Loss of 5-hydroxymethylcytosine is linked to gene body hypermethylation in kidney cancer

Both 5-methylcytosine (5mC) and its oxidized form 5-hydroxymethylcytosine (5hmC) have been proposed to be involved in tumorigenesis. Because the readout of the broadly used 5mC mapping method, bisulfite sequencing (BS-seq), is the sum of 5mC and 5hmC levels, the 5mC/5hmC patterns and relationship of these two modifications remain poorly understood. By profiling real 5mC (BS-seq corrected by Tet-assisted BS-seq, TAB-seq) and 5hmC (TAB-seq) levels simultaneously at single-nucleotide resolution, we here demonstrate that there is no global loss of 5mC in kidney tumors compared with matched normal tissues. Conversely, 5hmC was globally lost in virtually all kidney tumor tissues. The 5hmC level in tumor tissues is an independent prognostic marker for kidney cancer, with lower levels of 5hmC associated with shorter overall survival. Furthermore, we demonstrated that loss of 5hmC is linked to hypermethylation in tumors compared with matched normal tissues, particularly in gene body regions. Strikingly, gene body hypermethylation was significantly associated with silencing of the tumor-related genes. Downregulation of IDH1 was identified as a mechanism underlying 5hmC loss in kidney cancer. Restoring 5hmC levels attenuated the invasion capacity of tumor cells and suppressed tumor growth in a xenograft model. Collectively, our results demonstrate that loss of 5hmC is both a prognostic marker and an oncogenic event in kidney cancer by remodeling the DNA methylation pattern.     

A sensitive mass spectrometry method for simultaneous quantification of DNA methylation and hydroxymethylation levels in biological samples

The recent discovery of 5-hydroxymethyl-cytosine (5hmC) in embryonic stem cells and postmitotic neurons has triggered the need for quantitative measurements of both 5-methyl-cytosine (5mC) and 5hmC in the same sample. We have developed a method using liquid chromatography electrospray ionization tandem mass spectrometry with multiple reaction monitoring (LC–ESI–MS/MS–MRM) to simultaneously measure levels of 5mC and 5hmC in digested genomic DNA. This method is fast, robust, and accurate, and it is more sensitive than the current 5hmC quantitation methods such as end labeling with thin layer chromatography and radiolabeling by glycosylation. Only 50 ng of digested genomic DNA is required to measure the presence of 0.1% 5hmC in DNA from mouse embryonic stem cells. Using this procedure, we show that human induced pluripotent stem cells exhibit a dramatic increase in 5mC and 5hmC levels compared with parental fibroblast cells, suggesting a dynamic regulation of DNA methylation and hydroxymethylation during cellular reprogramming.     

Generation and replication-dependent dilution of 5fC and 5caC during mouse preimplantation development

One of the recent advances in the epigenetic field is the demonstration that the Tet family of proteins are capable of catalyzing conversion of 5-methylcytosine (5mC) of DNA to 5-hydroxymethylcytosine (5hmC). Interestingly, recent studies have shown that 5hmC can be further oxidized by Tet proteins to generate 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), which can be removed by thymine DNA glycosylase (TDG). To determine whether Tet-catalyzed conversion of 5mC to 5fC and 5caC occurs in vivo in zygotes, we generated antibodies specific for 5fC and 5caC. By immunostaining, we demonstrate that loss of 5mC in the paternal pronucleus is concurrent with the appearance of 5fC and 5caC, similar to that of 5hmC. Importantly, instead of being quickly removed through an enzyme-catalyzed process, both 5fC and 5caC exhibit replication-dependent dilution during mouse preimplantation development. These results not only demonstrate the conversion of 5mC to 5fC and 5caC in zygotes, but also indicate that both 5fC and 5caC are relatively stable and may be functional during preimplantation development. Together with previous studies, our study suggests that Tet-catalyzed conversion of 5mC to 5hmC/5fC/5caC followed by replication-dependent dilution accounts for paternal DNA demethylation during preimplantation development.     

Dynamic reprogramming of 5-hydroxymethylcytosine during early porcine embryogenesis

DNA active demethylation is an important epigenetic phenomenon observed in porcine zygotes, yet its molecular origins are unknown. Our results show that 5-methylcytosine (5mC) converts into 5-hydroxymethylcytosine (5hmC) during the first cell cycle in porcine in vivo fertilization (IVV), IVF, and SCNT embryos, but not in parthenogenetically activated embryos. Expression of Ten-Eleven Translocation 1 (TET1) correlates with this conversion. Expression of 5mC gradually decreases until the morula stage; it is only expressed in the inner cell mass, but not trophectoderm regions of IVV and IVF blastocysts. Expression of 5mC in SCNT embryos is ectopically distinct from that observed in IVV and IVF embryos. In addition, 5hmC expression was similar to that of 5mC in IVV cleavage-stage embryos. Expression of 5hmC remained constant in IVF and SCNT embryos, and was evenly distributed among the inner cell mass and trophectoderm regions derived from IVV, IVF, and SCNT blastocysts. Ten-Eleven Translocation 3 was highly expressed in two-cell embryos, whereas TET1 and TET2 were highly expressed in blastocysts. These data suggest that TET1-catalyzed 5hmC may be involved in active DNA demethylation in porcine early embryos. In addition, 5mC, but not 5hmC, participates in the initial cell lineage specification in porcine IVV and IVF blastocysts. Last, SCNT embryos show aberrant 5mC and 5hmC expression during early porcine embryonic development.     

Accurate Measurement of 5-Methylcytosine and 5-Hydroxymethylcytosine in Human Cerebellum DNA by Oxidative Bisulfite on an Array (OxBS-Array)

The Infinium 450K Methylation array is an established tool for measuring methylation. However, the bisulfite (BS) reaction commonly used with the 450K array cannot distinguish between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). The oxidative-bisulfite assay disambiguates 5mC and 5hmC. We describe the use of oxBS in conjunction with the 450K array (oxBS-array) to analyse 5hmC/5mC in cerebellum DNA. The “methylation” level derived by the BS reaction is the combined level of 5mC and 5hmC at a given base, while the oxBS reaction gives the level of 5mC alone. The level of 5hmC is derived by subtracting the oxBS level from the BS level. Here we present an analysis method that distinguishes genuine positive levels of 5hmC at levels as low as 3%. We performed four replicates of the same sample of cerebellum and found a high level of reproducibility (average r for BS = 98.3, and average r for oxBS = 96.8). In total, 114,734 probes showed a significant positive measurement for 5hmC. The range at which we were able to distinguish 5hmC occupancy was between 3% and 42%. In order to investigate the effects of multiple replicates on 5hmC detection we also simulated fewer replicates and found that decreasing the number of replicates to two reduced the number of positive probes identified by > 50%. We validated our results using qPCR in conjunction with glucosylation of 5hmC sites followed by MspI digestion and we found good concordance with the array estimates (r = 0.94). This experiment provides a map of 5hmC in the cerebellum and a robust dataset for use as a standard in future 5hmC analyses. We also provide a novel method for validating the presence of 5hmC at low levels, and highlight some of the pitfalls associated with measuring 5hmC and 5mC.     

Placental 5-methylcytosine and 5-hydroxymethylcytosine patterns associate with size at birth

Altered placental function as a consequence of aberrant imprinted gene expression may be one mechanism mediating the association between low birth weight and increased cardiometabolic disease risk. Imprinted gene expression is regulated by epigenetic mechanisms, particularly DNA methylation (5mC) at differentially methylated regions (DMRs). While 5-hydroxymethylcytosine (5hmC) is also present at DMRs, many techniques do not distinguish between 5mC and 5hmC. Using human placental samples, we show that the expression of the imprinted gene CDKN1C associates with birth weight. Using specific techniques to map 5mC and 5hmC at DMRs controlling the expression of CDKN1C and the imprinted gene IGF2, we show that 5mC enrichment at KvDMR and DMR0, and 5hmC enrichment within the H19 gene body, associate positively with birth weight. Importantly, the presence of 5hmC at imprinted DMRs may complicate the interpretation of DNA methylation studies in placenta; future studies should consider using techniques that distinguish between, and permit quantification of, both modifications.     

Dynamics of 5-carboxylcytosine during hepatic differentiation: Potential general role for active demethylation by DNA repair in lineage specification

Patterns of DNA methylation (5-methylcytosine, 5mC) are rearranged during differentiation contributing to the regulation of cell type-specific gene expression. TET proteins oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be recognized and excised from DNA by thymine-DNA glycosylase (TDG) followed by the subsequent incorporation of unmodified cytosine into the abasic site via the base excision repair (BER) pathway. We previously demonstrated that 5caC accumulates during lineage specification of neural stem cells (NSCs) suggesting that such active demethylation pathway is operational in this system; however, it is still unknown if TDG/BER-dependent demethylation is used during other types of cellular differentiation. Here we analyze dynamics of the global levels of 5hmC and 5caC during differentiation of human pluripotent stem cells toward hepatic endoderm. We show that, similar to differentiating NSCs, 5caC transiently accumulates during hepatic differentiation. The levels of 5caC increase during specification of foregut, peak at the stage of hepatic endoderm commitment, and drop in differentiating cells concurrently with the onset of expression of α fetoprotein, a marker of committed hepatic progenitors. Moreover, we show that 5caC accumulates at promoter regions of several genes expressed during hepatic specification at differentiation stages corresponding to the beginning of their expression. Our data indicate that transient 5caC accumulation is a common feature of 2 different types (neural/glial and endoderm/hepatic) of cellular differentiation. This suggests that oxidation of 5mC may represent a general mechanism of rearrangement of 5mC profiles during lineage specification of somatic cells in mammals.     

Lead exposure induces changes in 5-hydroxymethylcytosine clusters in CpG islands in human embryonic stem cells and umbilical cord blood

Prenatal exposure to neurotoxicants such as lead (Pb) may cause stable changes in the DNA methylation (5mC) profile of the fetal genome. However, few studies have examined its effect on the DNA de-methylation pathway, specifically the dynamic changes of the 5-hydroxymethylcytosine (5hmC) profile. Therefore, in this study, we investigate the relationship between Pb exposure and 5mC and 5hmC modifications during early development. To study the changes in the 5hmC profile, we use a novel modification of the Infinium™ HumanMethylation450 assay (Illumina, Inc.), which we named HMeDIP-450K assay, in an in vitro human embryonic stem cell model of Pb exposure. We model Pb exposure-associated 5hmC changes as clusters of correlated, adjacent CpG sites, which are co-responding to Pb. We further extend our study to look at Pb-dependent changes in high density 5hmC regions in umbilical cord blood DNA from 48 mother-infant pairs from the Early Life Exposure in Mexico to Environmental Toxicants (ELEMENT) cohort. For our study, we randomly selected umbilical cord blood from 24 male and 24 female children from the 1st and 4th quartiles of Pb levels. Our data show that Pb-associated changes in the 5hmC and 5mC profiles can be divided into sex-dependent and sex-independent categories. Interestingly, differential 5mC sites are better markers of Pb-associated sex-dependent changes compared to differential 5hmC sites. In this study we identified several 5hmC and 5mC genomic loci, which we believe might have some potential as early biomarkers of prenatal Pb exposure.     

5‐Hydroxymethylcytosine: a new kid on the epigenetic block?

The discovery of the Ten‐Eleven‐Translocation (TET) oxygenases that catalyze the hydroxylation of 5‐methylcytosine (5mC) to 5‐hydroxymethylcytosine (5hmC) has triggered an avalanche of studies aiming to resolve the role of 5hmC in gene regulation if any. Hitherto, TET1 is reported to bind to CpG‐island (CGI) and bivalent promoters in mouse embryonic stem cells, whereas binding at DNAseI hypersensitive sites (HS) had escaped previous analysis. Significant enrichment/accumulation of 5hmC but not 5mC can indeed be detected at bivalent promoters and at DNaseI‐HS. Surprisingly, however, 5hmC is not detected or present at very low levels at CGI promoters notwithstanding the presence of TET1. Our meta‐analysis of DNA methylation profiling points to potential issues with regard to the various methodologies that are part of the toolbox used to detect 5mC and 5hmC. Discrepancies between published studies and technical limitations prevent an unambiguous assignment of 5hmC as a ‘true’ epigenetic mark, that is, read and interpreted by other factors and/or as a transiently accumulating intermediary product of the conversion of 5mC to unmodified cytosines.     

5-羟甲基胞嘧啶的研究进展

CpG二核苷酸中胞嘧啶的甲基化形式5-甲基胞嘧啶(5-methylcytosine,5mC)在哺乳动物中是一种常见的表观遗传修饰,在基因表达调控、发育调节、基因组印迹等方面发挥重要作用。近3年来研究发现,除了5mC外,胞嘧啶碱基的另一种修饰—5-羟甲基胞嘧啶(5-hydroxymethylcytosine,5hmC)在哺乳动物的多种组织中有着丰富的表达,它可能与5mC有着不同的生物学功能。文章就近年来5hmC的研究进展进行了综述。     

Global 5-Hydroxymethylcytosine Levels Are Profoundly Reduced in Multiple Genitourinary Malignancies

Solid tumors are characterized by a plethora of epigenetic changes. In particular, patterns methylation of cytosines at the 5-position (5mC) in the context of CpGs are frequently altered in tumors. Recent evidence suggests that 5mC can get converted to 5-hydroxylmethylcytosine (5hmC) in an enzymatic process involving ten eleven translocation (TET) protein family members, and this process appears to be important in facilitating plasticity of cytosine methylation. Here we evaluated the global levels of 5hmC using a validated immunohistochemical staining method in a large series of clear cell renal cell carcinoma (n = 111), urothelial cell carcinoma (n = 55) and testicular germ cell tumors (n = 84) and matched adjacent benign tissues. Whereas tumor-adjacent benign tissues were mostly characterized by high levels of 5hmC, renal cell carcinoma and urothelial cell carcinoma showed dramatically reduced staining for 5hmC. 5hmC levels were low in both primary tumors and metastases of clear cell renal cell carcinoma and showed no association with disease outcomes. In normal testis, robust 5hmC staining was only observed in stroma and Sertoli cells. Seminoma showed greatly reduced 5hmC immunolabeling, whereas differentiated teratoma, embryonal and yolk sack tumors exhibited high 5hmC levels. The substantial tumor specific loss of 5hmC, particularly in clear cell renal cell carcinoma and urothelial cell carcinoma, suggests that alterations in pathways involved in establishing and maintaining 5hmC levels might be very common in cancer and could potentially be exploited for diagnosis and treatment.     

5-羟甲基胞嘧啶的研究进展

CpG二核苷酸中胞嘧啶的甲基化形式5-甲基胞嘧啶(5-methylcytosine, 5mC)在哺乳动物中是一种常见的表观遗传修饰, 在基因表达调控、发育调节、基因组印迹等方面发挥重要作用。近3年来研究发现, 除了5mC外, 胞嘧啶碱基的另一种修饰-5-羟甲基胞嘧啶(5-hydroxymethylcytosine, 5hmC)在哺乳动物的多种组织中有着丰富的表达, 它可能与5mC有着不同的生物学功能。文章就近年来5hmC的研究进展进行了综述。     

Decreased 5-hydroxymethylcytosine is associated with neural progenitor phenotype in normal brain and shorter survival in malignant glioma

Epigenetic modification of DNA by cytosine methylation to produce 5-methylcytosine (5mC) has become well-recognized as an important epigenetic process in human health and disease. Recently, further modification of 5mC by the ten eleven translocated (TET) family of enzymes to produce 5-hydroxymethylcytosine (5hmC) has been described. In the present study, we used immunohistochemistry to evaluate the distribution of 5hmC in human brain during different periods of development and in a large series of gliomas (n=225). We found that during development, 5hmC levels are high in more differentiated compartments like the fetal cortex, but low in the periventricular progenitor cell regions. In adults, we found 5hmC levels to be highest in the cortex, but present in all intrinsic cell types in the brain including stromal elements. In brain tumors, 5hmC levels were high in low grade tumors and reduced in malignant glioma, but did not exhibit any correlation with IDH1 mutation status. Additionally, we identified a significant relationship between low levels of 5hmC and reduced survival in malignant glioma. This observation was further supported by in silico analysis showing differential expression of genes involved in 5hmC homeostasis in aggressive subsets of glioblastoma. Finally, we show that several genes involved in regulating the levels of 5hmC are also prognostic in malignant glioma. These findings suggest that 5hmC regulation in malignant glioma may represent an important determinant of tumor differentiation and aggressive behavior, as well as a potential therapeutic target.     

Genome-wide regulation of 5hmC, 5mC, and gene expression by Tet1 hydroxylase in mouse embryonic stem cells

DNA methylation at the 5 position of cytosine (5mC) in the mammalian genome is a key epigenetic event critical for various cellular processes. The ten-eleven translocation (Tet) family of 5mC-hydroxylases, which convert 5mC to 5-hydroxymethylcytosine (5hmC), offers a way for dynamic regulation of DNA methylation. Here we report that Tet1 binds to unmodified C or 5mC- or 5hmC-modified CpG-rich DNA through its CXXC domain. Genome-wide mapping of Tet1 and 5hmC reveals mechanisms by which Tet1 controls 5hmC and 5mC levels in mouse embryonic stem cells (mESCs). We also uncover a comprehensive gene network influenced by Tet1. Collectively, our data suggest that Tet1 controls DNA methylation both by binding to CpG-rich regions to prevent unwanted DNA methyltransferase activity, and by converting 5mC to 5hmC through hydroxylase activity. This Tet1-mediated antagonism of CpG methylation imparts differential maintenance of DNA methylation status at Tet1 targets, ultimately contributing to mESC differentiation and the onset of embryonic development.     

表观遗传新标志物5hmC的研究进展

5-羟甲基胞嘧啶(5-hydroxymethylcytosine,5hmC)作为表观遗传的新标志物,已引起人们的极大兴趣.5hmC由TET家族酶催化氧化5-甲基胞嘧啶(5-methylcytosine,5mC)产生,被称为高等生物基因组DNA的"第六碱基".5hmC不仅可以影响基因组结构及功能,还在早期胚胎发育中发挥重要的作用.本文综述了5hmC的代谢通路、生物学功能、在基因组的分布及分析方法的研究进展.