TET3 dioxygenase modulates gene conversion at the avian immunoglobulin variable region via demethylation of non-CpG sites in pseudogene templates.

Diversification of the avian primary immunoglobulin (Ig) repertoire is achieved in developing B cells by somatic hypermutation (SHM) and gene conversion (GCV). GCV is a type of homologous recombination that unidirectionally transfers segments of Ig pseudogenes to Ig variable domains. It is regulated by epigenetic mechanisms like histone modifications, but the role of DNA methylation remains unclear. Here, we demonstrate that the chicken B-cell line DT40 lacking TET3, a member of the TET (Ten-eleven translocation) family dioxygenases that facilitate DNA demethylation, exhibited a marked reduction in GCV activity in Ig variable regions. This was accompanied by a drop in the bulk levels of 5-hydroxymethylcytosine, an oxidized derivative of 5-methylcytosine, whereas TET1-deficient or TET2-deficient DT40 strains did not exhibit such effects. Deletion of TET3 caused little effects on the expression of proteins required for SHM and GCV, but induced hypermethylation in some Ig pseudogene templates. Notably, the enhanced methylation occurred preferably on non-CpG cytosines. Disruption of both TET1 and TET3 significantly inhibited the expression of activation-induced cytidine deaminase (AID), an essential player in Ig diversification. These results uncover unique roles of TET proteins in avian Ig diversification, highlighting the potential importance of TET3 in maintaining hypomethylation In Ig pseudogenes.

Epigenetic Factors in Eutopic Endometrium in Women with Endometriosis and Infertility.

Eutopic endometrium in patients with endometriosis is characterized by aberrant expression of essential genes during the implantation window. It predisposes to disturbance of endometrial receptivity. The pathomechanism of implantation failures in women with endometriosis remains unclear. This paper aims to summarize the knowledge on epigenetic mechanisms in eutopic endometrium in the group of patients with both endometriosis and infertility. The impaired DNA methylation patterns of gene promoter regions in eutopic tissue was established. The global profile of histone acetylation and methylation and the analysis of selected histone modifications showed significant differences in the endometrium of women with endometriosis. Aberrant expression of the proposed candidate genes may promote an unfavorable embryonic implantation environment of the endometrium due to an immunological dysfunction, inflammatory reaction, and apoptotic response in women with endometriosis. The role of the newly discovered proteins regulating gene expression, i.e., TET proteins, in endometrial pathology is not yet completely known. The cells of the eutopic endometrium in women with endometriosis contain a stable, impaired methylation pattern and a histone code. Medication targeting critical genes responsible for the aberrant gene expression pattern in eutopic endometrium may help treat infertility in women with endometriosis.

Function determinants of TET proteins: the arrangements of sequence motifs with specific codes.

The ten-eleven translocation (TET) proteins play a crucial role in promoting locus-specific reversal of DNA methylation, a type of chromatin modification. Considerable evidences have demonstrated that the sequence motifs with specific codes are important to determine the functions of domains and active sites. Here, we surveyed major studies and reviews regarding the multiple functions of the TET proteins and established the patterns of the motif arrangements that determine the functions of TET proteins. First, we summarized the functional sequence basis of TET proteins and identified the new functional motifs based on the phylogenetic relationship. Next, we described the sequence characteristics of the functional motifs in detail and provided an overview of the relationship between the sequence motifs and the functions of TET proteins, including known functions and potential functions. Finally, we highlighted that sequence motifs with diverse post-translational modifications perform unique functions, and different selection pressures lead to different arrangements of sequence motifs, resulting in different paralogs and isoforms.

Association of TET1 expression with colorectal cancer progression.

OBJECTIVE: The ten-eleven translocation (TET) proteins, as methylcytosine dioxygenases, catalyze 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). The altered expression of TET1 disrupts the balance between DNA methylation and demethylation. This alteration has been reported to be associated with carcinogenesis in various malignancies. The aim of the present study was to investigate changes in expression and the role of TET1 in colorectal cancer (CRC). MATERIAL AND METHODS: A total of 109 CRC patients who underwent radical surgical colon resection were enrolled. The QuantiGene Plex Assay was used to detect the expression of TET1 in CRC tissues and matching adjacent normal tissues. We analyzed the associations between TET1 expression levels and various clinicopathologic features of CRC. TET1 overexpression and depletion cells were constructed to investigate its biological role in CRC. RESULTS: Compared to normal tissues, the expression level of TET1 in CRC was significantly lower. The ratio of TET1 in CRC tissues to that in adjacent normal tissues (C/N-TET1) was an independent overall survival predictive factor. Moreover, in vitro studies showed that TET1 could inhibit cell growth and promote cell metastasis and invasion. CONCLUSIONS: These findings indicated that TET1 played a multifaceted role in the pathogenesis of CRC, and thereby resulting in multiple effects on tumor progression.

Molecular Evolution of the TET Gene Family in Mammals.

Ten-eleven translocation (TET) proteins, a family of Fe(2+)- and 2-oxoglutarate-dependent dioxygenases, are involved in DNA demethylation. They also help regulate various cellular functions. Three TET paralogs have been identified (TET1, TET2, and TET3) in humans. This study focuses on the evolution of mammalian TET genes. Distinct patterns in TET1 and TET2 vs. TET3 were revealed by codon-based tests of positive selection. Results indicate that TET1 and TET2 genes have experienced positive selection more frequently than TET3 gene, and that the majority of codon sites evolved under strong negative selection. These findings imply that the selective pressure on TET3 may have been relaxed in several lineages during the course of evolution. Our analysis of convergent amino acid substitutions also supports the different evolutionary dynamics among TET gene subfamily members. All of the five amino acid sites that are inferred to have evolved under positive selection in the catalytic domain of TET2 are localized at the protein's outer surface. The adaptive changes of these positively selected amino acid sites could be associated with dynamic interactions between other TET-interacting proteins, and positive selection thus appears to shift the regulatory scheme of TET enzyme function.

TET protein inhibitors: Potential and limitations.

TET proteins (methylcytosine dioxygenases) play an important role in the regulation of gene expression. Dysregulation of their activity is associated with many serious pathogenic states such as oncological diseases. Regulation of their activity by specific inhibitors could represent a promising therapeutic strategy. Therefore, this review describes various types of TET protein inhibitors in terms of their inhibitory mechanism and possible applicability. The potential and possible limitations of this approach are thoroughly discussed in the context of TET protein functionality in living systems. Furthermore, possible therapeutic strategies based on the inhibition of TET proteins are presented and evaluated, especially in the field of oncological diseases.

Assessment of TET1 gene expression, DNA methylation and H3K27me3 level of its promoter region in eutopic endometrium of women with endometriosis and infertility.

Endometriosis is the cause of infertility. The eutopic endometrium of women with endometriosis showed an aberrant expression pattern of multitude genes. The role of TET1 protein in the pathogenesis of endometriosis and related infertility is not sufficiently known. Further, knowledge on TET1 transcriptional control still remains incomplete. The aim of the study was assessment of TET1 gene expression, DNA methylation and H3K27me3 level of its promoter region in eutopic endometrium of women with endometriosis and infertility. The study included 44 infertile patients with endometriosis (IWE) and 77 infertile (IW) and fertile (FW) patients without endometriosis. The research material was eutopic endometrium. The TET1 mRNA level was analyzed by qPCR. Western blot was used to evaluate the level of TET1 protein. The level of DNA methylation and H3K27me3 level of TET1 gene's promoter region were assessed using HRM and ChIP qPCR, respectively. The level of TET1 expression (TET1 mRNA; TET1 protein level) was lower in IWE during the implantation window (p < 0.001; p = 0.0329). The level of TET1 DNA methylation was higher in the secretory endometrium in mild and advanced IWE (p < 0.004; p < 0.008). H3K27me3 level did not differ between the study groups. The diminished expression of TET1 gene during the secretory phase, may account for the aberrant process of embryonic implantation in infertile endometriosis patients. DNA hypermethylation of TET1 gene is a potential relevant regulator of its expression. H3K27me3 occupancy does not affect the expression of TET1 gene in our study group.

Vaginal Aging-What We Know and What We Do Not Know.

The aging of the organism is a complex and multifactorial process. It can be viewed in the context of the whole organism, but also of individual tissues and organs. The problem of vaginal aging and the related genitourinary syndrome of menopause significantly reduces the quality of women's lives. The aging process of the vagina includes estrogen deficiencies, changes in the microbiome, and changes at the genetic level associated with DNA methylation. During the menopause, the number of Lactobacillus colonies decreases, and the number of pathological bacteria colonies increases. The decrease in estrogen levels results in a decrease in vaginal epithelial permeability, perfusion, and elastin levels, resulting in vaginal dryness and atrophy. Changes at the molecular level are the least clear. It can also be assumed that, similarly to the tissues studied so far, there are changes in cytosine methylation and TET (ten-eleven translocation) expression. The interrelationships between DNA methylation, hormonal changes, and the vaginal microbiome have not yet been fully elucidated.

[The expression and significance of TET gene and 5hmC in chronic rhinosinusitis].

Objective:To investigate the expression and clinical significance of TET gene and 5hmC in chronic sinusitis. Methods:Acquiring 20 cases of nasal polyps from chronic sinusitis with polyps（CW）, 20 cases of uncinate process tissues from chronic sinusitis without polyps（CS）, 10 cases of middle turbinate tissues from patients with nasal septum deviated as normal group（N）.The expression of TET gene and 5hmC in chronic sinusitis was measured by immunofluorescence, Western-Blot and Quantitative real-time PCR. Results:Both TET1/2/3 gene and 5 hmC were highly expressed in the CS group, but were lower in the CW group and the N group.There were statistically significant differences between the CS group and the CW group（P<0.05）, and between the CS group and the N group（P<0.05）.The expressions of TET1 and TET3 in the N group were higher than those in the CW group, and the difference was statistically significant（P<0.05）. Conclusion:DNA demethylation plays an important role in the formation of nasal polyps.High expression of TET1, TET2, TET3 and 5hmC May reduce the risk of nasal polyps.Increased DNA demethylation in chronic sinusitis may reduce the risk of nasal polyp formation.When the degree of DNA methylation in chronic sinusitis is high and the degree of DNA demethylation is low, the disease may develop to CRSwNP, and when the degree of DNA methylation is low and the degree of DNA demethylation is high, the disease may develop to CRSsNP.

The Battle between TET Proteins and DNA Methylation for the Right Cell.

DNA methylation is an important epigenetic barrier during somatic cell reprogramming. Yet, how genome-wide methylation is reprogrammed remains largely unknown. Sardina et al. (Cell Stem Cell 2018:https://doi.org/10.1016/j.stem.2018.08.016) address this question by mapping the DNA methylomes of cells undergoing reprogramming and show that TET proteins and transcription factors cooperate to orchestrate demethylation critical to reprogramming.

Ten-Eleven Translocation Proteins Modulate the Response to Environmental Stress in Mice.

5-hydroxymethylcytosine (5hmC) is enriched in brain and has been recognized as an important DNA modification. However, the roles of 5hmC and its writers, ten-eleven translocation (Tet) proteins, in stress-induced response have yet to be elucidated. Here, we show that chronic restraint stress (CRS) induced depression-like behavior in mice and resulted in a 5hmC reduction in prefrontal cortex (PFC). We found that loss of Tet1 (Tet1 KO) led to resistance to CRS, whereas loss of Tet2 (Tet2 KO) increased the susceptibility of mice to CRS. Genome-wide 5hmC profiling identified the phenotype-associated stress-induced dynamically hydroxymethylated loci (PA-SI-DhMLs), which are strongly enriched with hypoxia-induced factor (HIF) binding motifs. We demonstrated the physical interaction between TET1 and HIF1α induced by CRS and revealed that the increased HIF1α binding under CRS is associated with SI-DhMLs. These results suggest that TET1 could regulate stress-induced response by interacting with HIF1α.

Effects of intracerebral hemorrhage on 5-hydroxymethylcytosine modification in mouse brains.

The past decade has resulted in an increase in the knowledge of molecular mechanisms underlying brain injury induced by intracerebral hemorrhage (ICH). Recent advances have provided a link between epigenetic modification and the regulation of gene expression. 5-hydroxymethylcytosine (5hmC) converted from 5-methylcytosine by the ten-eleven translocation (TET) family of proteins has emerged as a new epigenetic modification. While the dynamics of 5hmC during cerebral ischemia have recently been reported, whether 5hmC is involved in ICH remains unexplored. In this study, we investigated the effects of ICH on DNA hydroxymethylation. We showed that the global level of 5hmC rapidly decreased as early as 24 hours after ICH and persisted until 72 hours. Furthermore, the level of 5hmC in the CpG-rich regions of Akt2, Pdpk1 and Vegf genes was significantly decreased with a minimum level observed at 48 hours or 72 hours. Decreased 5hmC was observed in parallel with an increase in 5-methylcytosine over this time course, and mRNA levels of Akt2, Pdpk1 and Vegf were downregulated upon ICH injury. Finally, Tet1, Tet2 and Tet3 mRNA levels were dramatically decreased in the ICH brain. Our study for the first time established the correlation between DNA hydroxymethylation and ICH injury. Further investigations should examine whether 5hmC modification could be a therapeutic target for the treatment of ICH injury.

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.

5-Hydroxymethylcytosine: A new player in brain disorders?

5-Hydroxymethylcytosine (5 hmC), a novel modified cytosine, is oxidized from 5-methylcytosine (5 mC) by the ten-eleven translocation (Tet) protein family. The specific distribution of 5 hmC in mammalian brain and its roles in gene regulation suggest that 5 hmC is important in brain development. 5 hmC may also contribute to the mechanisms underlying neurological diseases. Here, we summarize the current knowledge of 5 hmC, with an emphasis on its roles in neurodevelopmental and neurodegenerative disorders.