Phospho-H2AFX (Y142) Antibody

What is Phospho-H2AFX (Y142) and why is it significant in DNA damage research?

Phospho-H2AFX (Y142) refers to the phosphorylation of tyrosine residue 142 on histone variant H2AFX (also known as H2AX). This post-translational modification plays a pivotal role in determining whether cells undergo DNA repair or apoptosis following genotoxic stress. H2AFX is a variant histone that replaces conventional H2A in a subset of nucleosomes and is required for checkpoint-mediated arrest of cell cycle progression in response to DNA damage .

The Y142 residue is constitutively phosphorylated in unstressed cells by Williams-Beuren syndrome transcription factor (WSTF) . Following DNA damage, this residue undergoes dephosphorylation by EYA1 or EYA3 phosphatases, which is necessary to facilitate the formation of γ-H2AX (phosphorylation at S139) and the subsequent binding of MDC1 that initiates DNA repair responses . If Y142 remains phosphorylated during genotoxic stress, the cellular response shifts toward apoptosis rather than DNA repair .

The phosphorylation status of Y142 therefore functions as a molecular switch that determines cell fate decisions in response to DNA damage, making it a critical target for cancer research and studies of genomic stability.

How does Y142 phosphorylation interact with S139 phosphorylation (γ-H2AX) in the DNA damage response?

The interaction between Y142 and S139 phosphorylation represents a sophisticated phosphorylation code that regulates downstream protein recruitment and ultimately determines cell fate decisions:

• Dual Phosphorylation Status: While S139 phosphorylation (γ-H2AX) is widely recognized as a marker of DNA double-strand breaks, the phosphorylation status of Y142 critically modulates the downstream effects of γ-H2AX .

• Competitive Binding: Phosphorylation experiments using peptides corresponding to the C-terminal tail of H2AX have revealed that when both S139 and Y142 are phosphorylated, binding of DNA repair factors including MDC1, MRE11, and Rad50 is greatly reduced. Instead, this dual phosphorylation promotes binding of pro-apoptotic factors such as JNK1 .

• Impact on S139 Phosphorylation: The Y142F mutation (preventing Y142 phosphorylation) results in consistently reduced levels of S139 phosphorylation compared to wild-type H2AX after DNA damage, suggesting Y142 phosphorylation may promote or maintain serine phosphorylation by DNA damage response kinases .

• Temporal Dynamics: Y142 dephosphorylation by EYA phosphatases appears to be necessary before MDC1 can efficiently bind to γ-H2AX, creating a sequential phosphorylation/dephosphorylation process that regulates the DNA damage response .

This interplay demonstrates that while Y142 phosphorylation does not function as a prerequisite for S139 phosphorylation, it significantly influences the maintenance and downstream effects of γ-H2AX signaling.

What experimental applications are recommended for Phospho-H2AFX (Y142) antibodies?

Phospho-H2AFX (Y142) antibodies are valuable tools for studying DNA damage responses and cell fate decisions. Based on the available commercial antibodies, the following applications are recommended:

When conducting immunofluorescence studies, researchers should consider the following methodological approaches:

• Co-staining with γ-H2AX (S139) antibodies: This allows assessment of the relative levels of both phosphorylation marks and their colocalization at DNA damage sites.

• Time-course analysis: Given the dynamic nature of Y142 phosphorylation/dephosphorylation following DNA damage, temporal analysis is crucial for understanding the kinetics of this modification .

• Cell type considerations: The phosphorylation dynamics may vary between different cell types, particularly embryonic versus differentiated cells, based on the differential expression of WSTF and EYA proteins .

The antibodies are typically supplied in liquid form, containing preservatives like 0.03% Proclin 300 in a buffer of 50% Glycerol and 0.01M PBS at pH 7.4. For optimal results, store at -20°C or -80°C and avoid repeated freeze-thaw cycles .

How does the phosphorylation status of H2AFX Y142 determine the recruitment of DNA repair versus apoptotic factors?

The phosphorylation status of H2AFX Y142 serves as a molecular switch that dictates which protein complexes are recruited to DNA damage sites, ultimately determining whether cells undergo repair or apoptosis:

This phosphorylation-dependent recruitment mechanism represents a sophisticated cellular decision-making process that balances DNA repair with apoptosis based on the severity of DNA damage and cellular context.

What is the role of WSTF in H2AFX Y142 phosphorylation and transcription-coupled homologous recombination repair?

WSTF (Williams-Beuren syndrome transcription factor) plays a multifaceted role in H2AFX Y142 phosphorylation and transcription-coupled homologous recombination (TC-HR) repair:

• Constitutive Phosphorylation: WSTF is responsible for constitutive phosphorylation of H2AFX at Y142 in unstressed cells, maintaining this phosphorylation mark under normal conditions .

• Association with RNA Polymerase II: Research has shown that constitutive H2AFX-pY142 generated by WSTF interacts with RNA polymerase II (RNAPII) and is associated with RNAPII-mediated active transcription in proliferating cells .

• Dynamics During DNA Damage Response: Following DNA damage, pre-existing H2AFX-pY142 is removed by ATM-dependent EYA1/3 phosphatases, which disrupts the association with RNAPII and is required for transcriptional silencing at transcribed active damage sites .

• WSTF Translocation and TC-HR: After the initial dephosphorylation phase, WSTF translocates to DNA lesions where it facilitates the recovery of H2AFX-pY142. This recovery promotes transcription-coupled homologous recombination repair in the G1 phase .

• RNA-Templated Repair: The WSTF-mediated process facilitates RAD51 loading, which utilizes RNAPII-dependent active RNA transcripts as donor templates for repair. Interestingly, this process involves RAD51 but not RPA32 .

This mechanism reveals a novel layer of regulation involving transcription-coupled H2AFX-pY142 in the DNA damage response, demonstrating that the WSTF-H2AX-RNAPII axis regulates both transcription and TC-HR repair to maintain genome integrity.

How do EYA phosphatases regulate the balance between DNA repair and apoptosis through H2AFX Y142 dephosphorylation?

EYA (Eyes Absent) phosphatases play a crucial role in regulating cellular responses to DNA damage through their dephosphorylation activity on H2AFX Y142:

This regulatory mechanism represents a phosphorylation-dependent decision point that modulates survival/apoptotic decisions during mammalian organogenesis and in response to DNA damage in differentiated tissues.

What methodological approaches can researchers use to study the temporal dynamics of H2AFX Y142 phosphorylation during DNA damage response?

To effectively study the temporal dynamics of H2AFX Y142 phosphorylation during the DNA damage response, researchers can employ several complementary methodological approaches:

• Time-Course Western Blotting:

  • Treat cells with DNA damaging agents (e.g., ionizing radiation, etoposide)

  • Collect samples at multiple time points (e.g., 0, 1, 2, 4, 8, 24 hours)

  • Perform western blotting with antibodies specific to H2AFX-pY142, γH2AX (pS139), and total H2AFX

  • Quantify relative phosphorylation levels normalized to total H2AFX

• Immunofluorescence Microscopy:

  • Perform dual immunostaining for H2AFX-pY142 and γH2AX

  • Analyze colocalization patterns at different time points after damage

  • Quantify signal intensity and foci formation using image analysis software

  • This approach allows for single-cell analysis and assessment of nuclear localization

• Phospho-specific Antibody Validation:

  • Use H2AFX Y142F mutant cells as negative controls for antibody specificity

  • Compare staining patterns in wild-type versus mutant cells after damage

  • This control is critical given that Y142F mutation affects S139 phosphorylation levels

• Genetic and Pharmacological Interventions:

  • Manipulate EYA1/3 phosphatases (through knockdown, knockout, or overexpression)

  • Modulate WSTF activity to alter constitutive Y142 phosphorylation

  • Use ATM/ATR inhibitors to study the interplay between canonical damage signaling and Y142 phosphorylation

• Chromatin Immunoprecipitation (ChIP):

  • Perform ChIP with H2AFX-pY142 antibodies at different time points after damage

  • Analyze enrichment at specific genomic loci (e.g., actively transcribed regions)

  • Combine with RNA polymerase II ChIP to study the WSTF-H2AX-RNAPII axis

• Mass Spectrometry-Based Approaches:

  • Use quantitative phospho-proteomics to measure the stoichiometry of different H2AFX phosphorylation states

  • Analyze peptides containing both Y142 and S139 to assess dual phosphorylation dynamics

  • This approach provides unbiased detection of multiple post-translational modifications

By combining these methodological approaches, researchers can comprehensively characterize the spatiotemporal dynamics of H2AFX Y142 phosphorylation and its relationship with other histone modifications during the DNA damage response.

How can researchers distinguish between different phosphorylation states of H2AFX in experimental systems?

Distinguishing between the different phosphorylation states of H2AFX (particularly Y142 and S139) is critical for understanding the complex signaling events in the DNA damage response. Researchers can employ several specialized techniques to identify and quantify these distinct phosphorylation states:

• Phospho-specific Antibodies:

  • Use antibodies that specifically recognize H2AFX-pY142, γH2AX (pS139), or dual-phosphorylated H2AFX

  • Validate antibody specificity using phosphatase treatments, phospho-mimetic mutants, and phospho-null mutants (Y142F, S139A)

  • For western blotting, the recommended dilutions for Phospho-H2AFX (Y142) antibodies range from 1:50 to 1:200 for immunofluorescence applications

• Peptide Competition Assays:

  • Pre-incubate antibodies with phosphorylated peptides corresponding to different H2AFX phosphorylation states

  • A reduction in signal when pre-incubated with the specific phospho-peptide confirms antibody specificity

• Phospho-peptide Enrichment and Mass Spectrometry:

  • Enrich for phosphorylated peptides using titanium dioxide (TiO2) or immobilized metal affinity chromatography (IMAC)

  • Use multiple reaction monitoring (MRM) or parallel reaction monitoring (PRM) to quantify specific H2AFX phospho-peptides

  • This approach can determine the absolute stoichiometry of different phosphorylation states

• Site-specific Mutants:

  • Generate cell lines expressing H2AFX Y142F, S139A, or double mutants

  • Compare phosphorylation patterns and cellular responses to DNA damage

  • Use these mutants as negative controls for antibody validation

• Sequential Immunoprecipitation:

  • First immunoprecipitate with one phospho-specific antibody (e.g., γH2AX)

  • Then immunoprecipitate the supernatant with another antibody (e.g., H2AFX-pY142)

  • This approach can separate populations with different phosphorylation states

• Phosphatase Treatment Controls:

  • Treat samples with tyrosine-specific (e.g., PTP1B) or serine/threonine-specific (e.g., PP2A) phosphatases

  • Analyze the resulting changes in antibody recognition

  • This confirms the phospho-specificity of the observed signals

• Proximity Ligation Assays (PLA):

  • Use antibodies against different phosphorylation marks or interacting proteins

  • PLA generates a signal only when two proteins/modifications are in close proximity

  • This technique can identify dual-phosphorylated H2AFX populations in situ

These methodological approaches enable researchers to distinguish between the different phosphorylation states of H2AFX and determine how these states influence the recruitment of DNA repair and apoptotic factors during the DNA damage response.

What controls should be included when using Phospho-H2AFX (Y142) antibodies in DNA damage experiments?

When designing experiments utilizing Phospho-H2AFX (Y142) antibodies, researchers should incorporate several critical controls to ensure reliable and interpretable results:

• Phosphorylation State Controls:

  • Positive Control: Cells with constitutive Y142 phosphorylation (e.g., unstressed proliferating cells)

  • Negative Control: Cells expressing H2AFX Y142F mutant

  • Dynamic Control: Time course after DNA damage induction to capture dephosphorylation and re-phosphorylation events

• Antibody Specificity Controls:

  • Peptide Competition: Pre-incubation of antibody with phosphorylated and non-phosphorylated peptides corresponding to the Y142 region

  • Phosphatase Treatment: Sample treatment with tyrosine-specific phosphatases to remove the Y142 phosphorylation

  • Knockdown/Knockout: siRNA or CRISPR-mediated depletion of H2AFX to confirm signal specificity

• DNA Damage Response Controls:

  • ATM/ATR Inhibition: Treatment with specific inhibitors to modulate the canonical DNA damage response pathways

  • EYA1/3 Manipulation: Knockdown or overexpression of these phosphatases to alter Y142 dephosphorylation dynamics

  • WSTF Modulation: Manipulation of WSTF levels to affect constitutive Y142 phosphorylation

• Technical Controls:

  • Loading Control: Antibodies against total H2AFX or other histones to normalize phosphorylation signals

  • Secondary Antibody Only: To assess background staining in immunofluorescence experiments

  • Cross-reactivity Control: Testing against related histone variants to ensure specificity

• Biological Context Controls:

  • Cell Cycle Synchronization: As phosphorylation patterns may vary throughout the cell cycle

  • Different Cell Types: To account for tissue-specific differences in H2AFX regulation

  • DNA Damage Types: Compare responses to different genotoxic agents (IR, UV, replication stress)

By incorporating these controls, researchers can confidently interpret the dynamics and functional significance of H2AFX Y142 phosphorylation in their experimental systems, distinguishing genuine biological effects from technical artifacts.

How can researchers optimize detection of Phospho-H2AFX (Y142) in different experimental contexts?

Optimizing the detection of Phospho-H2AFX (Y142) requires careful consideration of sample preparation, antibody selection, and detection methods based on the specific experimental context:

• Sample Preparation Strategies:

  • Extraction Buffers: Use buffers containing phosphatase inhibitors (sodium orthovanadate for tyrosine phosphatases) to preserve Y142 phosphorylation

  • Acid Extraction: For histone-focused analyses, acid extraction methods (e.g., 0.2N HCl) can enrich for histones while preserving phosphorylation marks

  • Subcellular Fractionation: Nuclear isolation can concentrate the H2AFX signal and reduce cytoplasmic background

  • Fixation Methods: For immunofluorescence, compare paraformaldehyde (PFA) fixation with methanol fixation to determine optimal epitope preservation

• Antibody Selection and Validation:

  • Validate Across Applications: Test antibodies in multiple applications (western blot, IF, ChIP) as performance may vary

  • Titration Experiments: For immunofluorescence, test dilutions ranging from 1:50 to 1:200 to determine optimal signal-to-noise ratio

  • Epitope Mapping: Understand the exact epitope recognized by the antibody and potential cross-reactivity with other phosphorylated histones

• Signal Enhancement Techniques:

  • Tyramide Signal Amplification: For low-abundance epitopes in immunofluorescence

  • Polymer Detection Systems: For immunohistochemistry applications

  • Proximity Ligation Assay: To detect dual-modified H2AFX or interactions with binding partners

• Application-Specific Optimizations:

  • Western Blotting:

    • Use gradient gels (15-20%) for optimal histone resolution

    • Consider transferring with SDS in the transfer buffer to improve histone transfer

    • Short blocking times (30-60 minutes) may improve detection of phospho-epitopes

  • Immunofluorescence:

    • Pre-extraction with detergent to remove soluble nuclear proteins and reduce background

    • Antigen retrieval methods may be necessary for certain fixation protocols

    • Confocal microscopy for precise localization and colocalization analysis

  • ChIP:

    • Optimize sonication conditions for histone ChIP (typically shorter fragments)

    • Consider dual crosslinking with DSG (disuccinimidyl glutarate) followed by formaldehyde

    • Include input normalization and appropriate IgG controls

• Quantification Approaches:

  • Western Blot: Normalize phospho-signal to total H2AFX rather than loading controls like actin

  • Immunofluorescence: Use automated image analysis software to quantify nuclear foci number, size, and intensity

  • Flow Cytometry: Consider phospho-flow approaches for high-throughput quantification across cell populations

By systematically optimizing these parameters, researchers can enhance the detection sensitivity and specificity of Phospho-H2AFX (Y142) across diverse experimental contexts, enabling more robust analysis of this critical DNA damage response regulatory mechanism.

How does H2AFX Y142 phosphorylation connect to transcription regulation and genome stability?

The relationship between H2AFX Y142 phosphorylation, transcription regulation, and genome stability represents a sophisticated regulatory network that integrates multiple cellular processes:

• Association with Active Transcription:

  • Constitutive H2AFX-pY142 generated by WSTF interacts with RNA polymerase II (RNAPII) and is associated with RNAPII-mediated active transcription in proliferating cells

  • This association suggests that Y142 phosphorylation plays a role in normal transcriptional processes beyond its functions in DNA damage response

• Transcriptional Silencing at Damage Sites:

  • Following DNA damage, removal of pre-existing H2AFX-pY142 by ATM-dependent EYA1/3 phosphatases disrupts the association with RNAPII

  • This dissociation is required for transcriptional silencing at transcribed active damage sites, preventing transcription through damaged DNA regions

• Transcription-Coupled Homologous Recombination (TC-HR):

  • The recovery of H2AFX-pY142 via translocation of WSTF to DNA lesions facilitates TC-HR repair specifically in the G1 phase

  • This process involves RAD51 loading that utilizes RNAPII-dependent active RNA transcripts as donor templates for repair

  • This mechanism represents a novel pathway for DNA repair that is intimately connected with transcription

• Cell Cycle-Specific Functions:

  • The WSTF-H2AX-RNAPII axis regulates both transcription and TC-HR repair throughout the cell cycle

  • This suggests that Y142 phosphorylation dynamics contribute to genome stability in a cell cycle-dependent manner

• Balancing Repair and Apoptosis:

  • The phosphorylation status of Y142 determines whether cells undergo repair or apoptosis in response to DNA damage

  • This balance is critical for maintaining genomic integrity while eliminating cells with irreparable damage

• Impact on Chromatin Accessibility:

  • H2AFX is involved in nucleosome formation, which wraps and compacts DNA into chromatin, limiting DNA accessibility to cellular machineries

  • The phosphorylation status of Y142 may influence chromatin accessibility during transcription and repair processes

This integrated view demonstrates that H2AFX Y142 phosphorylation serves as a critical nexus connecting transcriptional regulation, DNA damage responses, and genome stability, highlighting the multifunctional nature of histone post-translational modifications in maintaining cellular homeostasis.

What are the implications of H2AFX Y142 phosphorylation research for cancer and neurological disorders?

Research on H2AFX Y142 phosphorylation has significant implications for understanding and potentially treating various diseases, particularly cancer and neurological disorders:

Cancer Implications:

• Therapeutic Targeting:

  • The dual phosphorylation status of H2AFX (S139/Y142) presents a potential target for cancer therapy by modulating the balance between DNA repair and apoptosis

  • Inhibiting EYA phosphatases could promote persistent Y142 phosphorylation, potentially sensitizing cancer cells to DNA-damaging therapies by favoring apoptosis over repair

• Biomarker Development:

  • The ratio of different H2AFX phosphorylation states could serve as a biomarker for DNA damage response capacity in tumors

  • This might predict responsiveness to radiation or chemotherapy and inform treatment decisions

• Resistance Mechanisms:

  • Alterations in the WSTF-H2AX-RNAPII axis might contribute to therapy resistance by enhancing DNA repair capacity

  • Understanding the role of transcription-coupled homologous recombination mediated by this pathway could explain repair mechanisms in G1 phase cancer cells

• Genomic Instability:

  • Dysregulation of H2AFX Y142 phosphorylation could contribute to genomic instability, a hallmark of cancer

  • The balance between repair and apoptosis influenced by Y142 phosphorylation may impact cancer evolution and heterogeneity

Neurological Disorder Implications:

• Williams-Beuren Syndrome:

  • Given WSTF's role in constitutive Y142 phosphorylation, its haploinsufficiency in Williams-Beuren syndrome might affect DNA damage responses in neural cells

  • This could potentially contribute to neurodevelopmental aspects of the syndrome

• Neurodegeneration:

  • Neurons are postmitotic cells predominantly in G0/G1 phase, making them reliant on specific repair pathways

  • The WSTF-dependent transcription-coupled homologous recombination in G1 phase may be particularly important for neuronal genome maintenance

• Developmental Disorders:

  • EYA proteins play crucial roles in organogenesis, and their function in H2AFX Y142 dephosphorylation connects developmental signaling with DNA damage responses

  • This link may explain certain aspects of developmental disorders associated with EYA mutations

• Aging and Neurodegeneration:

  • Accumulation of DNA damage is associated with neurodegeneration and aging

  • Age-related changes in H2AFX phosphorylation dynamics might contribute to reduced repair capacity and increased neuronal vulnerability

Future Research Directions:

• Developing specific inhibitors or activators of the enzymes regulating H2AFX Y142 phosphorylation

• Investigating tissue-specific differences in Y142 phosphorylation dynamics, particularly in brain regions vulnerable to neurodegeneration

• Exploring the interaction between H2AFX Y142 phosphorylation and other genetic risk factors for cancer and neurological disorders

• Examining how environmental factors and cellular stressors influence Y142 phosphorylation and subsequent cell fate decisions

By deepening our understanding of H2AFX Y142 phosphorylation in disease contexts, researchers may uncover novel therapeutic approaches targeting the fundamental mechanisms of DNA damage response and cell fate decisions.

What are the current limitations and future directions in Phospho-H2AFX (Y142) research?

Current research on Phospho-H2AFX (Y142) has revealed its critical role in DNA damage response regulation, but several limitations remain and important future directions deserve attention:

Current Limitations:

• Antibody Specificity and Sensitivity:

  • Challenges in developing antibodies that specifically recognize Y142 phosphorylation without cross-reactivity

  • Limited sensitivity for detecting low levels of Y142 phosphorylation, particularly in complex tissue samples

• Temporal and Spatial Resolution:

  • Difficulty in tracking real-time dynamics of Y142 phosphorylation in living cells

  • Limited understanding of the spatial distribution of different H2AFX phosphorylation states within chromatin domains

• Mechanistic Gaps:

  • Incomplete understanding of how Y142 phosphorylation physically prevents binding of DNA repair factors

  • Limited knowledge about additional proteins that recognize and bind specifically to phosphorylated Y142

• Tissue and Context Specificity:

  • Most studies have focused on a limited range of cell types, with less understanding of tissue-specific regulation

  • Developmental context of Y142 phosphorylation dynamics remains poorly characterized

• Therapeutic Translation:

  • Lack of specific modulators of Y142 phosphorylation for therapeutic applications

  • Challenges in selectively targeting the Y142 phosphorylation pathway without disrupting essential cellular functions

Future Directions:

• Methodological Advances:

  • Development of live-cell imaging techniques for tracking Y142 phosphorylation dynamics

  • Creation of more specific and sensitive antibodies or alternative detection methods

  • Implementation of single-cell analysis approaches to capture cellular heterogeneity

• Integrated Multi-omics Approaches:

  • Combining phospho-proteomics, transcriptomics, and genomics to understand the broader impact of Y142 phosphorylation

  • Mapping global changes in chromatin accessibility and structure influenced by Y142 phosphorylation status

• Disease Relevance:

  • Systematic investigation of Y142 phosphorylation patterns across cancer types and stages

  • Exploration of Y142 phosphorylation in neurodegenerative diseases and during aging

  • Assessment of Y142 phosphorylation as a biomarker for disease diagnosis or treatment response

• Therapeutic Development:

  • Design of small molecules or peptides that specifically modulate Y142 phosphorylation

  • Development of targeted approaches to modify the WSTF-H2AX-RNAPII axis in disease contexts

  • Exploration of combination therapies targeting both Y142 and S139 phosphorylation pathways

• Evolutionary and Comparative Studies:

  • Investigation of Y142 phosphorylation across species to understand evolutionary conservation

  • Comparative analysis of this mechanism across different cell types and developmental stages