InteractiveFly: GeneBrief

Ten-Eleven Translocation (TET) family protein: Biological Overview | References

Enzymes of the ten-eleven translocation (TET) family play a key role in the regulation of gene expression by oxidizing 5-methylcytosine (5mC), a prominent epigenetic mark in many species. Yet, TET proteins also have less characterized noncanonical modes of action, notably in Drosophila, whose genome is devoid of 5mC. This study shows that Drosophila TET activates the expression of genes required for larval central nervous system (CNS) development mainly in a catalytic-independent manner. Genome-wide profiling shows that TET is recruited to enhancer and promoter regions bound by Polycomb group complex (PcG) proteins. TET interacts and colocalizes on chromatin preferentially with Polycomb repressor complex 1 (PRC1) rather than PRC2. Furthermore, PRC1 but not PRC2 is required for the activation of TET target genes. Last, these results suggest that TET and PRC1 binding to activated genes is interdependent. These data highlight the importance of TET noncatalytic function and the role of PRC1 for gene activation in the Drosophila larval CNS (Gilbert, 2024).

Epigenetic enzymes are involved in reversible modifications of DNA or histones, which affect chromatin structure and recruitment of chromatin binding factors. Thereby, they play an important role in genome biology notably by regulating gene transcription, DNA replication, or genome stability. Accordingly, the balanced action of writers, which deposit epigenetic modifications and erasers, which remove these marks, controls cell fate determination and differentiation in multicellular organisms. Mutations affecting these enzymes can cause developmental defects and various pathologies, including cancers and neurodegenerative disorders. Drugs targeting the enzymatic activity of these factors have thus emerged as promising therapeutics. Yet, recent studies revealed that several epigenetic enzymes also have crucial catalytic-independent functions, calling for a careful reexamination of the bases for their loss-of-function phenotypes (Gilbert, 2024).

Since 2009, members of the ten-eleven translocation (TET) family have emerged as key players in the epigenetic regulation of gene expression. TET proteins are Fe2+- and 2-oxoglutarate–dependent dioxygenases capable of oxidizing 5-methylcytosine (5mC) on DNA into 5-hydroxymethylcytosine (5hmC) and further oxidized derivatives, which serve as intermediate products in the cytosine demethylation cascade or as stable epigenetic marks with distinct regulatory roles. As 5mC is a prevalent epigenetic mark in many species, the discovery of 5mC "demethylases" has sparked much interest. TET enzymes are conserved across evolution in metazoans, and their functions have been particularly well studied in mammals, where the three TET paralogs (TET1 to TET3) are implicated in a variety of biological processes including regulation of embryonic stem cell pluripotency, blood cell differentiation, and immune response or nervous system development and neuronal plasticity. Their role is also highlighted in human pathologies as somatic mutations affecting TET genes are frequently associated with the development of various cancers, in particular leukemia, and germline mutations have been linked to blood cell transformation or neurodevelopmental and neurodegenerative disorders (Gilbert, 2024).

So far, TET protein activity has been largely associated with their capacity to oxidize 5mC, thereby regulating transcription by promoting DNA hypomethylation. Consistently, most studies focused on the link between TET and 5mC/5hmC levels, in particular in pathological contexts such as leukemia or neurodevelopmental and neurodegenerative disorders. Still, less-studied noncanonical modes of actions have been described that contribute to TET functions. First, TET can oxidize methylcytosines on RNA (m5C), and it was proposed that TET-mediated hydroxymethylation of mRNA or tRNA regulates gene expression at the posttranscriptional level. Second, it was shown in vertebrates that some TET functions are independent of their enzymatic activity. TET proteins also control gene expression by interacting with other factors implicated in chromatin regulation. For instance, recent evidence showed that TET1 can repress gene expression in a catalytic-independent manner by recruiting the Polycomb repressive complex 2 (PRC2) and the Sin3A deacetylase to target histone H3 lysine 27 (H3K27) modifications in embryonic stem cells (Gilbert, 2024).

The Drosophila genome contains a single and well-conserved Tet gene although it does not contain 5mC DNA methyltransferase genes and is largely devoid of this epigenetic mark. This insect thus stands as a valuable model system to investigate TET noncanonical functions. The complete loss of Tet expression is lethal at the pupal stage, indicating that TET plays a vital role in this organism. Further analyses showed that Tet loss or knockdown affects ovarian development, zygotic genome activation in the early embryo, larval locomotion, as well as larval and adult brain development. At the molecular level, TET function was attributed either to the oxidation of m5C on mRNA to promote translation or to the oxidation of 6-methyladenine (6mA) on DNA to control transcription. However, the direct impact of TET enzymes on mRNA modifications remains disputed notably because hm5C is essentially detected on tRNAs. In addition, the presence and potential significance of 6mA in metazoan genomes remain highly controversial, and the capacity of Drosophila TET to oxidize 6mA is at odds with the conserved structure of its catalytic domain (CD), which lacks crucial amino acids necessary for 6mA recognition and oxidation in more distant TET homologs or the related alkane hydroxylase AlkB family of 6mA demethylase. Along that line, recent results support the conclusion that TET does not act as a 6mA demethylase in Drosophila and showed that, in contrast to TET expression, its catalytic activity is dispensable for adult fly emergence, survival, and reproduction. Thus, TET essentially acts in a catalytic-independent manner in Drosophila, but the underlying molecular mechanisms remain unknown (Gilbert, 2024).

To fill this gap, this study investigated the TET mode of action in the larval central nervous system (CNS). By comparing the phenotypes associated with the absence of TET expression or only of its catalytic activity and using a combination of genetics, transcriptomics, and chromatin profiling, this study showed that TET can directly activate gene transcription independently of its catalytic activity. The results show that TET collaborates with Polycomb components for its gene regulatory activity. Polycomb group (PcG) proteins are organized into two main complexes, the Polycomb repressor complex 1 (PRC1) and PRC2, which are well known for their function in the maintenance of a repressive chromatin state, with PRC2 depositing the H3K27me3 repressive mark and PRC1 the H2AK118ub mark. PcG complexes are also found in active chromatin regions, and several lines of evidence indicate that PRC1 can act independently of PRC2 to activate gene expression, in particular in Drosophila. The data show that TET preferentially colocalizes with PRC1 rather than with PRC2 on chromatin and that TET interacts with PRC1 but not with PRC2 in the larval CNS. Moreover, it appears that PRC1 is required for the activation of TET target genes, while PRC2 is dispensable. In addition, TET and PRC1 facilitate each other’s recruitment to chromatin on TET-activated target genes. These results highlight a hitherto unknown mode of gene expression regulation by TET, which does not require its catalytic activity, and support a role for PRC1 in the activation of gene transcription independently of PRC2 (Gilbert, 2024).

Previous work using Tet null or hypomorphic alleles as well as RNAi-mediated knockdown showed that TET expression is critical for larval CNS development. However, the functional importance of TET enzymatic activity was not assessed in these studies. This study, shows that abolishing TET catalytic activity does not recapitulate the CNS phenotypes observed in Tet null mutant larvae. The large majority of genes deregulated in Tet^null was not affected in Tet^CD. Moreover, gene set enrichment analysis revealed that TET expression, but not its enzymatic activity, is important for the activation of CNS development–associated processes. In particular, several genes implicated in axon guidance were only deregulated in Tet^null, which is consistent with the axonal projection defects observed specifically in this condition. Similarly, the EGFR pathway, which controls the development of ventral midline glial cells, was also affected only in Tet^null. Recent single-cell transcriptomic experiments indicate that Tet is enriched in larval and adult astrocytes, and several astrocyte markers, such as Gaba transporter (Gat), heartless (htl), and stumps are down-regulated specifically in Tet^null, suggesting that TET could control the development of this glial cell population in a catalytic-independent manner. The fibroblast growth factor receptor Htl and its adaptor Stumps are required for astrocyte development, and the down-regulation of Gat or astrocyte ablation leads to crawling and muscular contraction defects as observed in Tet mutant larvae. Moreover, astrocytes are required for axon pruning in the brain mushroom bodies during pupariation, and TET loss is associated with mushroom body abnormalities in the adult. Hence, essential processes underlying CNS formation are controlled by TET independently of its enzymatic activity (Gilbert, 2024).

Yet, many genes were deregulated in the CNS of Tet^CD larvae. It cannot be ruled out that the HRD to YRA mutation in the TET CD might cause unexpected alterations, but it does not seem to modify TET recruitment to DNA, consistent with similar analyses on TET1 in mouse embryonic stem cells. Thus, TET probably exerts some of its functions via a catalytic-dependent mechanism. Although one might have expected that most genes deregulated in Tet^CD were also affected in Tet^null, there was a limited overlap between the two conditions. This suggests that CNS cells in Tet^CD larvae have followed a different developmental trajectory as compared to the null condition. Alternatively, it could reflect a neomorphic behavior of the Tet^CD protein, leading to spurious gene deregulation in the CNS. Still, further experiments will be necessary to decipher the TET catalytic mode of action in Drosophila. In contrast with previous reports, the recent results indicate that TET does not act as a 6mA demethylase. Along that line, the capacity of a distant fungal TET homolog to target 6mA requires specific amino acids, which are not conserved among metazoan TETs. It is thus doubtful that TET catalytic function in the larval CNS is mediated by 6mA demethylation. One hypothesis is that it may involve m5C on RNA. Drosophila and mammalian TETs can oxidize m5C into hm5C and thereby control gene expression at the posttranscriptional level by regulating transcript stability, mRNA translation, or tRNA maturation. Besides, it remains possible that TET targets 5mC DNA in Drosophila, as very low levels of 5mC (0.001 to 0.03%) have been observed in its genome. However, the impact, if any, of 5mC on Drosophila genome biology is still elusive. Last, TET might target other yet unknown substrates at the DNA, RNA, or potentially protein levels (Gilbert, 2024).

Focusing on TET enzymatic-independent mode of action, this study highlights an unexpected link between TET and PRC1. The integration of RNA-seq and ChIP-seq results indicates that TET binding mostly promotes gene expression. This conclusion agrees with a study in BG3-c2 cells and adult brains showing that TET is important for neurodevelopmental gene activation and H3K4me3 accumulation. At the molecular level, it has been proposed that TET interacts with Will die slowly (Wds, WDR5 in mammals), a component of the H3K4 methylation Complex Proteins Associated with Set1 (COMPASS), to activate transcription and demethylate 6mA in gene bodies to prevent Polycomb recruitment and gene repression. While Wds might contribute to TET-induced gene activation in the larval CNS, TET can activate gene expression in an enzymatic-independent manner and that the PcG protein Ph is required in this process. Furthermore, the data show that TET promotes Ph recruitment (and vice versa) and H2AK118ub deposition at the genes it activates. Previous work in mammals showed that TET1 interacts with PRC2 to repress transcription. In the larval CNS, TET less frequently colocalized with the PRC2 component E(z) than with Ph and Psc, two components of PRC1. Moreover, contrary to Ph, E(z) did not coprecipitate TET, and its knockdown did not impair the expression of TET targets or its recruitment to chromatin. Conversely, TET loss did not seem to modify E(z) binding nor to affect H3K27me3 deposition. It thus appears that TET does not functionally interact with PRC2 in this context. The presence of high levels of both H3K27me3 and H2AK118ub at TET-activated targets could reflect CNS cell type heterogeneity, whereby TET and Ph/PRC1 promote gene expression in some cells, whereas E(z)/PRC2 might repress the same genes in other cells. As E(z) knockdown was not sufficient to induce an overexpression of TET targets that were tested (except for CenG1A and otk), other transcriptional regulators are probably required to activate these genes (Gilbert, 2024).

Since ~50% of TET peaks colocalized with PRC1 and ph knockdown reduced TET binding, TET could be recruited to chromatin together with PRC1 by the combinatorial action of PcG recruiters such as GAF or Cg, which are strong PRC1 interactors. However, other determinants could be involved. For instance, interactions with ncRNAs might be important for TET/PRC1 recruitment. In mammals, both TET proteins and PRC1 interact with ncRNAs and are recruited to R-loop. Furthermore, other transcription factors likely contribute to TET recruitment. Notably, binding sites for Tango are present in ~6% of TET peaks, including in slit. As Tango directly activates the expression of slit in the ventral midline glia , it could help target TET/PRC1 to this gene (Gilbert, 2024).

These data bring further evidence that PRC1 can function independently of PRC2 and contribute to gene activation. Besides the typical implication of both complexes in gene repression, PRC1 was shown to bind active genes and promote their transcription independently of PRC2. Yet, how PRC1 mediates gene activation is still unclear. It was shown that PRC1 can assist transcription by modulating RNA polymerase II phosphorylation or the pausing-elongation factor Spt5 occupancy and can also contribute to specific chromatin loops favoring promoter/enhancer interactions . It is tempting to speculate that similar mechanisms underlie the Ph-dependent activation of TET target genes. However, further investigations along those lines are hindered by larval CNS cellular heterogeneity and the accumulation of defects over time. It is anticipated that the development of lineage-restricted chromatin profiling approaches and acute depletion techniques will help gain a finer resolution of TET and PRC1 mode of action in gene activation in vivo (Gilbert, 2024).

In conclusion, this work brings strong evidence that TET acts in an enzymatic-independent process to control Drosophila CNS development and reveals an unexpected link between TET and PRC1 for gene activation in vivo. Given the conservation of these factors across evolution and their multiple roles during normal development and in diseases, it will be interesting to decipher more precisely the molecular mechanisms underlying their mode(s) of cooperation. This study also underlines the necessity to consider the noncatalytic functions of epigenetic enzymes to fully embrace their mode of action in normal and pathological situations (Gilbert, 2024).

Tet controls axon guidance in early brain development through glutamatergic signaling

Mutations in ten-eleven translocation (TET) proteins are associated with human neurodevelopmental disorders. This study found a function of Tet in regulating Drosophila early brain development. The Tet DNA-binding domain Tet^AXXC is required for axon guidance in the mushroom body (MB). Glutamine synthetase 2 (Gs2), a key enzyme in glutamatergic signaling, is significantly down-regulated in the Tet^AXXC brains. Loss of Gs2 recapitulates the Tet^AXXC phenotype. Surprisingly, Tet and Gs2 act in the insulin-producing cells (IPCs) to control MB axon guidance, and overexpression of Gs2 in IPCs rescues the defects of Tet^AXXC. Feeding Tet^AXXC with metabotropic glutamate receptor antagonist MPEP rescues the phenotype while glutamate enhances it. Mutants in Tet and Drosophila Fmr1, the homolog of human FMR1, have similar defects, and overexpression of Gs2 in IPCs also rescues the Fmr1 phenotype. This study provides the first evidence that Tet controls the guidance of developing brain axons by modulating glutamatergic signaling (Tran, 2024).

TET is essential for neurodevelopment, but how it functions in the developing brain is not well understood. For instance, TET3 is upregulated and required for axon regeneration in adult dorsal root ganglion (DRG) neurons after injury, and Tet is involved in neuronal functions including commissure axon patterning and glial homeostasis in Drosophila. However, the requirement of Tet and its DNA-binding domain in regulating axon guidance in the developing brain has not been studied. The current results establish that Tet regulates the guidance of β axons in the MB, a prominent structure in the brain that controls different neurological functions, and this regulation is dependent on its DNA-binding domain (Tran, 2024).

The results also show that the requirement for Tet function in the MB β axon guidance is limited to the first 48 h of pupal brain development, at a time when β axons grow to form the mature MB β lobes. When Tet levels are reduced β axons are not restricted to one hemisphere, rather they grow across the brain midline. Careful inspection of the Tet RNAi axons that cross the midline showed that these axons formed synaptic boutons in the "wrong" brain hemisphere suggesting that the circuits of the hemispheres are misconnected. Together these observations suggest that treatment for neurodevelopmental disorders that are due to axon guidance problems and the ensuing misconnection of synapses needs to be applied at early stages of nervous system development (Tran, 2024).

Transcriptomic studies demonstrated that Glutamine synthetase 2 (Gs2) and GABA transporter (Gat) are generally regulated by Tet in the brain, but this study concentrated on their requirement in the MB. Gs2 is a key enzyme in regulating glutamate levels in glutamatergic signaling and Gat is the neurotransporter that controls GABA levels in the GABAergic signaling suggesting that Tet is required in both pathways. However, knockdown of Gat did not result in an β lobe phenotype suggesting that Gat is not required for normal axon guidance. In contrast, inducing Gs2 RNAi by the 201Y-GAL4 driver resulted in axon defects suggesting that MB axon guidance is regulated by glutamatergic signaling via Gs2. In addition, treating Tet^AXXC mutants with MPEP, a metabotropic glutamate receptor (mGluR) antagonist, can partially rescue the axon guidance defects while feeding mutants glutamate enhances the TetAXXC phenotype. These data demonstrate a new function of Tet in regulating glutamatergic signaling in the brain and support the notion that the pathway is mis-regulated in Tet mutant brains. Glutamatergic signaling is a major excitatory signal in the human brain and the pathway is also known to act in neural developmental events such as proliferation, differentiation, migration, and synaptogenesis. Interestingly, elevated levels of glutamatergic signaling have also been proposed as the underlying mechanism of the neurodevelopmental disorder in FXS.50 Based on the conserved functional domains of Tet in humans and flies, the mis-regulation of the glutamatergic signaling observed in Drosophila brain during early development may also occur in developing human brains harboring Tet mutations that lead to neurodevelopmental disorders (Tran, 2024).

Previousl work has mapped Tet-DNA -binding sites in the genome by ChIP-seq and mapped Tet-mediated 5hmrC RNA modification throughout the transcriptome by hMeRIP-seq. There is no Tet binding detected by ChIP-seq on the gene body or promoter region of Gs2 and Gat. In addition, the Gs2 and Gat mRNAs do not carry the Tet-dependent 5hmrC modification. These observations suggest that Tet regulates Gs2 and Gat indirectly. This study investigated if robo2/slit, which were identified as RNA modification targets of Tet, could regulate MB axon guidance. However, no significant effects were observed on MB axon guidance upon robo2 knockdown in the MB. Therefore, it is proposed that one or several Tet target genes are responsible for controlling the transcription or RNA levels of Gs2 and Gat (Tran, 2024).

Alternatively, Tet may have a novel, so far unknown function. Since Gs2 and Gat are also down-regulated in the Fmr1³ mutant, Tet and Fmr1 may function together to control the mRNA levels of these two and potentially additional genes. Fmr1 was previously found in the regulatory protein complex Not1/CCR4 which is known to control all steps of gene expression from transcription in the nucleus to mRNA degradation in the cytoplasm depending on the composition of proteins that interact with the complex. In this case the DNA-binding domain of Tet may be responsible for protein-protein interaction(s) necessary to form the complex. Previous work has identified Fmr1 in a complex with the zinc finger protein RP-8 (Zfrp8), which functions in the assembly of mRNA ribonucleoprotein (mRNP) complexes. Interestingly, by co-immunoprecipitation this study found Zfrp8 and Tet in the same complex. Thus, Tet and Fmr1 may both coordinate with the Not1/CCR4 or other mRNP complexes to regulate the expression levels of Gs2 and Gat. Indeed, understanding these complexes and their functions in controlling mRNA levels is of great importance, especially since RNA processing is not well understood (Tran, 2024).

IPCs in Drosophila are a group of neurons located at the dorsal midline of the central brain that produce and secrete insulin-like peptides into the hemolymph to regulate growth and metabolism. Previous studies have shown that IPCs can also regulate neuronal functions. For instance, overexpressing Fmr1 in IPCs could rescue the circadian defects observed in Fmr1 mutants. But the involvement of IPCs in regulating MB axon guidance has not previously been shown. In RNA-seq experiments, Gs2 is the most significantly reduced transcript in the Tet^>AXXC mutant brain. Gs2 is expressed in the IPCs but not in MB neurons. Tet is also expressed in IPCs and in agreement with this expression the axon midline-crossing phenotype is detected when either Tet or Gs2 in IPCs specifically knocked down. In addition, the axon midline-crossing phenotype was rescued when expressing Gs2 in the IPCs of Tet and Fmr1 mutants. Hence, the results point to a previously unknown function of IPCs in regulating MB axon guidance via glutamatergic signaling in the developing brain (Tran, 2024).

In addition to IPCs, Gs2 is also expressed in glial cells where it is responsible for converting glutamate to glutamine to regulate the levels of the neurotransmitter glutamate in the brain. Thus, the reduction of Gs2 levels in Tet^AXXC mutants may lead to the accumulation of glutamate and a deficiency of glutamine in the IPCs. However, the mechanism by which Tet and Gs2 regulate axon guidance via IPCs needs to be further elucidated. One possible mechanism is that in Tet mutant brains Gs2 is reduced in the IPCs leading to the accumulation of glutamate in the IPCs that is then secreted at the brain midline. The elevated levels of glutamate at the midline could cause MB β axons to cross over the midline. In support of this possible mechanism, glutamate has been reported to reduce the responsiveness of axonal growth cones to repellent cues such as Slit-2 and Sema-3A/C23 and also to enhance the growth rate and branching of dopaminergic axons. In addition, glutamate can be released by pancreatic cells, which are the insulin-secreting cells in mammals, via excitatory amino acid transporters (EAAT) by uptake reversal. The uptake reversal activity of EAAT has also been reported in astrocytes and it depends on the relative intracellular and extracellular glutamate concentration. Further studies are needed to test the secretion of glutamate by IPCs and to investigate whether the MB β axon crossing phenotype is due to elevated glutamate levels at the midline of Tet^AXXC mutant brains (Tran, 2024).

Previously, the glutamate receptor antagonist MPEP was successfully used to suppress the axonal defect in Fmr¹³ mutants while high glutamate concentration can enhance the phenotype. The observation that MPEP and glutamate have similar effects on the TetAXXC phenotype suggests that Tet and Fmr1 have overlapping functions. This conclusion is further strengthened by the finding that Gs2 is down-regulated in the brain of both Tet^AXXC and Fmr¹³ mutants and that increasing Gs2 levels by overexpressing it from a transgene reduced the occurrence of the β lobe fusion phenotype in both Tet^AXXC and Fmr¹³ mutants. The rescue is partial, suggesting that the expression levels of Gs2 are essential additional targets of Tet and Fmr1 may also contribute to the phenotype. As Tet and Fmr1 can regulate a broad spectrum of genes, it is impressive that overexpression of only one gene, Gs2, encoding a key enzyme in the glutamatergic pathway, can rescue the axonal defect in about half of both Tet and Fmr1 mutants (Tran, 2024).

Dysfunction of glutamatergic signaling is involved in a variety of human diseases including FXS, autism spectrum disorder, schizophrenia, neurodegenerative diseases, and gliomas. The current results suggest that glutamine synthetase is a potential target for early intervention. Recently, an increasing number of studies have shown that Tet mutations are associated with neurodevelopmental disorders in humans. The mechanism by which human TET mutations cause these disorders needs to be elucidated, but the highly conserved functional domains of human and Drosophila Tet suggest that the Tet function in axon guidance that was found in Drosophila may be conserved in humans. Thus, this study provides initial evidence that glutamatergic signaling is altered in the Tet mutant brains and the mutant may serve as a Drosophila model for drug screening and study of neurodevelopmental disorders caused by TET mutations (Tran, 2024).

This study described the new function of Tet in controlling axon guidance in the Drosophila brain by regulating glutamatergic signaling via Gs2. Tet was shown to indirectly regulates Gs2 levels and suggest a possible underlying mechanism. However further studies need to be conducted to confirm how Tet regulates Gs2 transcript levels. This study also demonstrated that Tet and Gs2 are required in the IPCs to control axon guidance. How glutamate is secreted from IPCs and becomes localized to the brain midline and how the outgrowth of the MB axons is misguided by excess glutamate is not known. Identifying a transporter (e.g., EAAT) in the IPCs that is responsible for the secretion of glutamate and a receptor on the MB axons that is responsible for the guidance is necessary to understand the mechanism of how the IPCs regulate MB axons via the neurotransmitter glutamate (Tran, 2024).

Drosophila Tet Is Required for Maintaining Glial Homeostasis in Developing and Adult Fly Brains

Ten-eleven translocation (TET) proteins are crucial epigenetic regulators highly conserved in multicellular organisms. TETs' enzymatic function in demethylating 5-methyl cytosine in DNA is required for proper development and TETs are frequently mutated in cancer. Recently, Drosophila melanogaster Tet (dTet) was shown to be highly expressed in developing fly brains and discovered to play an important role in brain and muscle development as well as fly behavior. Furthermore, dTet was shown to have different substrate specificity compared with mammals. However, the exact role dTet plays in glial cells and how ectopic TET expression in glial cells contributes to tumorigenesis and glioma is still not clear (Frey, 2022).

This study reports a novel role for dTet specifically in glial cell organization and number. Loss of dTet was shown to affect the organization of a specific glia population in the optic lobe, the "optic chiasm" glia. Additionally, irregularities were found in axon patterns in the ventral nerve cord (VNC) both, in the midline and longitudinal axons. These morphologic glial and axonal defects were accompanied by locomotor defects in developing larvae escalating to immobility in adult flies. Furthermore, glia homeostasis was disturbed in dTet-deficient brains manifesting in gain of glial cell numbers and increased proliferation. Finally, this study established a Drosophila model to understand the impact of human TET3 in glia, and ectopic expression of hTET3 in dTet-expressing cells was found to cause glia expansion in larval brains and affects sleep/rest behavior and the circadian clock in adult flies (Frey, 2022).

Expression of mammalian TETs has been mainly reported in neuronal cells. While one study reported that TET1 is expressed in astrocytes in adult mouse hippocampus at low levels, another study analyzed TET protein expression during oligodendrocyte development in vivo and in vitro and found a dynamic pattern of TET protein expression that is accompanied by dynamic changes in 5hmC levels during oligodendrocyte maturation. In addition, knock-down experiments from the same study demonstrated that all three TET enzymes are required for normal oligodendrocyte development. To date, data available on TET protein expression and function in glial cells remains scarce and this study on dTet requirement in glia of the adult/larval optic chiasm and midline of developing larvae may provide some clues for similar functions of TETs in glia in higher organisms as both glia types are anatomically conserved in vertebrates. The current findings indicate the importance of dTet in glia hemostasis as glial cell numbers were significantly increased in brain lobes of Tet[MI03920]/Tet[null] larvae coinciding with an increase in the mitotic index. CyE patterns in brain lobes was furthe investigated. CycE is a downstream target of the hippo pathway and is the most important cyclin in G1 to S phase transition in Drosophila. This is of importance as it may explain the observed expansion in glial cell population in Tet[MI03920]/Tet[null] flies. The hippo pathway has been shown to maintain the quiescence in Drosophila neural stem cells and any perturbation in this pathway may affect glia as well as neuronal differentiation and proliferation. Although neoplastic brain is one of the standard brain phenotypes assessed in Drosophila glioblastoma models deficiency of dTet did not cause neoplasia as is the case when expressing common glioblastoma EGFR-Ras and PI3K mutations in Drosophila glia ; however, it caused an expansion in a differentiated glia population (Repo positive cells) in larval brain lobes. It remains interesting to identify whether this increase in glia numbers is accompanied by loss of neurons, since an increase was detected in apoptotic cells that was not of glial origin. There are different glial cells in the developing Drosophila brain, such as cortex glia, surface glia, neuropil glia and peripheral glia. Each responsible for specific functions as is the case in vertebrate astrocytes, oligodendrocytes, microglia, and Schwann cells, respectively. Assessing which glia population is proliferating on loss of dTet, will determine which glia processes dTet safeguards in the Drosophila brain. This study shows that dTet is playing a more general role in axon guidance in the VNC and that dTet is required for proper glial cell differentiation in the brain lobes. Additionally, a recent study described defects in mushroom bodies, a region in the fly brain that is well characterized and associated with olfactory learning and memory, on neuron-specific knock-down of dTet (Yao , 2018). These phenotypes were confirmed and analyzed in more detail with the dTet-deficient allele further reinforcing a role for dTet in olfactory learning and memory. In adult mammalian brains, TET proteins have emerged as important players in modulating neuronal plasticity, behavior and memory; however, their exact roles in brain function appear to be somehow distinct and region-dependent. TET1, for example, has been reported to be regulated by neuronal activity in mice, where it positively regulates several genes implicated in learning and memory. Moreover, its overexpression impaired hippocampus-dependent long-term associative memory independent from its catalytic activity. TET2 on the other hand has been linked to neurogenic rejuvenation. Conditional knock-out of TET2 within the hippocampal neurogenic niche of young mice led to decreased neurogenesis and impairment of learning and memory, whereas overexpression of TET2 in the same neurogenic niche of mature adults reversed age-related decline in neurogenesis and enhanced learning and memory (Frey, 2022).

Based on the described glia phenotypes in Tet[MI03920]/Tet[null] flies, it was asked whether expressing TET3, the closest human homologues of dTet, in Drosophila, would recapitulate some glioma like phenotypes. hTET3 expression in combination with dTet knock-down, did show a negative effect on survival and hTET3 was not able to rescue the dTet knock-down associated locomotion defects. It is therefore possible, that the amount of TET3 expressed, and cell-specific expression, might be key factors in restoring some of the functional defects observed in dTet-deficient flies. Expressing human TET3 in dTet-expressing cells caused a general increase in glial cells in the optic lobes. This increase was seen in both, dTet positive and negative cells, indicating that TET3 has not only an autonomous, but also a nonautonomous effect on glial cell proliferation. Interestingly, this increase in glia proliferation was not observed when TET3 was expressed only in differentiated glia populations (Repo driver) indicating that TET3 might have an earlier effect at the stem cell level, which is usually observed in cancer. Although all three TET enzymes are expressed in the central nervous system of mice and human, TET3 has been shown to be the most abundant transcript in different mammalian brain regions. A study on mouse embryonic stem cells (mESCs), either lacking Tet3 alone or with triple deficiency of Tet1/2/3, found that TET proteins, and in particular TET3, play a key role in modulating Wnt signaling and establishing the proper balance between neural and mesodermal cell fate specification in ESCs as well as in mouse embryos. Furthermore, a recent study in hepatoblastoma (embryonal liver tumor) re-emphasized that not only loss of TET function, but also aberrant expression of TET can lead to DNA hypomethylation and an increase in overall 5hmC level in these tumors. Another study reported that TET3 expression is activity-dependent in primary cortical neurons and mediates accumulation of 5-hmC, in turn promoting gene expression and rapid behavioral adaptation contributing to formation of fear extinction memory, an important form of reversal learning. In the Drosophila human TET3 model, expression of TET3 caused premature mortality in adult flies accompanied by an increase in glial cells in larval brain lobes, only when expressed in dTet expressing tissues. In contrast, defects in circadian behavior were observed on expression of TET3 in both, dTet expressing tissues as well as differentiated glial cells. Notably, dTet has previously been linked to the circadian rhythm. In particular, dTet has been shown to be required during embryonic and larval stages in PDF neurons to ensure proper circadian behavior in adult flies. In fact, the circadian rhythm also modulates the timing of preadult developmental events in Drosophila and thus, defects in the circadian clock might contribute to the observed lethality (Frey, 2022).

Overall, this study reports a yet undescribed role for dTet in normal glia homeostasis, proper arrangement in optic chiasm as well as behavior. Finally, a human TET3 Drosophila model was established, and it was show that ectopic expression of TET3 results in deregulation of glia proliferation in the optic lobe and affects fly survival and circadian rhythm (Frey, 2022).

Tet-dependent 5-hydroxymethyl-Cytosine modification of mRNA regulates axon guidance genes in Drosophila

Modifications of mRNA, especially methylation of adenosine, have recently drawn much attention. The much rarer modification, 5-hydroxymethylation of cytosine (5hmC), is not well understood and is the subject of this study. Vertebrate Tet proteins are 5-methylcytosine (5mC) hydroxylases and catalyze the transition of 5mC to 5hmC in DNA. These enzymes have recently been shown to have the same function in messenger RNAs in both vertebrates and in Drosophila. The Tet gene is essential in Drosophila as Tet knock-out animals do not reach adulthood. This study describes the identification of Tet-target genes in the embryo and larval brain by mapping one, Tet DNA-binding sites throughout the genome and two, the Tet-dependent 5hmrC modifications transcriptome-wide. 5hmrC modifications are distributed along the entire transcript, while Tet DNA-binding sites are preferentially located at the promoter where they overlap with histone H3K4me3 peaks. The identified mRNAs are preferentially involved in neuron and axon development and Tet knock-out led to a reduction of 5hmrC marks on specific mRNAs. Among the Tet-target genes were the robo2 receptor and its slit ligand that function in axon guidance in Drosophila and in vertebrates. Tet knock-out embryos show overlapping phenotypes with robo2 and both Robo2 and Slit protein levels were markedly reduced in Tet KO larval brains. These results establish a role for Tet-dependent 5hmrC in facilitating the translation of modified mRNAs primarily in cells of the nervous system (Singh, 2024).

Drosophila Tet Is Expressed in Midline Glia and Is Required for Proper Axonal Development

Ten-Eleven Translocation (TET) proteins are important epigenetic regulators that play a key role in development and are frequently deregulated in cancer. Drosophila melanogaster has a single homologous Tet gene (dTet) that is highly expressed in the central nervous system during development. This study examined the expression pattern of dTet in the third instar larval CNS and discovered its presence in a specific set of glia cells: midline glia (MG). Moreover, dTet knockdown resulted in significant lethality, locomotor dysfunction, and alterations in axon patterning in the larval ventral nerve cord. Molecular analyses on dTet knockdown larvae showed a downregulation in genes involved in axon guidance and reduced expression of the axon guidance cue Slit. These findings point toward a potential role for dTet in midline glial function, specifically the regulation of axon patterning during neurodevelopment (Ismail, 2019).

Tet protein function during Drosophila development

The TET (Ten-eleven translocation) 1, 2 and 3 proteins have been shown to function as DNA hydroxymethylases in vertebrates and their requirements have been documented extensively. Recently, the Tet proteins have been shown to also hydroxylate 5-methylcytosine in RNA. 5-hydroxymethylcytosine (5hmrC) is enriched in messenger RNA but the function of this modification has yet to be elucidated. Because Cytosine methylation in DNA is barely detectable in Drosophila, it serves as an ideal model to study the biological function of 5hmrC. This study characterized the temporal and spatial expression and requirement of Tet throughout Drosophila development. Tet is essential for viability as Tet complete loss-of-function animals die at the late pupal stage. Tet is highly expressed in neuronal tissues and at more moderate levels in somatic muscle precursors in embryos and larvae. Depletion of Tet in muscle precursors at early embryonic stages leads to defects in larval locomotion and late pupal lethality. Although Tet knock-down in neuronal tissue does not cause lethality, it is essential for neuronal function during development through its affects upon locomotion in larvae and the circadian rhythm of adult flies. Further, this study reports the function of Tet in ovarian morphogenesis. Together, these findings provide basic insights into the biological function of Tet in Drosophila, and may illuminate observed neuronal and muscle phenotypes observed in vertebrates (Wang, 2018).

Adenine methylation is very scarce in the Drosophila genome and not erased by the ten-eleven translocation dioxygenase

N6-methyladenine (6mA) DNA modification has recently been described in metazoans, including in Drosophila, for which the erasure of this epigenetic mark has been ascribed to the ten-eleven translocation (TET) enzyme. This study re-evaluated 6mA presence and TET impact on the Drosophila genome. Using axenic or conventional breeding conditions, traces of 6mA was found by LC-MS/MS with no significant increase in 6mA levels in the absence of TET, suggesting that this modification is present at very low levels in the Drosophila genome but not regulated by TET. Consistent with this latter hypothesis, further molecular and genetic analyses showed that TET does not demethylate 6mA but acts essentially in an enzymatic-independent manner. These results call for further caution concerning the role and regulation of 6mA DNA modification in metazoans and underline the importance of TET non-enzymatic activity for fly development (Boulet, 2023).

^ranscriptome-wide distribution and function of RNA hydroxymethylcytosine

Hydroxymethylcytosine, well described in DNA, occurs also in RNA. This study showed that hydroxymethylcytosine preferentially marks polyadenylated RNAs and is deposited by Tet in Drosophila. The transcriptome-wide hydroxymethylation landscape was mapped, revealing hydroxymethylcytosine in the transcripts of many genes, notably in coding sequences, and consensus sites were identified for hydroxymethylation. Hydroxymethylation was shown to favor mRNA translation. Tet and hydroxymethylated RNA are found to be most abundant in the Drosophila brain, and Tet-deficient fruitflies suffer impaired brain development, accompanied by decreased RNA hydroxymethylation. This study highlights the distribution, localization, and function of cytosine hydroxymethylation and identifies central roles for this modification in Drosophila (Delatte, 2016).

The mysterious presence of a 5-methylcytosine oxidase in the Drosophila genome: possible explanations

5-methylcytosine is an important epigenetic modification involved in gene control in vertebrates and many other complex living organisms. Its presence in Drosophila has been a matter of debate and recent bisulfite sequencing studies of early-stage fly embryos have concluded that the genome of Drosophila is essentially unmethylated. However, as outlined in this study, the Drosophila genome harbors a well-conserved homolog of the TET protein family. The mammalian orthologs TET1/2/3 are known to convert 5-methylcytosine into 5-hydroxymethylcytosine. Several possible explanations are discussed for these seemingly contradictory findings. One possibility is that the 2 modified cytosine bases (Dunwell, 2013)

Developmental DNA demethylation is a determinant of neural stem cell identity and gliogenic competence

DNA methylation is extensively reconfigured during development, but the functional significance and cell type-specific dependencies of DNA demethylation in lineage specification remain poorly understood. This study demonstrates that developmental DNA demethylation, driven by ten-eleven translocation 1/2/3 (TET1/2/3) enzymes, is essential for establishment of neural stem cell (NSC) identity and gliogenic potential. Loss of all three TETs during NSC specification is dispensable for neural induction and neuronal differentiation but critical for astrocyte and oligodendrocyte formation, demonstrating a selective loss of glial competence. Mechanistically, TET-mediated demethylation was essential for commissioning neural-specific enhancers in proximity to master neurodevelopmental and glial transcription factor genes and for induction of these genes. Consistently, loss of all three TETs in embryonic NSCs in mice compromised glial gene expression and corticogenesis. Thus, TET-dependent developmental demethylation is an essential regulatory mechanism for neural enhancer commissioning during NSC specification and is a cell-intrinsic determinant of NSC identity and gliogenic potential (MacArthur, 2024).

Vitamin C and MEK Inhibitor PD0325901 Synergistically Promote Oligodendrocytes Generation by Promoting DNA Demethylation

DNA methylation and demethylation are key epigenetic events that regulate gene expression and cell fate. DNA demethylation via oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) is typically mediated by TET (ten-eleven translocation) enzymes. The 5hmC modification is considered an intermediate state of DNA demethylation; it is particularly prevalent in the brain and is believed to play a role in the development of many cell types in the brain. Previous studies have identified that vitamin C (Vc) and MEK inhibitor PD0325901 could significantly promote OPC (oligodendrocyte progenitor cell)-to-OL (oligodendrocyte) differentiation. This study discovered that Vc and PD0325901 may promote OPC-to-OL differentiation by inducing DNA demethylation via hydroxymethylation. Blocking 5hmC formation almost totally blocked Vc- and PD0325901-stimulated OPC-to-OL differentiation. In addition, TET1 is not involved in Vc,- and PD0325901-promoted OL generation. A synergistic effect was found between the two compounds in inducing OL generation, suggesting the possibility of a combination therapy for demyelination diseases in the future (Rem. 2024).

Ten-eleven translocation 1 mediated-DNA hydroxymethylation is required for myelination and remyelination in the mouse brain

Ten-eleven translocation (TET) proteins, the dioxygenase for DNA hydroxymethylation, are important players in nervous system development and diseases. However, their role in myelination and remyelination after injury remains elusive. This study identified a genome-wide and locus-specific DNA hydroxymethylation landscape shift during differentiation of oligodendrocyte-progenitor cells (OPC). Ablation of Tet1 results in stage-dependent defects in oligodendrocyte (OL) development and myelination in the mouse brain. The mice lacking Tet1 in the oligodendrocyte lineage develop behavioral deficiency. TET1 was shown to be required for remyelination in adulthood. Transcriptomic, genomic occupancy, and 5-hydroxymethylcytosine (5hmC) profiling reveal a critical TET1-regulated epigenetic program for oligodendrocyte differentiation that includes genes associated with myelination, cell division, and calcium transport. Tet1-deficient OPCs exhibit reduced calcium activity, increasing calcium activity rescues the differentiation defects in vitro. Deletion of a TET1-5hmC target gene, Itpr2, impairs the onset of OPC differentiation. Together, these results suggest that stage-specific TET1-mediated epigenetic programming and intracellular signaling are important for proper myelination and remyelination in mice (Zhang, 2021).

TET1-mediated DNA hydroxymethylation regulates adult remyelination in mice

The mechanisms regulating myelin repair in the adult central nervous system (CNS) are unclear. This study identified DNA hydroxymethylation, catalyzed by the Ten-Eleven-Translocation (TET) enzyme TET1, as necessary for myelin repair in young adults and defective in old mice. Constitutive and inducible oligodendrocyte lineage-specific ablation of Tet1 (but not of Tet2), recapitulate this age-related decline in repair of demyelinated lesions. DNA hydroxymethylation and transcriptomic analyses identify TET1-target in adult oligodendrocytes, as genes regulating neuro-glial communication, including the solute carrier (Slc) gene family. Among them, this study shows that the expression levels of the Na(+)/K(+)/Cl(-) transporter, SLC12A2, are higher in Tet1 overexpressing cells and lower in old or Tet1 knockout. Both aged mice and Tet1 mutants also present inefficient myelin repair and axo-myelinic swellings. Zebrafish mutants for slc12a2b also display swellings of CNS myelinated axons. These findings suggest that TET1 is required for adult myelin repair and regulation of the axon-myelin interface (Moyon, 2021).

Boulet, M., Gilbert, G., Renaud, Y., Schmidt-Dengler, M., Plantie, E., Bertrand, R., Nan, X., Jurkowski, T., Helm, M., Vandel, L., Waltzer, L. (2023). Adenine methylation is very scarce in the Drosophila genome and not erased by the ten-eleven translocation dioxygenase. Elife, 12 PubMed ID: 38126351

Delatte, B., Wang, F., Ngoc, L. V., Collignon, E., Bonvin, E., Deplus, R., Calonne, E., Hassabi, B., Putmans, P., Awe, S., Wetzel, C., Kreher, J., Soin, R., Creppe, C., Limbach, P. A., Gueydan, C., Kruys, V., Brehm, A., Minakhina, S., Defrance, M., Steward, R., Fuks, F. (2016). RNA biochemistry. Transcriptome-wide distribution and function of RNA hydroxymethylcytosine. Science, 351(6270):282-285 PubMed ID: 26816380

Dunwell, T. L., McGuffin, L. J., Dunwell, J. M., Pfeifer, G. P. (2013). The mysterious presence of a 5-methylcytosine oxidase in the Drosophila genome: possible explanations. Cell Cycle, 12(21):3357-3365 PubMed ID: 24091536

Frey, F., Sandakly, J., Ghannam, M., Doueiry, C., Hugosson, F., Berlandi, J., Ismail, J. N., Gayden, T., Hasselblatt, M., Jabado, N., Shirinian, M. (2022). Drosophila Tet Is Required for Maintaining Glial Homeostasis in Developing and Adult Fly Brains. eNeuro, 9(2) PubMed ID: 35396259

Gilbert, G., Renaud, Y., Teste, C., Anglaret, N., Bertrand, R., Hoehn, S., Jurkowski, T. P., Schuettengruber, B., Cavalli, G., Waltzer, L., Vandel, L. (2024). Drosophila TET acts with PRC1 to activate gene expression independently of its catalytic activity. Sci Adv, 10(18):eadn5861 PubMed ID: 38701218

Ismail, J. N., Badini, S., Frey, F., Abou-Kheir, W., Shirinian, M. (2019). Drosophila Tet Is Expressed in Midline Glia and Is Required for Proper Axonal Development. Front Cell Neurosci, 13:252 PubMed ID: 31213988

MacArthur, I. C., Ma, L., Huang, C. Y., Bhavsar, H., Suzuki, M., Dawlaty, M. M. (2024). Developmental DNA demethylation is a determinant of neural stem cell identity and gliogenic competence. Sci Adv, 10(35):eado5424 PubMed ID: 39196941

Moyon, S., Frawley, R., Marechal, D., Huang, D., Marshall-Phelps, K. L. H., Kegel, L., Bostrand, S. M. K., Sadowski, B., Jiang, Y. H., Lyons, D. A., Mobius, W., Casaccia, P. (2021). TET1-mediated DNA hydroxymethylation regulates adult remyelination in mice. Nat Commun, 12(1):3359 PubMed ID: 34099715

Ren, X., Yang, Y., Wang, M., Yuan, Q., Suo, N., Xie, X. (2024). Vitamin C and MEK Inhibitor PD0325901 Synergistically Promote Oligodendrocytes Generation by Promoting DNA Demethylation. Molecules, 29(24) PubMed ID: 39770028

Singh, B. N., Tran, H., Kramer, J., Kirichenko, E., Changela, N., Wang, F., Feng, Y., Kumar, D., Tu, M., Lan, J., Bizet, M., Fuks, F., Steward, R. (2024). Tet-dependent 5-hydroxymethyl-Cytosine modification of mRNA regulates axon guidance genes in Drosophila. PLoS One, 19(2):e0293894 PubMed ID: 38381741

Tran, H., Le, L., Singh, B. N., Kramer, J., Steward, R. (2024). Tet controls axon guidance in early brain development through glutamatergic signaling. iScience, 27(5):109634 PubMed ID: 38655199

Tu, R., Ping, Z., Liu, J., Tsoi, M. L., Song, X., Liu, W., Xie, T. (2024). Niche Tet maintains germline stem cells independently of dioxygenase activity. The EMBO journal, 43(8):1570-1590 PubMed ID: 38499787

Wang, F., Minakhina, S., Tran, H., Changela, N., Kramer, J., Steward, R. (2018). Tet protein function during Drosophila development. PLoS One, 13(1):e0190367 PubMed ID: 29324752

Yao, B., Li, Y., Wang, Z., Chen, L., Poidevin, M., Zhang, C., Lin, L., Wang, F., Bao, H., Jiao, B., Lim, J., Cheng, Y., Huang, L., Phillips, B. L., Xu, T., Duan, R., Moberg, K. H., Wu, H., Jin, P. (2018). Active N(6)-Methyladenine Demethylation by DMAD Regulates Gene Expression by Coordinating with Polycomb Protein in Neurons. Mol Cell, 71(5):848-857 e846 PubMed ID: 30078725

Zhang, G., Huang, H., Liu, D., Cheng, Y., Liu, X., Zhang, W., Yin, R., Zhang, D., Zhang, P., Liu, J., Li, C., Liu, B., Luo, Y., Zhu, Y., Zhang, N., He, S., He, C., Wang, H., Chen, D. (2015). N6-methyladenine DNA modification in Drosophila. Cell, 161(4):893-906 PubMed ID: 25936838

Zhang, M., Wang, J., Zhang, K., Lu, G., Liu, Y., Ren, K., Wang, W., Xin, D., Xu, L., Mao, H., Xing, J., Gao, X., Jin, W., Berry, K., Mikoshiba, K., Wu, S., Lu, Q. R., Zhao, X. (2021). Ten-eleven translocation 1 mediated-DNA hydroxymethylation is required for myelination and remyelination in the mouse brain. Nat Commun, 12(1):5091 PubMed ID: 34429415 Biological Overview

date revised: 10 April 2025

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