Abstract
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.
Highlights
DNA methylation at the five position of the cytosine base (5mC) is the primary epigenetic modification on the mammalian genomic DNA (Jaenisch and Bird, 2003), and dysregulation of DNA methylation is a hallmark of various diseases and cancer (Sharma et al, 2010)
Western blot analysis and immunocytochemistry staining confirmed that the endogenous Kruppel-like factor 4 (KLF4) level in non-transfected U87 cells was barely detectable; after 48 hr Dox induction, both KLF4 wild type (WT) and R458A proteins showed a dose-dependent increase without an impact on cell proliferation (Figure 1A, Figure 1—figure supplement 1A,B)
Immunofluorescence staining for F-actin and vinculin further confirmed the formation of stress actin fibers and focal adhesion, respectively, in KLF4 WT-expressing GBM cells, but not in R458A-expressing cells (Figure 2C)
Summary
DNA methylation at the five position of the cytosine base (5mC) is the primary epigenetic modification on the mammalian genomic DNA (Jaenisch and Bird, 2003), and dysregulation of DNA methylation is a hallmark of various diseases and cancer (Sharma et al, 2010). CpG methylation in cisregulatory elements is generally believed to repress gene expression by disrupting transcription factor (TF)-DNA interactions directly or indirectly via the recruitment of proteins containing methylCpG-binding domain (MBD), which are largely sequence independent (Boyes and Bird, 1991) This dogma has been challenged by several recent studies in which many TFs were identified as a new class of sequence-specific methylated DNA readers (Filion et al, 2006; Mann et al, 2013; Rishi et al, 2010; Sasai et al, 2010; Serra et al, 2014; Spruijt et al, 2013; Zhu et al, 2016).
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