Altered DNA methylation status is associated with human diseases and cancer; however, the underlying molecular mechanisms remain elusive. We previously identified many human transcription factors, including Krüppel-like factor 4 (KLF4), as sequence-specific DNA methylation readers that preferentially recognize methylated CpG (mCpG), here we report the biological function of mCpG-dependent gene regulation by KLF4 in glioblastoma cells. We show that KLF4 promotes cell adhesion, migration, and morphological changes, all of which are abolished by R458A mutation. Surprisingly, 116 genes are directly activated via mCpG-dependent KLF4 binding activity. In-depth mechanistic studies reveal that recruitment of KLF4 to the methylated cis-regulatory elements of these genes result in chromatin remodeling and transcription activation. Our study demonstrates a new paradigm of DNA methylation-mediated gene activation and chromatin remodeling, and provides a general framework to dissect the biological functions of DNA methylation readers and effectors.DOI: http://dx.doi.org/10.7554/eLife.20068.001

UDP-Glucose 6-dehydrogenase (UGDH) produces UDP-α-D-glucuronic acid, the precursors for glycosaminoglycans (GAGs) and proteoglycans (PGs) of the extracellular matrix. Elevated GAG formation has been implicated in a variety of human diseases, including glioblastoma (GBM). In our previous study, we found that krÜppel-like factor 4 (KLF4) promotes GBM cell migration by binding to methylated DNA, mainly methylated CpGs (mCpG) and transactivating gene expression. We identified UDGH as one of the downstream targets of KLF4-mCpG binding activity. In this study, we show that KLF4 upregulates UGDH expression in a mCpG-dependent manner, and UGDH is required for KLF4 induced cell migration in vitro. UGDH knockdown decreases glycosaminoglycan (GAG) abundance in GBM cells, as well as cell proliferation and migration in vitro. In intracranial xenografts, reduced UGDH inhibits tumor growth and migration, accompanied by a decrease in the expression of extracellular matrix proteins such as tenascin C, brevican. Our studies demonstrate a novel DNA methylation-dependent UGDH upregulation by KLF4. Developing UGDH antagonists to decrease the synthesis of extracellular matrix components will be a useful strategy for GBM therapy.

Mutations in the isocitrate dehydrogenase 1 (IDH1) gene have been identified in a number of cancer types, including brain cancer. The Cancer Genome Atlas project has revealed that IDH1 mutations occur in 70-80% of grade II and grade III gliomas. Until recently, most of the functional studies of IDH1 mutations in cellular models have been conducted in overexpression systems with the IDH1 wild type background. In this study, we employed a modified CRISPR/Cas9 genome editing technique called "single base editing", and efficiently introduced heterozygous IDH1 R132H mutation (IDH1) in human astroglial cells. Global DNA methylation profiling revealed hypermethylation as well as hypomethylation induced by IDH1. Global gene expression analysis identified molecular targets and pathways altered by IDH1, including cell proliferation, extracellular matrix (ECM), and cell migration. Our phenotype analysis indicated that compared with IDH1 wild type cells, IDH1 promoted cell migration by upregulating integrin β4 (ITGB4); and significantly inhibited cell proliferation. Using our mutated IDH1 models generated by "single base editing", we identified novel molecular targets of IDH1, namely Yes-associated protein (YAP) and its downstream signaling pathway Notch, to mediate the cell growth-inhibiting effect of IDH1. In summary, the "single base editing" strategy has successfully created heterozygous IDH1 R132H mutation that recapitulates the naturally occurring IDH1 mutation. Our isogenic cellular systems that differ in a single nucleotide in one allele of the IDH1 gene provide a valuable model for novel discoveries of IDH1-driven biological events.