Targeted TET oxidase activity through methyl‐CpG‐binding domain extensively suppresses cancer cell proliferation

DNA methyltransferase (DNMT) inhibitors are epigenetic drugs used to treat myelodysplastic syndrome. They not only induce DNA demethylation but also have significant cytostatic and cytotoxic effects; however, the relationships between these characteristics have not been established yet due to the lack of a method to induce only DNA demethylation. Herein, we show that a fusion protein comprised of the methyl‐CpG‐binding domain (MBD) and the catalytic domain of Ten‐eleven translocation protein 1 (TET1‐CD) globally demethylates and upregulates a number of methylated genes. These upregulated genes frequently contained CpG islands (CGIs) within ± 1000 bp of the transcription start site (TSS). Interestingly, 65% of the genes upregulated fivefold or more by MBD‐TET1‐CDwt were also reactivated after treatment with a DNMT inhibitor, 5‐azacytidine (Aza‐CR), suggesting that gene reactivation by both methods primarily shares the same mechanism, DNA demethylation. In order to examine whether DNA demethylation affects the growth of cancer cells, we have established a tetracycline inducible system that can regulate the expression of MBD‐TET1‐CDwt in a prostate cancer cell line, LNCaP. The induction of MBD‐TET1‐CDwt demethylated and upregulated glutathione S‐transferase pi 1 (GSTP1), one of the hypermethylated genes in prostate cancer. In accordance with the reactivation of methylated genes, induction of MBD‐TET1‐CDwt extensively suppressed the growth of LNCaP cells through G1/S arrest. These results clearly indicate that TET oxidase activity recruited at methyl‐CpG sites through MBD induces reactivation of hypermethylated genes by DNA demethylation and allows us to analyze the effect of only global DNA demethylation in a wide variety of cancer cells.

[1]  S. Baylin,et al.  DNMT1 binds HDAC2 and a new co-repressor, DMAP1, to form a complex at replication foci , 2000, Nature Genetics.

[2]  Yi Zhang,et al.  Mechanisms and functions of Tet protein-mediated 5-methylcytosine oxidation. , 2011, Genes & development.

[3]  Andrew P. Feinberg,et al.  Cancer as a dysregulated epigenome allowing cellular growth advantage at the expense of the host , 2013, Nature Reviews Cancer.

[4]  A. Horii,et al.  The human PMS2L proteins do not interact with hMLH1, a major DNA mismatch repair protein. , 1999, Journal of biochemistry.

[5]  A. Horii,et al.  Methyl-CpG targeted recruitment of p300 reactivates tumor suppressor genes in human cancer cells. , 2009, Biochemical and biophysical research communications.

[6]  F. Lyko,et al.  Modes of action of the DNA methyltransferase inhibitors azacytidine and decitabine , 2008, International journal of cancer.

[7]  A. Horii,et al.  Methyl-CpG targeted transcriptional activation allows re-expression of tumor suppressor genes in human cancer cells. , 2008, Biochemical and biophysical research communications.

[8]  F. Motoi,et al.  Identification of epigenetically silenced genes in human pancreatic cancer by a novel method "microarray coupled with methyl-CpG targeted transcriptional activation" (MeTA-array). , 2011, Biochemical and biophysical research communications.

[9]  M. Ishida,et al.  DCK is frequently inactivated in acquired gemcitabine-resistant human cancer cells. , 2012, Biochemical and biophysical research communications.

[10]  J. Herman,et al.  Gene silencing in cancer in association with promoter hypermethylation. , 2003, The New England journal of medicine.

[11]  M. Frommer,et al.  CpG islands in vertebrate genomes. , 1987, Journal of molecular biology.

[12]  A. Horii,et al.  Microarray coupled with methyl-CpG targeted transcriptional activation (MeTA-array) identifies hypermethylated genes containing the stringent criteria of CpG islands at high frequency , 2011, Epigenetics.

[13]  E. Ballestar,et al.  Methyl-CpG-binding proteins. Targeting specific gene repression. , 2001, European journal of biochemistry.

[14]  Haikuo Zhang,et al.  TET1 is a DNA-binding protein that modulates DNA methylation and gene transcription via hydroxylation of 5-methylcytosine , 2010, Cell Research.

[15]  Chun-nan Lin,et al.  Identification of Kazinol Q, a Natural Product from Formosan Plants, as an Inhibitor of DNA Methyltransferase , 2014, Phytotherapy research : PTR.

[16]  B. Schutte,et al.  Optimized conditions for cloning PCR products into an XcmI T-vector. , 1997, BioTechniques.

[17]  R. Meehan,et al.  Genomic insights into cancer-associated aberrant CpG island hypermethylation , 2013, Briefings in functional genomics.

[18]  M. Ishida,et al.  RNA interference targeting against S100A4 suppresses cell growth and motility and induces apoptosis in human pancreatic cancer cells. , 2009, Biochemical and biophysical research communications.

[19]  G. Ming,et al.  Hydroxylation of 5-Methylcytosine by TET1 Promotes Active DNA Demethylation in the Adult Brain , 2011, Cell.

[20]  A. Horii,et al.  The Thymine DNA Glycosylase MBD4 Represses Transcription and Is Associated with Methylated p16INK4a and hMLH1 Genes , 2005, Molecular and Cellular Biology.

[21]  M. Shirakawa,et al.  Cell Cycle-Dependent Turnover of 5-Hydroxymethyl Cytosine in Mouse Embryonic Stem Cells , 2013, PloS one.

[22]  Keith D. Robertson,et al.  DNA Methylation Inhibitor 5-Aza-2′-Deoxycytidine Induces Reversible Genome-Wide DNA Damage That Is Distinctly Influenced by DNA Methyltransferases 1 and 3B , 2007, Molecular and Cellular Biology.

[23]  J. Issa,et al.  TET1 is a maintenance DNA demethylase that prevents methylation spreading in differentiated cells , 2014, Nucleic acids research.

[24]  J. Christman,et al.  5-Azacytidine and 5-aza-2′-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy , 2002, Oncogene.

[25]  J. Issa.  DNA methylation as a therapeutic target in cancer. , 2008, Clinical cancer research : an official journal of the American Association for Cancer Research.

[26]  G. Garcia-Manero,et al.  Therapy with azanucleosides for myelodysplastic syndromes , 2010, Nature Reviews Clinical Oncology.

[27]  William A. Pastor,et al.  TETonic shift: biological roles of TET proteins in DNA demethylation and transcription , 2013, Nature Reviews Molecular Cell Biology.

[28]  Michael R. Green,et al.  A KRAS-directed transcriptional silencing pathway that mediates the CpG island methylator phenotype , 2014, eLife.

[29]  K. Shiga,et al.  A novel G106D alteration of the SDHD gene in a pedigree with familial paraganglioma , 2006, American journal of medical genetics. Part A.

[30]  Daiya Takai,et al.  Comprehensive analysis of CpG islands in human chromosomes 21 and 22 , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[31]  A. Horii,et al.  Alternative splicing of hMSH2 in normal human tissues , 1997, Human Genetics.

[32]  Jeffry D Sander,et al.  Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins , 2013, Nature Biotechnology.

[33]  S. Baylin,et al.  Dnmt3a and Dnmt3b Are Transcriptional Repressors That Exhibit Unique Localization Properties to Heterochromatin* , 2001, The Journal of Biological Chemistry.