GO:0140864 histone H2AK129 ubiquitin ligase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0140864 describes the enzymatic activity that transfers ubiquitin specifically to lysine 129 of histone H2A, a molecular function central to chromatin regulation.
• The best-characterized H2AK129 ubiquitin ligase is RNF168, which together with the HDAC6-RNF168 axis orchestrates H2A/H2A.X ubiquitination at DNA double-strand breaks.
• H2AK129 ubiquitination is a prerequisite for downstream chromatin events including H2AK119 ubiquitination, heterochromatin maintenance, and DNA damage repair.
• Dysregulation of H2AK129 ubiquitin ligase activity is linked to cancer, genomic instability, and impaired DNA damage responses.
• CRISPR knockout, point-mutation, and knock-in models are essential to dissect the causal role of H2AK129 ubiquitin ligases in chromatin biology and disease.
• Studying this activity requires integrated methods such as ubiquitin proteomics, ChIP-seq, and DNA damage repair assays.
Description
Histone H2AK129 ubiquitin ligase activity (GO:0140864) is a molecular function defined as the catalysis of ubiquitin transfer to histone H2A at lysine 129. This post-translational modification is a key epigenetic mark that influences chromatin structure, DNA repair, and gene expression. The activity is carried out by E3 ubiquitin ligases, with RNF168 being the most extensively studied enzyme for this specific site. Understanding GO:0140864 is critical because H2AK129 ubiquitination serves as a signaling platform for the recruitment of downstream factors that maintain genome stability. Research on this term has expanded beyond DNA damage response to include heterochromatin inheritance and epigenetic crosstalk. For example, UHRF1 ubiquitin ligase activity supports the maintenance of low-density CpG methylation, which is linked to H2A ubiquitination pathways. Similarly, the HDAC6-RNF168 axis regulates H2A/H2A.X ubiquitination to enable double-strand break repair, directly implicating GO:0140864 in genome maintenance. These findings highlight the broad biological significance of this enzymatic activity. For researchers, GO:0140864 provides a precise annotation for functional studies of chromatin-modifying enzymes. It distinguishes H2AK129-specific ligases from other histone ubiquitin ligases, such as those targeting H3K23 or H3K14. This specificity is essential for accurate experimental design and data interpretation in epigenetics and cancer biology.
histone H2AK129 ubiquitin ligase activity At A Glance
| GO ID | GO:0140864 |
|---|---|
| GO term | histone H2AK129 ubiquitin ligase activity |
| Ontology | molecular_function |
| Synonym | histone H2A-K129 ubiquitin ligase activity; histone ubiquitin ligase activity (H2A-K129 specific) |
| Major function | Catalyzes ubiquitin transfer to histone H2A at lysine 129, regulating chromatin dynamics and DNA repair |
| Representative enzyme | RNF168, an E3 ubiquitin ligase that monoubiquitinates H2A/H2A.X at K129 |
| Associated processes | DNA double-strand break repair, heterochromatin maintenance, epigenetic crosstalk |
| Disease relevance | Cancer, genomic instability, and developmental disorders |
What Is GO:0140864?
GO:0140864, histone H2AK129 ubiquitin ligase activity, is defined as the catalysis of the transfer of a ubiquitin molecule to histone 2A at the lysine-129 residue. In other words, it is the enzymatic function that attaches a single ubiquitin moiety to a specific lysine on histone H2A, thereby altering chromatin structure and function.
Why Is histone H2AK129 ubiquitin ligase activity Important in Cell Biology?
GO:0140864 is important because histone H2AK129 ubiquitination is a critical epigenetic mark that orchestrates DNA damage response, chromatin remodeling, and gene silencing. Dysregulation of this activity leads to genomic instability and is implicated in cancer and other diseases. Understanding this term enables researchers to target specific ubiquitin ligases for therapeutic intervention and to interpret chromatin biology data accurately.
• DNA double-strand break repair: H2AK129 ubiquitination by RNF168 is essential for recruiting repair factors such as 53BP1 and BRCA1.
• Heterochromatin maintenance: H2AK129 ubiquitination contributes to the inheritance of heterochromatic states.
• Epigenetic crosstalk: It coordinates with H3K9me3 and DNA methylation pathways.
• Cancer: Altered H2AK129 ligase activity is associated with tumorigenesis and therapy resistance.
• Genome stability: Loss of this activity leads to accumulation of DNA damage and chromosomal aberrations.
• Therapeutic target: RNF168 and related ligases are potential targets for cancer therapy.
• Chromatin architecture: H2AK129 ubiquitination influences higher-order chromatin compaction.
• Developmental biology: It plays roles in cell differentiation and embryonic development.
• Immunity: DNA damage response pathways involving H2AK129 ubiquitination are linked to immune signaling.
• Aging: Defective DNA repair due to impaired H2AK129 ubiquitination may contribute to aging.
Molecular Mechanism of histone H2AK129 ubiquitin ligase activity
Substrate recognition and E3 ligase recruitment
In simple terms: The enzyme first finds and binds to histone H2A at the site of damage or specific chromatin regions.
H2AK129 ubiquitin ligases, such as RNF168, are recruited to DNA double-strand breaks through interactions with phosphorylated H2A.X and other damage sensors. The HDAC6-RNF168 axis facilitates this recruitment and regulates the subsequent ubiquitination of H2A/H2A.X. Substrate specificity is determined by the E3 ligase's RING domain and its ability to recognize the H2A C-terminal tail containing lysine 129.
Catalytic transfer of ubiquitin
In simple terms: The enzyme attaches a single ubiquitin molecule to lysine 129 of histone H2A.
The catalytic mechanism involves the transfer of ubiquitin from an E2 conjugating enzyme to the epsilon-amino group of H2A lysine 129. This monoubiquitination event is a prerequisite for downstream polyubiquitination and the recruitment of repair proteins. The reaction is ATP-dependent and requires the coordinated action of E1, E2, and E3 enzymes.
Cofactors and regulatory modifications
In simple terms: Other proteins and chemical marks help or hinder the enzyme's activity.
HDAC6 deacetylase activity is required for efficient RNF168-mediated H2A ubiquitination, as acetylation of H2A may inhibit ubiquitination. Additionally, UHRF1 ubiquitin ligase activity supports the maintenance of low-density CpG methylation, which crosstalks with H2A ubiquitination pathways. The SWI/SNF-related protein SMARCA3 is a histone H3K23 ubiquitin ligase that regulates H3K9me3, illustrating the broader network of histone ubiquitination crosstalk.
Downstream signaling and chromatin remodeling
In simple terms: The ubiquitin mark acts as a signal for other proteins to come and modify chromatin.
H2AK129 ubiquitination serves as a docking site for proteins containing ubiquitin-binding domains, such as RNF168 itself and 53BP1, leading to chromatin relaxation and repair factor assembly. This mark also promotes H2AK119 ubiquitination by PRC1, reinforcing heterochromatin states. The conserved H3K14ub-driven H3K9me3 pathway further demonstrates how histone ubiquitination contributes to chromatin compartmentalization.
Reversibility and regulation
In simple terms: The ubiquitin mark can be removed by deubiquitinating enzymes, making the process reversible.
Deubiquitinating enzymes (DUBs) such as USP48 and BRCC36 counteract H2AK129 ubiquitination to maintain dynamic chromatin states. The balance between ligase and DUB activity is critical for proper DNA repair and heterochromatin inheritance. Stress conditions can control heterochromatin inheritance via histone H3 ubiquitylation, indicating that environmental factors modulate ubiquitin signaling.
Key Genes Involved in GO:0140864 histone H2AK129 ubiquitin ligase activity
The following genes encode proteins directly involved in histone H2AK129 ubiquitin ligase activity or its regulatory network.
| Gene | Major Role | Research Relevance |
|---|---|---|
| RNF168 | E3 ubiquitin ligase that monoubiquitinates H2A/H2A.X at K129 | Central enzyme for GO:0140864; knockout impairs DNA repair |
| HDAC6 | Deacetylase that regulates RNF168 activity and H2A ubiquitination | Modulates H2AK129 ubiquitination; target for cancer therapy |
| UHRF1 | Ubiquitin ligase involved in CpG methylation maintenance and crosstalk with H2A ubiquitination | Links DNA methylation to histone ubiquitination |
| SMARCA3 | Histone H3K23 ubiquitin ligase regulating H3K9me3 | Provides crosstalk context for histone ubiquitination |
| H2AX | Histone variant phosphorylated at damage sites; substrate for RNF168 | Essential for recruiting H2AK129 ligases |
| 53BP1 | DNA repair protein recruited by H2AK129 ubiquitination | Downstream effector of H2AK129 signaling |
| BRCA1 | DNA repair protein that counteracts 53BP1 at breaks | Balances repair pathway choice influenced by H2AK129ub |
| PRC1 | Polycomb repressive complex that ubiquitinates H2AK119 | Cooperates with H2AK129ub in heterochromatin |
| SUV39H1 | Histone methyltransferase for H3K9me3 | Crosstalks with H3K18ub and H2A ubiquitination |
| SUV39H2 | Histone methyltransferase for H3K9me3 | Crosstalks with H3K18ub and H2A ubiquitination |
| USP48 | Deubiquitinating enzyme that removes H2A ubiquitin | Regulates reversibility of H2AK129ub |
| BRCC36 | Deubiquitinating enzyme in DNA damage response | Counteracts H2AK129 ubiquitination |
| RNF8 | E3 ligase that initiates ubiquitination cascade at breaks | Upstream of RNF168 in H2A ubiquitination |
| ATM | Kinase that phosphorylates H2AX and coordinates repair | Upstream regulator of H2AK129 ligase recruitment |
| MDC1 | Mediator of DNA damage checkpoint | Binds phosphorylated H2AX to recruit RNF168 |
| PKM2 | Pyruvate kinase M2 involved in metabolic regulation | March2 regulates PKM2 polymerization in aortic aneurysm |
| March2 | E3 ubiquitin ligase that regulates PKM2 | Example of ubiquitin ligase in non-histone pathways |
How Is histone H2AK129 ubiquitin ligase activity Regulated?
Histone H2AK129 ubiquitin ligase activity is regulated at multiple levels. Recruitment of RNF168 to DNA damage sites depends on ATM-mediated phosphorylation of H2A.X and MDC1. HDAC6 deacetylase activity is required for efficient RNF168 function, and its inhibition reduces H2A ubiquitination. UHRF1 ubiquitin ligase activity supports the maintenance of low-density CpG methylation, which crosstalks with H2A ubiquitination. Additionally, stress conditions control heterochromatin inheritance via histone H3 ubiquitylation, indicating that environmental factors modulate ubiquitin signaling. The balance between ligases and deubiquitinating enzymes such as USP48 and BRCC36 further fine-tunes the levels of H2AK129 ubiquitination.
histone H2AK129 ubiquitin ligase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| RNF168 | RIDDLE syndrome, cancer, immunodeficiency | RNF168 knockout cell lines and mouse models |
| HDAC6 | Cancer, neurodegeneration | HDAC6 knockout and point-mutation models |
| UHRF1 | Cancer, DNA methylation disorders | UHRF1 knockout and overexpression models |
| SMARCA3 | Cancer, chromatin remodeling disorders | SMARCA3 knockout and point-mutation models |
| March2 | Aortic aneurysm/dissection | March2 knockout and overexpression models |
Cancer and genomic instability
Dysregulation of H2AK129 ubiquitin ligase activity leads to defective DNA double-strand break repair, contributing to genomic instability and cancer predisposition. RNF168 mutations are associated with RIDDLE syndrome, an immunodeficiency characterized by radiosensitivity and cancer susceptibility. Targeting the HDAC6-RNF168 axis is a potential therapeutic strategy for cancers with defective H2A ubiquitination.
Aortic aneurysm and dissection
The E3 ubiquitin ligase March2 alleviates aortic aneurysm/dissection by regulating PKM2 polymerization, highlighting the broader role of ubiquitin ligases in vascular disease. Although not directly linked to H2AK129, this illustrates how ubiquitin signaling pathways impact cardiovascular health.
Heterochromatin-related disorders
Disruption of H2AK129 ubiquitination affects heterochromatin maintenance and can lead to aberrant gene silencing, which is implicated in developmental disorders and aging. The crosstalk between H3K18ub and H3K9me3 reinforces heterochromatin states, and its perturbation may contribute to disease.
From histone H2AK129 ubiquitin ligase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does RNF168 loss impair H2AK129 ubiquitination? | RNF168 knockout cell lines (e.g., HEK293T, U2OS) |
| What is the role of HDAC6 in H2A ubiquitination? | HDAC6 knockout or catalytic-dead point mutant |
| How does UHRF1 ubiquitin ligase activity affect CpG methylation? | UHRF1 knockout and knock-in of ligase-dead mutant |
| Does SMARCA3 regulate H3K23 ubiquitination? | SMARCA3 knockout and point-mutation models |
| What is the impact of H2AK129 ubiquitination on heterochromatin? | Knock-in of H2AK129R mutant histone |
| Can overexpression of RNF168 enhance DNA repair? | RNF168 overexpression cell lines |
How to Study the histone H2AK129 ubiquitin ligase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Mass spectrometry | Ubiquitination site identification and quantification | Detecting H2AK129ub changes |
| ChIP-seq | Genomic localization of ubiquitinated H2A | Mapping H2AK129ub across the genome |
| Comet assay | DNA double-strand breaks | Assessing repair defects in knockout cells |
| Gamma-H2AX foci | DNA damage response activation | Measuring repair factor recruitment |
| CRISPR knockout screens | Gene essentiality and pathway discovery | Identifying regulators of H2AK129ub |
| Western blot | Protein expression and ubiquitination levels | Validating ligase activity |
| Immunoprecipitation | Protein-protein interactions | Identifying E3 ligase complexes |
| Laser microirradiation | Real-time recruitment kinetics | Live-cell imaging of repair factors |
Ubiquitin proteomics and mass spectrometry
Mass spectrometry-based proteomics can identify and quantify ubiquitination sites on histones, including H2AK129. This method is essential for validating the specificity of H2AK129 ligases and for detecting changes in ubiquitination patterns upon genetic perturbation.
Chromatin immunoprecipitation and sequencing (ChIP-seq)
ChIP-seq using antibodies against ubiquitinated H2A or tagged ligases can map the genomic distribution of H2AK129 ubiquitination. This approach reveals how the mark correlates with DNA damage sites, heterochromatin domains, and gene regulatory elements.
DNA damage repair assays
Comet assays, gamma-H2AX foci formation, and laser microirradiation are used to assess the functional impact of H2AK129 ubiquitination on DNA repair. These assays measure recruitment kinetics of repair factors such as 53BP1 and BRCA1.
CRISPR-based genetic screens
Genome-wide CRISPR knockout screens can identify genes that regulate H2AK129 ubiquitination and its downstream effects. Such screens are powerful for discovering novel components of the ubiquitination pathway and for identifying therapeutic targets.
How CRISPR Can Be Used to Study GO:0140864 histone H2AK129 ubiquitin ligase activity
Knockout
CRISPR knockout of RNF168 or HDAC6 abolishes H2AK129 ubiquitin ligase activity, leading to defective DNA repair and chromatin changes. Knockout models are essential for establishing causality and for identifying downstream effectors.
Point Mutation
Point mutations in the catalytic domain of RNF168 (e.g., C16A) or in the ubiquitin acceptor site of H2A (K129R) can dissect the specific contribution of H2AK129 ubiquitination without affecting other functions. These models are valuable for separating enzymatic activity from scaffolding roles.
Knock-in
Knock-in of tagged or mutant histones (e.g., H2A-K129R) allows precise tracking of ubiquitination dynamics and its impact on heterochromatin inheritance. Tagged knock-in of RNF168 enables live-cell imaging of ligase recruitment.
Overexpression
Overexpression of wild-type or constitutively active RNF168 can enhance H2AK129 ubiquitination and DNA repair, while overexpression of dominant-negative mutants can inhibit the pathway. Overexpression models are useful for testing therapeutic hypotheses.
How EDITGENE Supports histone H2AK129 ubiquitin ligase activity Research
Researchers studying histone H2AK129 ubiquitin ligase activity-related genes often need to determine whether a candidate gene is causally involved in chromatin regulation, DNA repair, or disease. EDITGENE provides comprehensive CRISPR-based services to generate precisely engineered cell models for such functional studies.
Contact EDITGENE today to design your custom CRISPR model for histone H2AK129 ubiquitin ligase activity research.
Frequently Asked Questions About histone H2AK129 ubiquitin ligase activity
What is histone H2AK129 ubiquitin ligase activity?
It is the enzymatic activity (GO:0140864) that transfers ubiquitin to histone H2A at lysine 129, regulating chromatin and DNA repair.
What genes are involved in histone H2AK129 ubiquitin ligase activity?
Key genes include RNF168, HDAC6, UHRF1, and SMARCA3, which encode ligases or regulators of this activity.
Which enzyme is the main H2AK129 ubiquitin ligase?
RNF168 is the best-characterized E3 ligase that monoubiquitinates H2A/H2A.X at K129.
How is H2AK129 ubiquitination linked to DNA repair?
It recruits repair factors such as 53BP1 and BRCA1 to DNA double-strand breaks, facilitating repair.
What diseases are associated with H2AK129 ubiquitin ligase dysfunction?
Cancer, RIDDLE syndrome, genomic instability, and heterochromatin-related disorders.
What methods are used to study H2AK129 ubiquitination?
Mass spectrometry, ChIP-seq, DNA damage assays, and CRISPR screens.
Can CRISPR knockout help study H2AK129 ubiquitin ligase activity?
Yes, knockout of RNF168 or HDAC6 abolishes the activity and reveals its cellular functions.
What is the difference between H2AK129 and H2AK119 ubiquitination?
H2AK129 is monoubiquitination by RNF168 in DNA repair, while H2AK119 is mediated by PRC1 in gene silencing.
How does HDAC6 regulate H2AK129 ubiquitination?
HDAC6 deacetylase activity is required for efficient RNF168-mediated H2A ubiquitination.
What model systems are available for studying H2AK129 ubiquitin ligase activity?
Knockout, point-mutation, knock-in, and overexpression cell models, as well as mouse models.
Conclusion
Histone H2AK129 ubiquitin ligase activity (GO:0140864) is a fundamental molecular function that governs chromatin dynamics, DNA repair, and heterochromatin maintenance. Its dysregulation is implicated in cancer and genomic instability, making it a compelling target for basic and translational research. By leveraging CRISPR-based models and advanced proteomics, researchers can dissect the precise roles of H2AK129 ubiquitination in health and disease.
References
- 1. Tiedemann RL et al.. 2024. UHRF1 ubiquitin ligase activity supports the maintenance of low-density CpG methylation.. Nucleic Acids Res 52(22):13733-13756 PMID: 39607687
- 2. Li YE et al.. 2025. March2 Alleviates Aortic Aneurysm/Dissection by Regulating PKM2 Polymerization.. Circ Res 136(8):e73-e93 PMID: 40079144
- 3. Akano I et al.. 2025. The SWI/SNF-related protein SMARCA3 is a histone H3K23 ubiquitin ligase that regulates H3K9me3 in cancer.. Mol Cell 85(15):2885-2899.e8 PMID: 40680746
- 4. Huang Y et al.. 2025. A conserved H3K14ub-driven H3K9me3 for chromatin compartmentalization.. Nature 647(8090):786-797 PMID: 41094145
- 5. Feng LL et al.. 2024. Ubiquitin-induced RNF168 condensation promotes DNA double-strand break repair.. Proc Natl Acad Sci U S A 121(28):e2322972121 PMID: 38968116
- 6. Qiu L et al.. 2023. The HDAC6-RNF168 axis regulates H2A/H2A.X ubiquitination to enable double-strand break repair.. Nucleic Acids Res 51(17):9166-9182 PMID: 37503842
- 7. Bhatt B et al.. 2026. Stress controls heterochromatin inheritance via histone H3 ubiquitylation.. Nature 650(8102):768-778 PMID: 41501458
- 8. Liu Y et al.. 2025. DNA hypomethylation promotes UHRF1-and SUV39H1/H2-dependent crosstalk between H3K18ub and H3K9me3 to reinforce heterochromatin states.. Mol Cell 85(2):394-412.e12 PMID: 39631394
