GO:0141167 chromosomal 5-methylcytosine DNA demethylation, oxidation pathway: Mechanism, Genes, Functions and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0141167 describes the active, oxidation-dependent pathway that removes DNA methylation marks by converting 5-methylcytosine (5meC) to 5-hydroxymethylcytosine (5hmC), then 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC).
• The TET family enzymes (TET1, TET2, TET3) catalyze the successive oxidation steps, while the DNA glycosylase TDG excises 5fC and 5caC to initiate base excision repair and restore unmethylated cytosine.
• This pathway is essential for embryonic development, gastrulation, and the regulation of signaling genes such as Lefty-Nodal.
• Loss of TET function leads to myeloid malignancy, readthrough transcription, and heterochromatin-to-euchromatin switching.
• TET2-mediated 5hmC accumulation can induce genetic instability and mutagenesis, linking the pathway to cancer risk.
• The pathway is dynamically regulated by chromatin context, with cyclic remethylation occurring after active demethylation in euchromatic regions.
Description
DNA methylation at the 5-position of cytosine (5-methylcytosine, 5meC) is a major epigenetic mark that regulates gene expression, genome stability, and development. The removal of this mark can occur through an active, oxidation-dependent process known as chromosomal 5-methylcytosine DNA demethylation, oxidation pathway (GO:0141167). This pathway is initiated by the TET family of dioxygenases, which convert 5meC to 5-hydroxymethylcytosine (5hmC) and then to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). The modified bases 5fC and 5caC are recognized and excised by the DNA glycosylase TDG, followed by base excision repair (BER) to restore an unmethylated cytosine. This process is critical for epigenetic reprogramming during development and for maintaining normal cellular function. Dysregulation of this pathway has been implicated in myeloid malignancies, genetic instability, and altered chromatin states. Understanding the molecular players and regulatory mechanisms of GO:0141167 is therefore essential for researchers in epigenetics, cancer biology, and developmental biology.
chromosomal 5-methylcytosine DNA demethylation, oxidation pathway At A Glance
| GO ID | GO:0141167 |
|---|---|
| GO term | chromosomal 5-methylcytosine DNA demethylation, oxidation pathway |
| Ontology | biological_process |
| Synonym | epigenetic 5-methylcytosine DNA demethylation, oxidation pathway; epigenetic DNA demethylation, oxidation pathway |
| Major function | Active DNA demethylation via oxidation of 5meC and base excision repair |
| Key enzymes | TET1, TET2, TET3 (oxidation); TDG (excision) |
| Key intermediates | 5hmC, 5fC, 5caC |
| Cellular context | Chromosomal DNA, euchromatic and heterochromatic regions |
| Pathway outcome | Restoration of unmethylated cytosine |
What Is GO:0141167?
GO:0141167 is a biological process that defines an epigenetic cytosine DNA demethylation pathway starting with the enzymatic oxidation of 5-methylcytosine (5meC) to 5-hydroxymethylcytosine (5hmC), which is successively converted to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). A DNA glycosylase, such as TDG, recognizes the intermediate bases 5fC and 5caC and excises the modified base to initiate its replacement with unmethylated cytosine through base excision repair.
Why Is chromosomal 5-methylcytosine DNA demethylation, oxidation pathway Important in Cell Biology?
The oxidation pathway of DNA demethylation is a fundamental mechanism for resetting epigenetic marks, enabling dynamic gene regulation during development and in response to environmental cues. It is essential for embryonic stem cell pluripotency, gastrulation, and the proper expression of developmental signaling genes such as Lefty-Nodal. Disruption of this pathway leads to severe consequences, including myeloid malignancy, transcriptional readthrough, and large-scale chromatin reorganization. Moreover, aberrant TET2 activity and 5hmC accumulation can cause genetic instability and mutagenesis, highlighting its role in cancer initiation and progression. The pathway also exhibits crosstalk with chromatin structure, as TET1 preferentially oxidizes 5meC in linker regions of nucleosomes, and local chromatin microenvironments can influence DNMT and TET activities. Understanding GO:0141167 is therefore critical for researchers studying epigenetics, cancer, and developmental biology.
• Essential for embryonic development and gastrulation through regulation of Lefty-Nodal signaling.
• Loss of TET function causes myeloid malignancy and heterochromatin-to-euchromatin switching.
• TET2-mediated 5hmC accumulation induces genetic instability and mutagenesis.
• Dynamic cyclic DNA remethylation occurs after active demethylation in euchromatic regions.
• Chromatin microenvironment determines whether DNMT enzymes act as methyltransferases or demethylases.
• TET1 preferentially oxidizes 5meC in linker regions of positioned nucleosomes.
• Genome-wide TET and TDG-dependent oxidation dynamics shape DNA methylation patterns.
• The pathway is a target for therapeutic intervention in cancers with TET mutations.
What Happens During chromosomal 5-methylcytosine DNA demethylation, oxidation pathway?
Oxidation of 5-methylcytosine by TET enzymes
In simple terms: TET enzymes act like molecular erasers that start removing methyl marks from DNA.
The pathway begins when TET family dioxygenases (TET1, TET2, TET3) oxidize 5-methylcytosine (5meC) to 5-hydroxymethylcytosine (5hmC) in a reaction that requires oxygen, iron(II), and alpha-ketoglutarate. This initial oxidation is the rate-limiting step and is influenced by the local chromatin environment, as TET1 preferentially targets 5meC in linker regions of reconstituted positioned nucleosomes. The activity of TET enzymes can be modulated by the chromatin microenvironment, which also affects DNMT enzymes.
Successive oxidation to 5-formylcytosine and 5-carboxylcytosine
In simple terms: The TET enzymes continue to modify the mark, turning it into two more intermediate forms.
Following the formation of 5hmC, TET enzymes catalyze further oxidation to 5-formylcytosine (5fC) and then to 5-carboxylcytosine (5caC). These oxidized bases are recognized as intermediates in the demethylation pathway and serve as substrates for downstream excision. Genome-wide analysis has revealed that TET and TDG-dependent oxidation dynamics are tightly coordinated to shape DNA methylation patterns.
Excision of 5fC and 5caC by TDG
In simple terms: A DNA repair enzyme cuts out the modified bases so they can be replaced.
The DNA glycosylase TDG specifically recognizes and excises 5fC and 5caC from DNA, creating an abasic site. This excision step is a critical commitment to demethylation, as it initiates the base excision repair (BER) process. TDG activity is essential for the completion of the oxidation pathway, and its loss leads to accumulation of oxidized bases.
Base excision repair restores unmethylated cytosine
In simple terms: The cell's repair machinery fills the gap with a fresh, unmethylated cytosine.
After TDG excision, the base excision repair (BER) machinery processes the abasic site and inserts an unmethylated cytosine, completing the demethylation event. This restoration of unmethylated cytosine allows the gene to regain its potential for active transcription or reset its epigenetic state. The entire process is dynamically regulated, with cyclic DNA remethylation observed following active demethylation in euchromatic regions of mouse embryonic stem cells.
Key Genes Involved in GO:0141167 chromosomal 5-methylcytosine DNA demethylation, oxidation pathway
The following genes and proteins are central to the chromosomal 5-methylcytosine DNA demethylation, oxidation pathway (GO:0141167).
| Gene | Major Role | Research Relevance |
|---|---|---|
| TET1 | Oxidizes 5meC to 5hmC, 5fC, 5caC; preferentially targets linker regions | Embryonic stem cell pluripotency, epigenetic reprogramming |
| TET2 | Oxidizes 5meC to 5hmC, 5fC, 5caC | Myeloid malignancy, genetic instability, mutagenesis |
| TET3 | Oxidizes 5meC to 5hmC, 5fC, 5caC | Zygotic reprogramming, developmental gene regulation |
| TDG | Excises 5fC and 5caC to initiate BER | Base excision repair, demethylation completion |
| DNMT1 | Maintains DNA methylation; can be influenced by chromatin | Epigenetic inheritance, chromatin crosstalk |
| DNMT3A | De novo DNA methylation | Development, cancer |
| DNMT3B | De novo DNA methylation | Development, cancer |
| IDH1 | Produces alpha-ketoglutarate for TET activity | Metabolism-epigenetics crosstalk |
| IDH2 | Produces alpha-ketoglutarate for TET activity | Metabolism-epigenetics crosstalk |
| TDG | DNA glycosylase | Demethylation and repair |
| MBD4 | Methyl-CpG binding domain protein, potential backup glycosylase | DNA repair, demethylation |
| GADD45A | Recruits TET and TDG to demethylation sites | Active demethylation |
| UBE2B | Ubiquitin-conjugating enzyme, may regulate TDG turnover | Protein stability |
| SMUG1 | Uracil-DNA glycosylase, potential backup for TDG | Base excision repair |
| XRCC1 | Scaffold protein in BER | Repair of abasic sites |
| LIG3 | DNA ligase in BER | Completion of repair |
| PARP1 | Poly(ADP-ribose) polymerase, detects DNA breaks | BER regulation |
| APEX1 | AP endonuclease in BER | Processing abasic sites |
How Is chromosomal 5-methylcytosine DNA demethylation, oxidation pathway Regulated?
The oxidation pathway is regulated at multiple levels. TET enzyme activity depends on oxygen, iron(II), and alpha-ketoglutarate, making it sensitive to cellular metabolism. Chromatin context plays a key role: TET1 preferentially oxidizes 5meC in linker regions of positioned nucleosomes, and the local chromatin microenvironment can determine whether DNMT enzymes act as methyltransferases or demethylases. Additionally, TET and TDG expression levels are dynamically controlled during development and in response to signaling cues, as shown by TET-mediated regulation of Lefty-Nodal signaling during gastrulation. Cyclic DNA remethylation following active demethylation further indicates feedback regulation that maintains epigenetic balance.
chromosomal 5-methylcytosine DNA demethylation, oxidation pathway and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| TET2 | Myeloid malignancy, genetic instability | Tet2 knockout mouse model, AML cell lines |
| TET1 | Developmental defects, cancer | Tet1 knockout embryonic stem cells |
| TET3 | Gastrulation defects | Tet3 knockout zebrafish or mouse |
| TDG | Base excision repair deficiency, cancer predisposition | TDG knockout cell lines |
| IDH1/2 | Glioma, AML via altered alpha-ketoglutarate | IDH mutant cell models |
Myeloid malignancies
Inducible disruption of Tet genes in mice results in myeloid malignancy, readthrough transcription, and a heterochromatin-to-euchromatin switch, demonstrating that loss of the oxidation pathway directly contributes to hematopoietic cancers. TET2 mutations are frequently observed in myeloid disorders, and the resulting loss of 5hmC production disrupts normal epigenetic regulation.
Genetic instability and mutagenesis
TET2-mediated 5-hydroxymethylcytosine accumulation can induce genetic instability and mutagenesis, suggesting that dysregulated oxidation intermediates may promote DNA damage and cancer progression. This links the pathway to the maintenance of genome integrity.
Developmental disorders
TET-mediated DNA demethylation controls gastrulation by regulating Lefty-Nodal signaling, and disruption of this pathway leads to severe developmental defects. Proper regulation of GO:0141167 is therefore essential for embryonic patterning and tissue differentiation.
From chromosomal 5-methylcytosine DNA demethylation, oxidation pathway-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of TET2 drive myeloid malignancy? | TET2 knockout mouse model or human AML cell lines |
| How does TET1 target linker DNA? | TET1 knockout or catalytic-dead knock-in in embryonic stem cells |
| What is the role of TDG in demethylation? | TDG knockout cell lines with 5fC/5caC detection |
| How does TET3 regulate gastrulation? | TET3 knockout zebrafish or mouse embryos |
| Does 5hmC accumulation cause mutagenesis? | TET2 overexpression or mutant cell models |
| How does chromatin context affect demethylation? | Reconstituted nucleosomes with TET1 |
How to Study the chromosomal 5-methylcytosine DNA demethylation, oxidation pathway Process
| Method | What It Measures | Typical Application |
|---|---|---|
| TAB-seq | 5hmC at single-base resolution | Genome-wide mapping of oxidation intermediates |
| oxBS-seq | 5hmC and 5meC distinction | Epigenetic profiling |
| ChIP-seq | TET/TDG binding sites | Chromatin association studies |
| LC-MS/MS | Quantification of 5meC, 5hmC, 5fC, 5caC | Direct pathway activity measurement |
| RNA-seq | Transcriptional changes | Functional impact of demethylation |
| CRISPR knockout | Gene function loss | Causal testing of TET/TDG |
| Bisulfite sequencing | DNA methylation patterns | Global methylation analysis |
| Base excision repair assays | TDG excision activity | Enzymatic mechanism studies |
Genome-wide mapping of oxidized bases
Techniques such as TAB-seq, oxBS-seq, and reduced bisulfite sequencing allow genome-wide mapping of 5hmC, 5fC, and 5caC at single-base resolution. These methods have been used to reveal TET- and TDG-dependent oxidation dynamics across the genome.
Chromatin immunoprecipitation and sequencing
ChIP-seq for TET1, TET2, TET3, and TDG can identify their binding sites and correlate them with histone modifications and chromatin states. This approach has shown preferential TET1 binding to linker regions.
Mass spectrometry and quantitative analysis
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) enables precise quantification of 5meC, 5hmC, 5fC, and 5caC levels in genomic DNA, providing a direct readout of pathway activity.
Functional assays in knockout models
CRISPR-Cas9 knockout of TET genes or TDG followed by phenotypic analysis, RNA-seq, and methylation profiling can determine the causal role of the pathway in development and disease.
How CRISPR Can Be Used to Study GO:0141167 chromosomal 5-methylcytosine DNA demethylation, oxidation pathway
Knockout
CRISPR-Cas9 knockout of TET1, TET2, TET3, or TDG allows researchers to study the loss-of-function consequences of the oxidation pathway. For example, inducible disruption of Tet genes in mice results in myeloid malignancy and chromatin changes. Knockout of TDG can reveal its role in excising 5fC and 5caC.
Point Mutation
Introducing catalytic-dead point mutations in TET enzymes (e.g., in the iron-binding or alpha-ketoglutarate-binding domains) can separate enzymatic activity from non-catalytic functions. Such models help determine whether oxidation activity is required for specific developmental or disease phenotypes.
Knock-in
Knock-in of tagged TET or TDG alleles (e.g., FLAG, HA, or GFP) enables chromatin immunoprecipitation, imaging, and proteomic studies to map binding sites and interacting partners. This approach has been used to study TET1 localization to linker DNA.
Overexpression
Overexpression of TET2 or TET1 can induce 5hmC accumulation and genetic instability, providing a model to study the mutagenic potential of the pathway. Overexpression in cell lines also allows biochemical characterization of oxidation kinetics.
How EDITGENE Supports chromosomal 5-methylcytosine DNA demethylation, oxidation pathway Research
Researchers studying chromosomal 5-methylcytosine DNA demethylation, oxidation pathway-related genes often need to determine whether a candidate gene is causally involved in epigenetic regulation, development, or disease. EDITGENE provides a comprehensive suite of CRISPR-based services to generate precisely engineered cell models for such investigations.
Contact EDITGENE today to design your custom CRISPR model for chromosomal 5-methylcytosine DNA demethylation, oxidation pathway research.
Frequently Asked Questions About chromosomal 5-methylcytosine DNA demethylation, oxidation pathway
What is GO:0141167?
GO:0141167 is the Gene Ontology term for chromosomal 5-methylcytosine DNA demethylation, oxidation pathway, a biological process that removes DNA methylation via TET-mediated oxidation and TDG-initiated base excision repair.
What genes are involved in chromosomal 5-methylcytosine DNA demethylation, oxidation pathway?
Key genes include TET1, TET2, TET3, and TDG, along with base excision repair factors such as APEX1, XRCC1, and LIG3.
How does TET2 contribute to DNA demethylation?
TET2 oxidizes 5-methylcytosine to 5-hydroxymethylcytosine and further to 5-formylcytosine and 5-carboxylcytosine, which are then excised by TDG. TET2 loss is linked to myeloid malignancy.
What is the role of TDG in the oxidation pathway?
TDG is a DNA glycosylase that recognizes and excises 5-formylcytosine and 5-carboxylcytosine, initiating base excision repair to restore unmethylated cytosine.
What diseases are associated with defects in this pathway?
Defects are associated with myeloid malignancies, genetic instability, and developmental disorders due to impaired gastrulation.
How is the oxidation pathway regulated?
It is regulated by oxygen, iron(II), alpha-ketoglutarate availability, chromatin context, and TET/TDG expression levels.
What are the intermediates of 5-methylcytosine oxidation?
The intermediates are 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC).
Can CRISPR be used to study this pathway?
Yes, CRISPR knockout, point mutation, knock-in, and overexpression models are widely used to dissect the function of TET and TDG in DNA demethylation.
What methods measure DNA demethylation intermediates?
TAB-seq, oxBS-seq, and LC-MS/MS are commonly used to quantify 5hmC, 5fC, and 5caC.
Why is this pathway important for development?
It controls epigenetic reprogramming during gastrulation by regulating Lefty-Nodal signaling, and its disruption causes severe developmental defects.
Conclusion
The chromosomal 5-methylcytosine DNA demethylation, oxidation pathway (GO:0141167) is a central mechanism for active DNA demethylation, driven by TET-mediated oxidation and TDG-initiated base excision repair. Its proper regulation is essential for embryonic development, epigenetic stability, and prevention of malignancies. Dysregulation of this pathway contributes to myeloid malignancies, genetic instability, and developmental disorders, making it a key area of biomedical research. Understanding the molecular players and regulatory mechanisms of GO:0141167 will continue to inform cancer biology, epigenetics, and developmental biology.
References
- 1. Yuita H et al.. 2023. Inducible disruption of Tet genes results in myeloid malignancy, readthrough transcription, and a heterochromatin-to-euchromatin switch.. Proc Natl Acad Sci U S A 120(6):e2214824120 PMID: 37406303
- 2. Ficz G. 2015. New insights into mechanisms that regulate DNA methylation patterning.. J Exp Biol 218(Pt 1):14-20 PMID: 25568447
- 3. van der Wijst MG et al.. 2015. Local chromatin microenvironment determines DNMT activity: from DNA methyltransferase to DNA demethylase or DNA dehydroxymethylase.. Epigenetics 10(8):671-6 PMID: 26098813
- 4. Kubiura-Ichimaru M et al.. 2021. Cyclic DNA remethylation following active demethylation at euchromatic regions in mouse embryonic stem cells.. Chromosome Res 29(2):145-157 PMID: 33205231
- 5. Mahfoudhi E et al.. 2016. TET2-mediated 5-hydroxymethylcytosine induces genetic instability and mutagenesis.. DNA Repair (Amst) 43:78-88 PMID: 27289557
- 6. Dai HQ et al.. 2016. TET-mediated DNA demethylation controls gastrulation by regulating Lefty-Nodal signalling.. Nature 538(7626):528-532 PMID: 27760115
- 7. Kizaki S et al.. 2016. Preferential 5-Methylcytosine Oxidation in the Linker Region of Reconstituted Positioned Nucleosomes by Tet1 Protein.. Chemistry 22(46):16598-16601 PMID: 27689340
- 8. Shen L et al.. 2013. Genome-wide analysis reveals TET- and TDG-dependent 5-methylcytosine oxidation dynamics.. Cell 153(3):692-706 PMID: 23602152
