TET1 Catalytic Domain Research Articles

Tet3 regulates synaptic transmission and homeostatic plasticity via DNA oxidation and repair.

Contrary to the long-held belief that DNA methylation of terminally differentiated cells is permanent and essentially immutable, post-mitotic neurons exhibit extensive DNA demethylation. The cellular function of active DNA demethylation in neurons, however, remains largely unknown. Tet family proteins oxidize 5-methylcytosine to initiate active DNA demethylation through the base-excision repair pathway. Here, we show that synaptic activity bi-directionally regulates neuronal Tet3 expression. Functionally, knockdown of Tet or inhibition of base-excision repair in hippocampal neurons elevates excitatory glutamatergic synaptic transmission, whereas overexpressing Tet3 or Tet1 catalytic domain decreases it. Furthermore, dysregulation of Tet3 signalling prevents homeostatic synaptic plasticity. Mechanistically, Tet3 dictates neuronal surface GluR1 levels. RNA-seq analyses further revealed a pivotal role of Tet3 in regulating gene expression in response to global synaptic activity changes. Thus, Tet3 serves as a synaptic activity sensor to epigenetically regulate fundamental properties and meta-plasticity of neurons via active DNA demethylation.

The hypomethylating agent Decitabine causes a paradoxical increase in 5-hydroxymethylcytosine in human leukemia cells.

The USFDA approved “epigenetic drug”, Decitabine, exerts its effect by hypomethylating DNA, demonstrating the pivotal role aberrant genome-wide DNA methylation patterns play in cancer ontology. Using sensitive technologies in a cellular model of Acute Myeloid Leukemia, we demonstrate that while Decitabine reduces the global levels of 5-methylcytosine (5mC), it results in paradoxical increase of 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) levels. Hitherto, the only biological mechanism known to generate 5hmC, 5fC and 5caC, involving oxidation of 5mC by members of Ten-Eleven-Translocation (TET) dioxygenase family, was not observed to undergo any alteration during DAC treatment. Using a multi-compartmental model of DNA methylation, we show that partial selectivity of TET enzymes for hemi-methylated CpG dinucleotides could lead to such alterations in 5hmC content. Furthermore, we investigated the binding of TET1-catalytic domain (CD)-GFP to DNA by Fluorescent Correlation Spectroscopy in live cells and detected the gradual increase of the DNA bound fraction of TET1-CD-GFP after treatment with Decitabine. Our study provides novel insights on the therapeutic activity of DAC in the backdrop of the newly discovered derivatives of 5mC and suggests that 5hmC has the potential to serve as a biomarker for monitoring the clinical success of patients receiving DAC.

Abstract 396: TET oxidase activity accumulated on methyl-CpG sites extensively upregulates methylated genes through DNA demethylation

Abstract Ten-Eleven Translocation (TET) proteins play an important role in regulating DNA methylation fidelity and their inactivation contributes to the DNA hypermethylation phenotype in cancer; they are Fe²+- and 2-oxoglutarate-dependent dioxygenases that successively oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) and these oxidized methylcytosines are important intermediates in the process of 5mC demethylation. TET proteins convert 5mC to 5hmC in vitro, but it is difficult to explore the process of DNA demethylation and their effects on endogenous hypermethylated genes in cells. In this study, we tried to develop a system that accumulates TET oxidase activity at hypermethylated promoters. To accomplish this, we constructed a fusion protein-producing vector consisting of methyl-CpG binding domain (MBD) and TET1 catalytic domain (TET1 CD) and asked whether this fusion protein (MBD-TET1 CD) could lead to DNA demethylation and gene reactivation. We first prepared DNA constructs that contain wild-type (wt) TET1 CD as well as catalytically inactive TET1 CD mutant (mut) with or without MBD from MBD2 protein and then transfected each construct into human embryonic kidney cell line 293T. We isolated stably transfected 293T cell lines and analyzed the expression of genes transcriptionally silenced by DNA hypermethylation using RT-PCR. Surprisingly, only plasmids containing TET1 CDwt reactivated silenced MAL, SOX17, TRH, MT1M, and MLH1 genes in a MBD dependent manner. To clarify the mechanisms of the antisilencing activities in clones stably expressed MBD-TET1 CDwt, we examined the CpG islands in MAL, TRH, and MT1M promoters by genomic sequencing after bisulfite modifications. Almost all CpG sites were methylated in parental cell line 293T, a TET1 CDwt clone, a TET1 CDmut clone, and a MBD-TET1 CDmut clone. On the other hand, MBD-TET1 CDwt clones showed an extensive demethylation at CpG islands of all three genes. In addition, because bisulfite sequencing does not distinguish between 5mC and 5hmC, we converted 5hmC to glucosylated form and discriminated between 5mC and 5hmC using restriction enzymes MspI and HpaII. Quantitative real time PCR analysis clearly demonstrated that MBD-TET1 CDwt promoted demethylation at CpG sites with some 5hmC bases. We further examined the changes in global gene expression in stable cell lines containing different TET constructs by microarray analyses. All top 50 genes upregulated by MBD-TET1 CDwt-stably expressing cell lines contained CpG islands and interestingly, 43 of top 50 genes were also upregulated after treatment with DNA demethylating agent, 5-azacytidine. These results suggest that specific recruitment of TET oxidase activity on hypermethylated gene promoters induces DNA demethylation and promotes reactivation of epigenetically silenced genes. Citation Format: Shinichi Fukushige, Yasuhiko Mizuguchi, Akira Horii. TET oxidase activity accumulated on methyl-CpG sites extensively upregulates methylated genes through DNA demethylation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 396. doi:10.1158/1538-7445.AM2014-396

TET1 is a maintenance DNA demethylase that prevents methylation spreading in differentiated cells.

TET1 is a 5-methylcytosine dioxygenase and its DNA demethylating activity has been implicated in pluripotency and reprogramming. However, the precise role of TET1 in DNA methylation regulation outside of developmental reprogramming is still unclear. Here, we show that overexpression of the TET1 catalytic domain but not full length TET1 (TET1-FL) induces massive global DNA demethylation in differentiated cells. Genome-wide mapping reveals that 5-hydroxymethylcytosine production by TET1-FL is inhibited as DNA methylation increases, which can be explained by the preferential binding of TET1-FL to unmethylated CpG islands (CGIs) through its CXXC domain. TET1-FL specifically accumulates 5-hydroxymethylcytosine at the edges of hypomethylated CGIs, while knockdown of endogenous TET1 induces methylation spreading from methylated edges into hypomethylated CGIs. We also found that gene expression changes after TET1-FL overexpression are relatively small and independent of its dioxygenase function. Thus, our results identify TET1 as a maintenance DNA demethylase that does not purposely decrease methylation levels, but specifically prevents aberrant methylation spreading into CGIs in differentiated cells.

Hydroxylation of 5-Methylcytosine by TET1 Promotes Active DNA Demethylation in the Adult Brain

Cytosine methylation is the major covalent modification of mammalian genomic DNA and plays important roles in transcriptional regulation. The molecular mechanism underlying the enzymatic removal of this epigenetic mark, however, remains elusive. Here, we show that 5-methylcytosine (5mC) hydroxylase TET1, by converting 5mCs to 5-hydroxymethylcytosines (5hmCs), promotes DNA demethylation in mammalian cells through a process that requires the base excision repair pathway. Though expression of the 12 known human DNA glycosylases individually did not enhance removal of 5hmCs in mammalian cells, demethylation of both exogenously introduced and endogenous 5hmCs is promoted by the AID (activation-induced deaminase)/APOBEC (apolipoprotein B mRNA-editing enzyme complex) family of cytidine deaminases. Furthermore, Tet1 and Apobec1 are involved in neuronal activity-induced, region-specific, active DNA demethylation and subsequent gene expression in the dentate gyrus of the adult mouse brain in vivo. Our study suggests a TET1-induced oxidation-deamination mechanism for active DNA demethylation in mammals.