpG-containing motifs and transactivate gene expression. Using a site-specific KLF4 mutant (R458A) that abolishes its binding activity to mCpG, but has no impact on binding to its canonical unmethylated motif, we investigated biological function of mCpG-dependent gene regulation by KLF4 in glioblastoma cells. Our studies showed that KLF4 promotes cell adhesion, migration, and morphological changes, all of which are abolished by R458A mutation. We identified over a 100 genes were directly activated via mCpG-dependent KLF4 binding activity. These targets were associated with multiple pathways including pathways involved in cytoskeletal organization, cell adhesion, cytoskeleton, and extracellular matrix. Genes such as NGEF, UGDH, PHLDB2, LIMS2, LM07 and Rabex-5/RABGEF1involved in migration and cytoskeletal organization were highly induced. We validated these targets by Bisulfite Sequencing, ChIP-PCR, RT-PCR and western blot to confirm that they are indeed KLF4-mCpG direct targets. To understand the biological implication of KLF4-mCpG activity and broaden the current understanding of the role of DNA methylation in transcriptional regulation, we utilized one of the direct KLF4-mCpG targets UGDH to investigate this novel epigenetic regulation. UGDH (UDP-α-D-glucose 6-dehydrogenase) is involved in the biosynthesis of the glycosaminoglycan precursor UDP-α-D-glucuronic. Glycosaminoglycans (GAGs) are one of the major components of the cellular environment. Certain GAGs such as Hyaluronic acid (HA) participate in numerous cellular phenomena, including adhesion, motility, angiogenesis and wound healing. UGDH, although upregulated in GBMs has not been implicated in GBM tumor biology. We show that UGDH is required for KLF4-mCpG dependent increase in migration and UGDH knockdown decreases GBM cell proliferation, migration and the abundance of GAGs. Elevated glycosaminoglycan formation is implicated in a variety of human diseases, including the progression of tumors. The inhibition of synthesis of UDP-α-D-glucuronic acid using UGDH antagonists might therefore be a useful strategy for therapy. Note: This abstract was not presented at the meeting. Citation Format: Olutobi Oyinlade, Jun Wan, Shuang Wei, Jiang Qian, Heng Zhu, Shuli Xia. UGDH is required for KLF4-mCpG dependent increase in GBM cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5884. doi:10.1158/1538-7445.AM2017-5884