Purification of TET Proteins.

The 5-methylcytosine (5mC) oxidation pathway mediated by TET proteins involves step-wise oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC can be removed from DNA by base excision repair and the completion of this pathway results in "demethylation" of 5mC by converting the modified base back into cytosine. In vitro studies with TET proteins aimed at analyzing their DNA substrate specificities and their activity within defined chromatin templates are relatively limited. Here we describe purification methods for mammalian TET proteins based on expression in insect cells or in 293T cells. We also briefly summarize a method that can be used to monitor 5-methylcytosine oxidase activity of the purified TET proteins in vitro.

Quantification of global DNA methylation level using 5-methylcytosine dioxygenase.

DNA methylation is one of the best studied epigenetic modifications. Alteration of the global DNA methylation level occurs in abnormal cells, such as those associated with cancers and Alzheimer's disease. Several assays are used to determine the global DNA methylation level, including the bisulfite-based assay, high-performance liquid chromatography (HPLC)-based assay, enzyme-linked immunosorbent assay (ELISA), and methyl acceptance assay. However, these assays require several cumbersome steps to detect methylation levels. We developed a simpler enzymatic assay for the quantification of the global DNA methylation level using the Ten-eleven translocation (TET) protein. TET proteins mediate DNA demethylation through the oxidation of 5-methylcytosine (5mC) in CpG in mammalian cells. Succinate is produced during this oxidation reaction, and the amount of succinate produced correlates to the global DNA methylation level. The catalytic domain of the TET2 was expressed in Escherichia coli (E. coli), and the purified TET2 catalytic domain was reacted with human genomic DNA. The reaction solution was used for enzymatic succinate quantification with no purification step. The results showed that the succinate produced through TET-mediated oxidation increased with increasing global DNA methylation levels in human genomic DNA, which was determined using the bisulfite method. These results show that the global DNA methylation level is quantifiable by measuring the amount of succinate produced by the TET2-mediated 5mC oxidation reaction. Graphical abstract.

Generative Models for Quantification of DNA Modifications.

There are multiple chemical modifications of cytosine that are important to the regulation and ultimately the functional expression of the genome. To date no single experiment can capture these separate modifications, and integrative experimental designs are needed to fully characterize cytosine methylation and chemical modification. This chapter describes a generative probabilistic model, Lux, for integrative analysis of cytosine methylation and its oxidized variants. Lux simultaneously analyzes partially orthogonal bisulfite sequencing data sets to estimate proportions of different cytosine methylation modifications and estimate multiple cytosine modifications for a single sample by integrating across experimental designs composed of multiple parallel destructive genomic measurements. Lux also considers the variation in measurements introduced by different imperfect experimental steps; the experimental variation can be quantified by using appropriate spike-in controls, allowing Lux to deconvolve the measurements and recover accurately the underlying signal.

A probabilistic generative model for quantification of DNA modifications enables analysis of demethylation pathways.

We present a generative model, Lux, to quantify DNA methylation modifications from any combination of bisulfite sequencing approaches, including reduced, oxidative, TET-assisted, chemical-modification assisted, and methylase-assisted bisulfite sequencing data. Lux models all cytosine modifications (C, 5mC, 5hmC, 5fC, and 5caC) simultaneously together with experimental parameters, including bisulfite conversion and oxidation efficiencies, as well as various chemical labeling and protection steps. We show that Lux improves the quantification and comparison of cytosine modification levels and that Lux can process any oxidized methylcytosine sequencing data sets to quantify all cytosine modifications. Analysis of targeted data from Tet2-knockdown embryonic stem cells and T cells during development demonstrates DNA modification quantification at unprecedented detail, quantifies active demethylation pathways and reveals 5hmC localization in putative regulatory regions.

Functions of TET Proteins in Hematopoietic Transformation.

DNA methylation is a well-characterized epigenetic modification that plays central roles in mammalian development, genomic imprinting, X-chromosome inactivation and silencing of retrotransposon elements. Aberrant DNA methylation pattern is a characteristic feature of cancers and associated with abnormal expression of oncogenes, tumor suppressor genes or repair genes. Ten-eleven-translocation (TET) proteins are recently characterized dioxygenases that catalyze progressive oxidation of 5-methylcytosine to produce 5-hydroxymethylcytosine and further oxidized derivatives. These oxidized methylcytosines not only potentiate DNA demethylation but also behave as independent epigenetic modifications per se. The expression or activity of TET proteins and DNA hydroxymethylation are highly dysregulated in a wide range of cancers including hematologic and non-hematologic malignancies, and accumulating evidence points TET proteins as a novel tumor suppressor in cancers. Here we review DNA demethylation-dependent and -independent functions of TET proteins. We also describe diverse TET loss-of-function mutations that are recurrently found in myeloid and lymphoid malignancies and their potential roles in hematopoietic transformation. We discuss consequences of the deficiency of individual Tet genes and potential compensation between different Tet members in mice. Possible mechanisms underlying facilitated oncogenic transformation of TET-deficient hematopoietic cells are also described. Lastly, we address non-mutational mechanisms that lead to suppression or inactivation of TET proteins in cancers. Strategies to restore normal 5mC oxidation status in cancers by targeting TET proteins may provide new avenues to expedite the development of promising anti-cancer agents.

TET proteins and 5-methylcytosine oxidation in hematological cancers.

DNA methylation has pivotal regulatory roles in mammalian development, retrotransposon silencing, genomic imprinting, and X-chromosome inactivation. Cancer cells display highly dysregulated DNA methylation profiles characterized by global hypomethylation in conjunction with hypermethylation of promoter CpG islands that presumably lead to genome instability and aberrant expression of tumor suppressor genes or oncogenes. The recent discovery of ten-eleven-translocation (TET) family dioxygenases that oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) in DNA has led to profound progress in understanding the mechanism underlying DNA demethylation. Among the three TET genes, TET2 recurrently undergoes inactivating mutations in a wide range of myeloid and lymphoid malignancies. TET2 functions as a bona fide tumor suppressor particularly in the pathogenesis of myeloid malignancies resembling chronic myelomonocytic leukemia (CMML) and myelodysplastic syndromes (MDS) in human. Here we review diverse functions of TET proteins and the novel epigenetic marks that they generate in DNA methylation/demethylation dynamics and normal and malignant hematopoietic differentiation. The impact of TET2 inactivation in hematopoiesis and various mechanisms modulating the expression or activity of TET proteins are also discussed. Furthermore, we also present evidence that TET2 and TET3 collaborate to suppress aberrant hematopoiesis and hematopoietic transformation. A detailed understanding of the normal and pathological functions of TET proteins may provide new avenues to develop novel epigenetic therapies for treating hematological malignancies.

Mechanism and function of oxidative reversal of DNA and RNA methylation.

The importance of eukaryotic DNA methylation [5-methylcytosine (5mC)] in transcriptional regulation and development was first suggested almost 40 years ago. However, the molecular mechanism underlying the dynamic nature of this epigenetic mark was not understood until recently, following the discovery that the TET proteins, a family of AlkB-like Fe(II)/α-ketoglutarate-dependent dioxygenases, can oxidize 5mC to generate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Since then, several mechanisms that are responsible for processing oxidized 5mC derivatives to achieve DNA demethylation have emerged. Our biochemical understanding of the DNA demethylation process has prompted new investigations into the biological functions of DNA demethylation. Characterization of two additional AlkB family proteins, FTO and ALKBH5, showed that they possess demethylase activity toward N(6)-methyladenosine (m(6)A) in RNA, indicating that members of this subfamily of dioxygenases have a general function in demethylating nucleic acids. In this review, we discuss recent advances in this emerging field, focusing on the mechanism and function of TET-mediated DNA demethylation.