DNA Demethylation Pathway Research Articles

A novel protein complex that regulates active DNA demethylation in Arabidopsis.

Active DNA demethylation is critical for altering DNA methylation patterns and regulating gene expression. The 5-methylcytosine DNA glycosylase/lyase ROS1 initiates a base-excision repair pathway for active DNA demethylation and is required for the prevention of DNA hypermethylation at 1 000s of genomic regions in Arabidopsis. How ROS1 is regulated and targeted to specific genomic regions is not well understood. Here, we report the discovery of an Arabidopsis protein complex that contains ROS1, regulates ROS1 gene expression, and likely targets the ROS1 protein to specific genomic regions. ROS1 physically interacts with a WD40 domain protein (RWD40), which in turn interacts with a methyl-DNA binding protein (RMB1) as well as with a zinc finger and homeobox domain protein (RHD1). RMB1 binds to DNA that is methylated in any sequence context, and this binding is necessary for its function in vivo. Loss-of-function mutations in RWD40, RMB1, or RHD1 cause DNA hypermethylation at several tested genomic regions independently of the known ROS1 regulator IDM1. Because the hypermethylated genomic regions include the DNA methylation monitoring sequence in the ROS1 promoter, plants mutated in RWD40, RMB1, or RHD1 show increased ROS1 expression. Importantly, ROS1 binding to the ROS1 promoter requires RWD40, RMB1, and RHD1, suggesting that this complex dictates ROS1 targeting to this locus. Our results demonstrate that ROS1 forms a protein complex with RWD40, RMB1, and RHD1, and that this novel complex regulates active DNA demethylation at several endogenous loci in Arabidopsis.

Experimental eye research / short communication format characterization of DNA hydroxymethylation in the ocular choroid

DNA methylation and hydroxymethylation represent important epigenetic modifications involved in cell differentiation. DNA hydroxymethylation can be used to classify independent biological samples by tissue type. Relatively little is known regarding the genomic abundance and function of 5-hydroxymethylcytosine (5-hmC) in ocular tissues. The choroid supplies oxygen and nutrients to the outer retina through its dense network of blood vessels. This connective tissue is mainly composed of pigmented melanocytes, and stromal fibroblasts. Since DNA hydroxymethylation level is relatively high in cutaneous melanocytes, we investigated the presence of 5-hmC in choroidal melanocytes, as well as the expression of ten-eleven translocation methylcytosine dioxygenases (TETs) and isocitrate dehydrogenases (IDHs) implicated in this DNA demethylation pathway. Immunofluorescence, DNA slot blots and liquid chromatography coupled to tandem mass spectrometry performed with choroidal tissues and melanocytes within these tissues revealed that they have a relatively high level of 5-hmC. We also examined the expression of TET1/2 and IDH1/2 in choroidal melanocytes by gene expression profiling, qPCR and Western blotting. In addition, we detected decreased levels of 5-hmC when choroidal melanocytes were exposed to a lower concentration of oxygen. Our study therefore demonstrates that DNA hydroxymethylation is present in choroidal melanocytes, and that the abundance of this epigenetic mark is impacted by hypoxia.

Direct decarboxylation of ten-eleven translocation-produced 5-carboxylcytosine in mammalian genomes forms a new mechanism for active DNA demethylation.

DNA cytosine methylation (5-methylcytosine, 5mC) is the most important epigenetic mark in higher eukaryotes. 5mC in genomes is dynamically controlled by writers and erasers. DNA (cytosine-5)-methyltransferases (DNMTs) are responsible for the generation and maintenance of 5mC in genomes. Active demethylation of 5-methylcytosine (5mC) is achieved by ten-eleven translocation (TET) dioxygenase-mediated oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC are further processed by thymine DNA glycosylase (TDG)-initiated base excision repair (BER) to restore unmodified cytosines. The TET-TDG-BER pathway could cause the production of DNA strand breaks and therefore jeopardize the integrity of genomes. Here, we investigated the direct decarboxylation of 5caC in mammalian genomes by using metabolic labeling with 2′-fluorinated 5caC (F-5caC) and mass spectrometry analysis. Our results clearly demonstrated the decarboxylation of 5caC occurring in mammalian genomes, which unveiled that, in addition to the TET-TDG-BER pathway, the direct decarboxylation of TET-produced 5caC constituted a new pathway for active demethylation of 5mC in mammalian genomes.

Transformation of 5-Carboxylcytosine to Cytosine Through C–C Bond Cleavage in Human Cells Constitutes a Novel Pathway for DNA Demethylation

Active demethylation of 5-methylcytosine (5mC) can be realized through ten-eleven translocation (TET) dioxygenase-mediated oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC)...

Precise genomic mapping of 5-hydroxymethylcytosine via covalent tether-directed sequencing.

5-hydroxymethylcytosine (5hmC) is the most prevalent intermediate on the oxidative DNA demethylation pathway and is implicated in regulation of embryogenesis, neurological processes, and cancerogenesis. Profiling of this relatively scarce genomic modification in clinical samples requires cost-effective high-resolution techniques that avoid harsh chemical treatment. Here, we present a bisulfite-free approach for 5hmC profiling at single-nucleotide resolution, named hmTOP-seq (5hmC-specific tethered oligonucleotide-primed sequencing), which is based on direct sequence readout primed at covalently labeled 5hmC sites from an in situ tethered DNA oligonucleotide. Examination of distinct conjugation chemistries suggested a structural model for the tether-directed nonhomologous polymerase priming enabling theoretical evaluation of suitable tethers at the design stage. The hmTOP-seq procedure was optimized and validated on a small model genome and mouse embryonic stem cells, which allowed construction of single-nucleotide 5hmC maps reflecting subtle differences in strand-specific CG hydroxymethylation. Collectively, hmTOP-seq provides a new valuable tool for cost-effective and precise identification of 5hmC in characterizing its biological role and epigenetic changes associated with human disease.

Expression and prognosis analysis of TET family in acute myeloid leukemia.

TET family members (TETs) encode proteins that represent crucial factors in the active DNA demethylation pathway. Evidence has proved that TET2 mutation is associated with leukemogenesis, drug response, and prognosis in acute myeloid leukemia (AML). However, few studies revealed the TETs expression and its clinical significance in AML. We conducted a detailed expression and prognosis analysis of TETs expression in human AML cell lines and patients by using public databases. We observed that TETs expression especially TET2 and TET3 was closely associated with AML among various human cancers. TET1 expression was significantly reduced in AML patients, whereas TET2 and TET3 expression was significantly increased. Kaplan-Meier analysis showed that only TET3 expression was associated with overall survival (OS) and disease-free survival (DFS) among both total AML as well as non-M3 AML, and was confirmed by another independent cohort. Moreover, Cox regression analysis revealed that TET3 expression may act as an independent prognostic factor for OS and DFS in total AML. Interestingly, patients that received hematopoietic stem cell transplantation (HSCT) did not show significantly longer OS and DFS than those who did not receive HSCT in TET3 high-expressed groups; whereas, in TET3 low-expressed groups, patients that accepted HSCT showed significantly longer OS and DFS than those who did not accept HSCT. By bioinformatics analysis, TET3 expression was found positively correlated with tumor suppressor gene including CDKN2B, ZIC2, miR-196a, and negatively correlated with oncogenes such as PAX2 and IL2RA. Our study demonstrated that TETs showed significant expression differences in AML, and TET3 expression acted as a potential prognostic biomarker in AML, which may guide treatment choice between chemotherapy and HSCT.

Involvement of MEM1 in DNA demethylation in Arabidopsis.

MEM1 participates in ROS1-mediated DNA demethylation pathway, and acts functionally as ROS3 to counteract the effects of RdDM pathway.mem1mutation leads to large numbers of hyper-DMRs inArabidopsisgenome. In higher plants, DNA methylation performs important functions in silencing transcribed genes and transposable elements (TEs). Active DNA demethylation mediated by REPRESSOR OF SILENCING 1 (ROS1) is able to antagonize the action of DNA methylation caused by RNA-directed DNA methylation (RdDM) pathway, which plays critical roles in keeping DNA methylation at a proper level. In this study, a new mutant named mem1 (for methylation elevated mutant 1) was isolated from a genetic screen of T-DNA insertional mutant population for lines with elevated DNA methylation at a particular locus through Chop-PCR method. MEM1 possesses a Zf-C3HC domain, and is localized in nucleus as well as highly expressed in cotyledons. Whole-genome bisulfite sequencing data showed that knockout mutation of MEM1 leads to 4519 CG, 1793 CHG and 12739 CHH hyper-DMRs (for differentially methylated regions). Further analysis indicated that there are 2751, 2216 and 2042 overlapped CG hyper-DMRs between mem1-1and three mutants, i.e. ros1-4, rdd and ros3-2, respectively; 797, 2514, and 6766 overlapped CHH hyper-DMRs were observed between mem1-1 and three such mutants, respectively; mem1 nrpd1-3 and mem1 rdm1 double mutants showed nearly complete or partial loss of hypermethylation at 4 tested loci, suggesting that MEM1 performs similar functions as DNA glycosylase/lyases in counteracting excessive DNA methylation, and MEM1 plays important roles as REPRESSOR OF SILENCING 3 (ROS3) in erasing CHH methylation caused by the RdDM pathway. Together, these data demonstrate the involvement of MEM1 in ROS1-mediated DNA demethylation pathway and functional connections between MEM1 and ROS3.

The mechanism and function of active DNA demethylation in plants.

DNA methylation is a conserved and important epigenetic mark in both mammals and plants. DNA methylation can be dynamically established, maintained, and removed through different pathways. In plants, active DNA demethylation is initiated by the RELEASE OF SILENCING 1 (ROS1) family of bifunctional DNA glycosylases/lyases. Accumulating evidence suggests that DNA demethylation is important in many processes in plants. In this review, we summarize recent studies on the enzymes and regulatory factors that have been identified in the DNA demethylation pathway. We also review the functions of active DNA demethylation in plant development as well as biotic and abiotic stress responses. Finally, we highlight those aspects of DNA demethylation that require additional research.

Photodamage repair pathways contribute to the accurate maintenance of the DNA methylome landscape upon UV exposure.

Plants are exposed to the damaging effect of sunlight that induces DNA photolesions. In order to maintain genome integrity, specific DNA repair pathways are mobilized. Upon removal of UV-induced DNA lesions, the accurate re-establishment of epigenome landscape is expected to be a prominent step of these DNA repair pathways. However, it remains poorly documented whether DNA methylation is accurately maintained at photodamaged sites and how photodamage repair pathways contribute to the maintenance of genome/methylome integrities. Using genome wide approaches, we report that UV-C irradiation leads to CHH DNA methylation changes. We identified that the specific DNA repair pathways involved in the repair of UV-induced DNA lesions, Direct Repair (DR), Global Genome Repair (GGR) and small RNA-mediated GGR prevent the excessive alterations of DNA methylation landscape. Moreover, we identified that UV-C irradiation induced chromocenter reorganization and that photodamage repair factors control this dynamics. The methylome changes rely on misregulation of maintenance, de novo and active DNA demethylation pathways highlighting that molecular processes related to genome and methylome integrities are closely interconnected. Importantly, we identified that photolesions are sources of DNA methylation changes in repressive chromatin. This study unveils that DNA repair factors, together with small RNA, act to accurately maintain both genome and methylome integrities at photodamaged silent genomic regions, strengthening the idea that plants have evolved sophisticated interplays between DNA methylation dynamics and DNA repair.

Sequential Assessment of Multiple Epigenetic Modifications of Cytosine in Whole Genomic DNA by Surface Plasmon Resonance.

Since the discovery of the active DNA demethylation pathway in mammals, numerous efforts have been made to distinguish epigenetic cytosine variants, including 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). However, the rapid discrimination of multiple cytosine variants in DNA remains challenging because the conventional assays require time-consuming DNA pretreatments, such as enzymatical digestion and chemical conversion. Here we demonstrated the high-throughput discrimination of four cytosine variants in DNA by using a sequential surface-plasmon-resonance (SPR)-based immunochemical assay. The target DNAs were biotinylated in one step with a bifunctional linker 1 and robustly immobilized on a streptavidin-coated sensor surface to hold them in place during an alkali washing designed to remove residual antibodies. By repeating the injection of antibodies and washing, we achieved a sequential assessment of cytosine variants in identical DNA and identified the yield of in vitro 5mC oxidation in genomic DNA by the ten-eleven translocation 1 (TET1) enzyme. These results demonstrated that our sequential SPR-based immunochemical assay was effective for evaluating multiple epigenetic modifications in a whole genome with a single row operation without time-consuming DNA pretreatments.

Characterization and fine mapping of Arabidopsis RLL3, a locus required for DNA demethylation pathway.

DNA methylation pattern is found to be established by the combined actions of DNA methylation and demethylation. Compared to the DNA methylation pathway, DNA demethylation pathway, however, remains largely unknown. To better understand the DNA demethylation pathway, we performed genetic screening for Arabidopsis mutants with increased genomic DNA methylation levels through a 2 × 35S:LUC (LUC, luciferase) reporter system. A mutant with reduced LUC luminescence was identified by such a system, therefore named rll3-1 (for reduced LUC luminescence 3-1). The rll3-1 mutant exhibited pleiotropic developmental defects, such as delayed bolting as well as flowering, more branches, etc. By map-based cloning approach, rll3 locus that contains a single nuclear recessive mutation as revealed by the genetic analysis was mapped to a region between molecular markers CL102_B1 M1 and CL102_B3M1, which are located in bacterial artificial chromosome (BAC) clones F9P14 and F12K11, respectively, on chromosome 1. Chop-PCR analysis indicated that a total of seven tested loci displayed elevated DNA methylation levels. Whole-genome bisulfite sequencing further revealed 1536 loci exhibiting increased DNA methylation levels relative to Col-LUC control, among which there are 507 such loci overlapping between the rll3-1 and ros1-7 mutants, suggestive of a functional association between RLL3 and REPRESSOR OF SILENCING 1 (ROS1). Further investigations demonstrated that the expression levels of a few genes (like ROS1, IDM1, etc.), which are involved in DNA demethylation pathway, remained unchanged in the rll3-1 mutant, indicating that the increased DNA methylation levels in rll3-1 mutant are not attributable to downregulation of such genes. Taken together, our studies provide a demonstration of the involvement of RLL3 in the DNA demethylation pathway.

DNA Base Excision Repair in Plants: An Unfolding Story With Familiar and Novel Characters.

Base excision repair (BER) is a critical genome defense pathway that deals with a broad range of non-voluminous DNA lesions induced by endogenous or exogenous genotoxic agents. BER is a complex process initiated by the excision of the damaged base, proceeds through a sequence of reactions that generate various DNA intermediates, and culminates with restoration of the original DNA structure. BER has been extensively studied in microbial and animal systems, but knowledge in plants has lagged behind until recently. Results obtained so far indicate that plants share many BER factors with other organisms, but also possess some unique features and combinations. Plant BER plays an important role in preserving genome integrity through removal of damaged bases. However, it performs additional important functions, such as the replacement of the naturally modified base 5-methylcytosine with cytosine in a plant-specific pathway for active DNA demethylation.

DNA Methylation Dynamics During the Differentiation of Retinal Progenitor Cells Into Retinal Neurons Reveal a Role for the DNA Demethylation Pathway.

To evaluate the contribution of the DNA methylation and DNA demethylation pathways in retinal development, we studied DNA methylation in retinal progenitor cells (RPCs) and retinal neurons using a combination of whole genome bisulfite sequencing (WGBS) data obtained in our study and WGBS data collected from previous studies. The data was analyzed using Hidden Markov Model- and change point-based methods to identify methylome states in different segments of the studied genomes following genome annotation. We found that promoters of rod and cone phototransduction genes and rod photoreceptor genes, but not genes required for the development and function of other retinal phenotypes, were highly methylated in DNA isolated from human and murine fetal retinas (which mostly contain RPCs) and postnatal murine RPCs. While these highly methylated genomic regions were inherited by non-photoreceptor phenotypes during RPC differentiation, the methylation of these promoters was significantly reduced during RPC differentiation into photoreceptors and accompanied by increased expression of these genes. Our analysis of DNA methylation during embryogenesis revealed low methylation levels in genomic regions containing photoreceptor genes at the inner cell mass stage, but a sharp increase in methylation at the epiblast stage, which remained the same later on (except for DNA demethylation in photoreceptors). Thus, our data suggest that the DNA demethylation pathway is required for photoreceptor phenotypes in the developing retina. Meanwhile, the role of the DNA methylation and DNA demethylation pathways during RPC differentiation into non-photoreceptor retinal phenotypes might be less important.

The Anticancer Role of Omega-3 Polyunsaturated Fatty Acids was Closely Associated with the Increase in Genomic DNA Hydroxymethylation.

Omega-3 polyunsaturated fatty acids (omega-3 PUFAs) have significant multiple antitumor roles. However, whether epigenetic DNA hydroxymethylation enrolls in the anticancer process of omega- 3 PUFAs is still not clear yet. To expound the interaction between the anti-tumor role of omega-3 PUFAs and the DNA demethylation pathway and thus provide a firm foundation for deepening our understanding on anticancer mechanism of omega-3 PUFAs. Colorectal Cancer (CRC) model rats were induced to generate tumor by N-methyl-N-nitrosourea and their counterparts treated with omega-3 PUFAs during the induction. The blood samples from different treatment groups of rats [Normal Control group (NC), colorectal cancer model group (CRC) and omega-3 PUFAs Medication Group (MG)] were used as experimental materials. Genomic 5-hydroxymethylocytosine (5hmC) content was quantified using LC-MS/MS, and the expression of ten-eleven translocation dioxygenase 1 (TET1), catalyzing the generation of 5hmC, was also evaluated by quantitative real-time PCR. We observed lower tumor incidence and small tumor size in MG group when compared with CRC group, supporting the effective anticancer role of omega-3 PUFAs. Due to the formation of CRC, 5hmC level was dramatically dropped in CRC group when compared with the NC group. Notably, 5hmC percentage in MG group remarkably increased close to NC group and was significantly higher than that in the CRC group. Consistent alteration pattern of TET1 expressions in mRNA was also observed in the tested groups of rats. The anticancer effect of omega-3 PUFAs was positively correlated with global 5hmC accumulation and TET1 expression, suggesting DNA hydroxymethylation pathway was factually involved in the anticancer process of omega-3 PUFAs.

Impact of polymorphisms within genes involved in regulating DNA methylation in patients with metastatic colorectal cancer enrolled in three independent, randomised, open-label clinical trials: a meta-analysis from TRIBE, MAVERICC and FIRE-3

BackgroundCpG island DNA hypermethylation and global DNA hypomethylation are hallmark characteristics of colorectal cancer (CRC). Therefore, we aim to explore the effect of genetic variations within the genes that regulate the DNA methylation and demethylation pathways on outcomes in patients with metastatic CRC (mCRC) treated with first-line therapy and enrolled in three independent, randomised, open-label clinical trials. MethodsA total of 884 patients with mCRC enrolled in TRIBE, MAVERICC and FIRE-3 trials were included. Single-nucleotide polymorphisms (SNPs) within genes involved in DNA methylation and demethylation pathways were analysed. The prognostic value of each SNP across all treatment arms was quantified using the inverse-variance–weighted effect size, a meta-analysis approach implemented in the METASOFT software. ResultsIn the meta-analysis, DNMT3A rs11681717 was significantly associated with overall survival (hazard ratio = 1.26; 95% confidence interval [CI] 1.08–1.46; P = 0.002; false discovery rate [FDR] = 0.016), accounting for seven tests in the DNA methylation pathway. In addition, there was suggestive evidence of association for ten-eleven translocation (TET) genes variance with tumour response (TET1 rs3814177, odds ratio [OR] = 0.76, 95% CI 0.59–0.97, P = 0.025, FDR = 0.087; TET3 rs7560668, OR = 1.44; 95% CI 1.10–1.89; P = 0.009; FDR = 0.062). ConclusionsWe showed that polymorphisms within the genes responsible for the DNA methylation and demethylation machineries are correlated with outcomes in patients with mCRC who were enrolled in three independent, randomised, open-label, phase II/III clinical trials. In addition, we demonstrated the feasibility of a meta-analysis approach to identify stronger and more convincing association between gene polymorphisms and outcome, potentially leading the way to a new method of analysis for similar data set.

Cover Image: DNA methylation and gene expression

Genome‐wide DNA methylation patterns in plants are dynamically regulated by the DNA methylation and demethylation pathways. In this issue, Xiao et al. (110–119) and Zhao et al. (120–139) published two studies back to back to show that a protein complex composed of Su(var)3–9 homologs (SUVHs) and SUVH‐interacting DNAJ (SDJ) proteins can recognize DNA methylation in the promoter of demethylase ROS1 and is required for activating the expression of ROS1 and other promoter‐methylated genes in Arabidopsis, thus dynamically regulate DNA methylation.

Thymine DNA glycosylase recognizes the geometry alteration of minor grooves induced by 5-formylcytosine and 5-carboxylcytosine.

The dynamic DNA methylation-demethylation process plays critical roles in gene expression control and cell development. The oxidation derivatives of 5-methylcytosine (5mC) generated by Tet dioxygenases in the demethylation pathway, namely 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), could impact biological functions by altering DNA properties or recognition by potential reader proteins. Hence, in addition to the fifth base 5mC, 5hmC, 5fC, and 5caC have been considered as the sixth, seventh, and eighth bases of the genome. How these modifications would alter DNA and be specifically recognized remain unclear, however. Here we report that formyl- and carboxyl-modifications on cytosine induce the geometry alteration of the DNA minor groove by solving two high-resolution structures of a dsDNA decamer containing fully symmetric 5fC and 5caC. The alterations are recognized distinctively by thymine DNA glycosylase TDG via its finger residue R275, followed by subsequent preferential base excision and DNA repair. These observations suggest a mechanism by which reader proteins distinguish highly similar cytosine modifications for potential differential demethylation in order to achieve downstream biological functions.

Reaction Mechanism and Substrate Specificity of Iso-orotate Decarboxylase: A Combined Theoretical and Experimental Study.

The C-C bond cleavage catalyzed by metal-dependent iso-orotate decarboxylase (IDCase) from the thymidine salvage pathway is of interest for the elucidation of a (hypothetical) DNA demethylation pathway. IDCase appears also as a promising candidate for the synthetic regioselective carboxylation of N-heteroaromatics. Herein, we report a joint experimental-theoretical study to gain insights into the metal identity, reaction mechanism, and substrate specificity of IDCase. In contrast to previous assumptions, the enzyme is demonstrated by ICPMS/MS measurements to contain a catalytically relevant Mn2+ rather than Zn2+. Quantum chemical calculations revealed that decarboxylation of the natural substrate (5-carboxyuracil) proceeds via a (reverse) electrophilic aromatic substitution with formation of CO2. The occurrence of previously proposed tetrahedral carboxylate intermediates with concomitant formation of could be ruled out on the basis of prohibitively high energy barriers. In contrast to related o-benzoic acid decarboxylases, such as γ-resorcylate decarboxylase and 5-carboxyvanillate decarboxylase, which exhibit a relaxed substrate tolerance for phenolic acids, IDCase shows high substrate fidelity. Structural and energy comparisons suggest that this is caused by a unique hydrogen bonding of the heterocyclic natural substrate (5-carboxyuracil) to the surrounding residues. Analysis of calculated energies also shows that the reverse carboxylation of uracil is impeded by a strongly disfavored uphill reaction.

Abstract B075: The role of TET1 and hydroxymethylation in high-risk prostate cancer

Abstract The Ten-eleven translocation (TET) proteins are dioxygenases that catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) as part of the DNA demethylation pathway. Gene silencing through cytosine methylation contributes to cancer formation, but how hydroxymethylation affects gene expression is largely unknown. In this work, TET1 and genomic hydroxymethylation were investigated in a high-risk prostate cancer cohort for which exome sequencing data are available (1). Immunohistochemical analysis of tumor versus nontumor prostate biopsies from high-risk prostate cancer showed a strong reduction of genome-wide 5hmC and milder but still significant reduction of 5mC levels in prostate cancer tissue compared to control tissue. Copy number analysis of high-risk prostate cancer showed a loss of the TET1 genomic region in 6 out of 39 samples of our cohort. As expected, the mRNA levels of TET1 were decreased in these tumor samples. Recently, an alternative promoter of TET1 was discovered. This promoter generates a novel isoform lacking the CXXC-domain, which is called TET1ALT (2). We therefore performed functional analyses of the two TET1 promoters in prostate cancer cells. Interestingly, the promoter controlling expression of the endogenous full-length TET1 is less active in prostate cancer cell line PC-3, while the alternative promoter generating TET1ALT is more active in these cells. This is shown by luciferase constructs driven by the two promoter regions. In conclusion, our findings indicate that there is a drop in (hydroxy)methylation levels in high-risk prostate cancer that correlates with decreased TET1 expression levels and in some cases by a copy number loss of TET1. We are currently working on a more detailed analysis of TET1 promoter usage and control. This will unravel the regulation of hydroxymethylation in prostate cancer, which is crucial for the understanding of prostate cancer development and progression.

Defining the impact of sumoylation on substrate binding and catalysis by thymine DNA glycosylase.

Thymine DNA glycosylase (TDG) excises thymine from mutagenic G·T mispairs generated by deamination of 5-methylcytosine (mC) and it removes two mC derivatives, 5−formylcytosine (fC) and 5−carboxylcytosine (caC), in a multistep pathway for DNA demethylation. TDG is modified by small ubiquitin-like modifier (SUMO) proteins, but the impact of sumoylation on TDG activity is poorly defined and the functions of TDG sumoylation remain unclear. We determined the effect of TDG sumoylation, by SUMO-1 or SUMO-2, on substrate binding and catalytic parameters. Single turnover experiments reveal that sumoylation dramatically impairs TDG base-excision activity, such that G·T activity is reduced by ≥45-fold and fC and caC are excised slowly, with a reaction half-life of ≥9 min (37°C). Fluorescence anisotropy studies reveal that unmodified TDG binds tightly to G·fC and G·caC substrates, with dissociation constants in the low nanomolar range. While sumoylation of TDG weakens substrate binding, the residual affinity is substantial and is comparable to that of biochemically-characterized readers of fC and caC. Our findings raise the possibility that sumoylation enables TDG to function, at least transiently, as reader of fC and caC. Notably, sumoylation could potentially facilitate TDG recruitment of other proteins, including transcription factors or epigenetic regulators, to these sites in DNA.