KRUPPLE LIKE FACTOR 4 (KLF4) BINDING TO METHYLATED DNA UNCOVERS NOVEL REGULATORY MECHANISM IN GLIOBLASTOMA MIGRATION
Embargo until
2020-05-01
Date
2017-11-06
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Johns Hopkins University
Abstract
Protein microarray technology provides a versatile platform for characterization of hundreds to thousands of proteins in a parallel and high-throughput manner. Over the last decade, applications of functional protein microarrays in particular have flourished in studying protein function at a systems level and has led to the construction of networks and pathways describing these functions. Relevant areas of research include the detection of various binding properties of proteins, the study of enzyme-substrate relationships, the analysis of host-microbe interactions, and profiling antibody specificity. In addition, discovery of novel biomarkers in autoimmune diseases and cancers is emerging as a major clinical application of functional protein microarrays. The Zhu lab used a protein microarray-based approach to systematically survey the entire human transcription factor (TF) family and found numerous purified TFs exhibit specific binding activity to methylated and unmethylated DNA motifs of distinct sequences. DNA methylation, especially CpG methylation at promoter regions, has been generally considered as a potent epigenetic modification that prohibits (TF) recruitment, resulting in transcription suppression. To elucidate the underlying mechanism, we focused on Kruppel-like factor 4 (KLF4), and decoupled its mCpG- and CpG-binding activities via site-directed mutagenesis. This study suggested that mCpG-dependent TF binding activity is a widespread phenomenon and provides a new framework to understand the role and mechanism of TFs in epigenetic regulation of gene transcription
Further studies from the Xia Lab showed that KLF4 promotes cell adhesion, migration, and morphological changes, all of which are abolished by a single mutation in R458 to an alanine. Surprisingly, 116 genes are directly activated via mCpG-dependent KLF4 binding activity. These studies demonstrate 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.
My thesis project focused on characterizing the KLF4 methylation dependent migration targets identified in previous studies. UDP-Glucose 6-dehydrogenase (UGDH) was identified as one of the downstream targets of KLF4-mCpG binding activity. This work shows that KLF4 upregulates UGDH expression in a mCpG-dependent manner, and UGDH is required for KLF4 induced cell migration in vitro. 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). 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 decreases expression of extracellular matrix, e.g. tenascin C, brevican. These 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. Further studies of KLF4 mCpG interaction revealed a novel KLF4 binding to enhancer regions to regulate transcription such as the BLK gene. Through Chromosome Conformation Capture (3C) analysis, we found BLK was activated by KLF4 binding to enhancers in a methylation dependent manner. In addition, we found that in genome scale, KLF4 binds to methylated enhancer regions and activates gene transcription.
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Keywords
DNA Methylation, Transcription factor binding, Glioblastoma, UGDH, glycosaminoglycans, ECM, Migration