DNMT3A Antibody

What is DNMT3A and why is it a significant target for antibody-based detection?

DNMT3A is a 130 kDa member of the C5-methyltransferase family that possesses DNA methyltransferase activity. It represents a critical epigenetic regulator expressed during development and in virtually all adult tissues except small intestine . As a nuclear protein associated with heterochromatin, DNMT3A forms functional complexes with proteins like DNMT3L in embryonic stem cells, creating a (3A)2:(3L)2 heterotetramer . Its interaction with SETDB1 also forms transcriptional repressor complexes .

The significance of DNMT3A as an antibody target stems from its pivotal roles in:

• Establishing DNA methylation patterns during development

• Regulating gene expression in differentiated tissues

• Contributing to disease pathogenesis when mutated or dysregulated

• Serving as a potential biomarker in conditions like acute myeloid leukemia (AML)

These diverse functions make DNMT3A antibodies essential tools for investigating epigenetic mechanisms in both normal development and disease states.

What are the major variants of DNMT3A and how do they differ functionally?

DNMT3A exists in multiple transcript variants with distinct functional properties:

• Full-length DNMT3A (transcript 1): The 912 amino acid protein contains a ProTrpTrpPro domain (aa 292-350) and an ADD-type zinc finger region (aa 494-586) .

• DNMT3A2 (transcript 2): A 100 kDa splice variant with an alternative start site at Met224 that preferentially interacts with euchromatin rather than heterochromatin .

• Additional variants (including transcript 4): Have distinct functions, particularly in hematopoietic differentiation .

Functionally, these variants demonstrate significant differences:

• Transcript 2 (DNMT3A2) activates cellular proliferation and induces loss of primitive immunophenotype in hematopoietic stem cells .

• Transcript 4 specifically interferes with colony formation of the erythroid lineage .

• Different variants establish complementary and transcript-specific DNA methylation patterns, suggesting non-redundant roles in cellular differentiation .

These functional differences make it crucial for researchers to specify which DNMT3A variant they are investigating and to select antibodies that can differentiate between these variants when necessary.

What cellular processes are regulated by DNMT3A that make it an important research target?

DNMT3A regulates several critical cellular processes that position it as a valuable research target:

• Epigenetic Programming: DNMT3A establishes de novo DNA methylation patterns essential for development and cellular differentiation .

• Hematopoietic Differentiation: DNMT3A variants have distinct impacts on blood cell development, with transcript 2 (DNMT3A2) particularly involved in driving differentiation of hematopoietic stem and progenitor cells .

• Genomic Integrity: DNMT3A contributes to preserving genome integrity during replication stress, with mutations associated with impaired recovery from replication fork arrest and accumulated DNA damage .

• Cardiac Function: In cardiomyocytes, DNMT3A knockout results in:

  • Gene expression changes in contractile proteins

  • Activation of glucose/lipid metabolism regulators

  • Hypoxia-inducible factor 1α protein instability

  • Impaired glucose metabolism under stress conditions

• Leukemogenesis: DNMT3A mutations in AML are associated with chemoresistance and poor prognosis, particularly in advanced-age patients, highlighting its role in malignancy .

Understanding these processes provides multiple entry points for antibody-based investigations of DNMT3A function in normal physiology and disease.

What are the optimal methods for detecting DNMT3A in different experimental contexts?

Detecting DNMT3A requires selecting appropriate methodologies based on experimental objectives:

Western Blot Detection:

• Use reducing conditions with PVDF membrane

• Optimal antibody dilution: ~0.1 μg/mL for monoclonal antibodies like MAB63151

• Expected molecular weight: approximately 120 kDa band

• Compatible cell types: HeLa, HEK293, U2OS (human); NIH-3T3 (mouse)

• Buffer recommendation: Immunoblot Buffer Group 1

Immunofluorescence/Immunocytochemistry:

• Fixation: Immersion fixation for adherent cells

• Optimal antibody concentration: 10-15 μg/mL for MAB6315

• Incubation time: 3 hours at room temperature

• Secondary antibody: NorthernLights™ 557-conjugated Anti-Mouse IgG

• Counterstain: DAPI for nuclear visualization

• Expected localization: Nuclear staining pattern

Flow Cytometry:
When utilizing flow cytometry, researchers should perform careful titration experiments with positive and negative control cell lines. HeLa cells show positive staining while Daudi human Burkitt's lymphoma cells demonstrate negative staining, making them useful controls .

The choice between polyclonal (AF6315) and monoclonal (MAB6315, MAB63151) antibodies should be based on the required specificity, with monoclonal antibodies offering greater epitope specificity but potentially more limited detection of variant forms.

How can researchers validate the specificity of DNMT3A antibodies in their experimental systems?

Validating DNMT3A antibody specificity is crucial for generating reliable data. Recommended validation approaches include:

Positive and Negative Control Cell Lines:

• Positive controls: HeLa, HEK293, U2OS, and OVCAR-3 cell lines demonstrate consistent DNMT3A expression

• Negative controls: Daudi human Burkitt's lymphoma cells show minimal DNMT3A expression

Genetic Controls:

• CRISPR/Cas9-mediated knockout models: Complete DNMT3A knockouts should show absence of antibody signal

• Heterozygous models (DNMT3A+/-): Should demonstrate approximately 40% reduction in protein signal compared to wild-type

• Consider using the three validated DNMT3A mutant cell lines described in the literature:

  • Heterozygous insertion of adenine (DNMT3A+/-)

  • Compound heterozygous insertion/deletion (DNMT3A-/- #1)

  • Homozygous insertion (DNMT3A-/- #2)

Peptide Competition Assays:
Pre-incubation of the antibody with the immunizing peptide (human DNMT3A Ala353-Lys486) should abolish specific signal .

Cross-Reactivity Testing:
When using antibodies across species, verify cross-reactivity through western blot analysis. MAB63151 has demonstrated reactivity with both human and mouse DNMT3A , while others like DF6795 are reported to react with human, mouse, and rat samples, with predicted cross-reactivity to bovine, horse, rabbit, and dog .

What are the key considerations for using DNMT3A antibodies in cancer research applications?

When applying DNMT3A antibodies in cancer research, investigators should consider:

Expression Pattern Analysis:

• DNMT3A shows differential expression across cancer types with notable detection in:

  • Ovarian carcinoma (OVCAR-3 cell line)

  • Cervical epithelial carcinoma (HeLa cell line)

  • Osteosarcoma (U2OS cell line)

• Negative/low expression in Burkitt's lymphoma (Daudi cell line)

Potential Therapeutic Applications:

• DNMT3A(R882)-mutant cells show increased sensitivity to replication stress inducers like cytarabine

• This vulnerability is characterized by:

  • Persistent intra-S-phase checkpoint activation

  • Impaired PARP1 recruitment

  • Elevated DNA damage

  • Higher rates of fork collapse after cytarabine washout

When designing experiments, researchers should determine whether to focus on detecting wild-type DNMT3A, specific mutations, or particular transcript variants, as each approach requires different antibody selection criteria and experimental controls.

How do mutations in DNMT3A affect DNA damage response, and what antibody-based approaches can best characterize these effects?

DNMT3A mutations, particularly R882 mutations commonly found in leukemia, significantly impact DNA damage response mechanisms through several interrelated pathways:

DNA Replication Stress Response:
DNMT3A(R882)-mutant cells show distinct responses to replication stress inducers like cytarabine:

• Persistent intra-S-phase checkpoint activation

• Impaired PARP1 recruitment to sites of DNA damage

• Elevated levels of unresolved DNA damage carried through mitosis

Fork Recovery Defects:

• Pulse-chase double-labeling experiments with EdU and BrdU demonstrate higher rates of fork collapse in DNMT3A(mut)-expressing cells after cytarabine removal

• This suggests fundamental defects in the machinery that protects and restores stalled replication forks

Transcriptional Consequences:
RNA-seq studies reveal that DNMT3A mutations lead to deregulation of:

• Cell-cycle progression pathways

• p53 activation networks

• Splicing mechanisms

• Ribosome biogenesis

• Metabolic processes

Recommended Antibody-Based Approaches:

• Co-immunoprecipitation Studies: Using validated DNMT3A antibodies like MAB6315 or AF6315 to pull down DNMT3A complexes and identify altered protein interactions in mutant vs. wild-type contexts

• Chromatin Immunoprecipitation (ChIP): To examine changes in DNMT3A chromatin association patterns in response to replication stress

• Dual Immunofluorescence: Combining DNMT3A antibodies with markers of DNA damage (γH2AX) or replication stress (pRPA) to visualize co-localization patterns

• Proximity Ligation Assays (PLA): To detect altered interactions between DNMT3A and DNA repair proteins like PARP1 in mutant cells

These approaches can help elucidate how DNMT3A mutations impact genome stability pathways and potentially identify novel therapeutic vulnerabilities in DNMT3A-mutant cancers.

How do different DNMT3A transcript variants contribute to lineage-specific differentiation, and what experimental strategies can distinguish their functions?

DNMT3A transcript variants play distinct roles in lineage-specific differentiation, particularly in hematopoiesis. Understanding these differences requires sophisticated experimental approaches:

Variant-Specific Functions:

• Transcript 2 (DNMT3A2): Activates proliferation and induces loss of primitive immunophenotype in hematopoietic stem and progenitor cells

• Transcript 4: Specifically interferes with colony formation of the erythroid lineage

• Each variant establishes unique DNA methylation and gene expression patterns

Experimental Strategies to Distinguish Variant Functions:

• Variant-Specific Knockdown/Overexpression:

  • Selectively manipulate individual transcript levels through targeted siRNA or overexpression constructs

  • Monitor effects on:

    • Cellular differentiation markers

    • DNA methylation landscapes

    • Gene expression profiles

    • Functional capabilities (colony formation, proliferation rates)

• Transcript-Specific Antibody Selection:

  • For DNMT3A2 (transcript 2) detection: Target the unique N-terminal region absent in this variant

  • For full-length DNMT3A: Target epitopes within the first 223 amino acids

  • Verify isoform specificity through western blotting of cells with known expression patterns

• Methylome Analysis Pipeline:

  • Perform whole-genome DNA methylation analysis after modulating specific variants

  • Identify differentially methylated regions (DMRs) associated with each variant

  • Correlate methylation changes with gene expression alterations

  • Connect molecular changes to phenotypic outcomes

• Chromatin Occupancy Mapping:

  • ChIP-seq using variant-specific antibodies (where available) or tagged constructs

  • Identify distinct genomic targeting preferences (euchromatin vs. heterochromatin)

  • Correlate binding patterns with downstream epigenetic effects

These approaches can help delineate the unique contributions of each DNMT3A variant to lineage specification and identify potential variant-specific therapeutic targets in diseases with dysregulated DNMT3A function.

What role does DNMT3A play in cardiac development and function, and how can researchers best investigate its tissue-specific effects?

DNMT3A serves critical functions in cardiac development and homeostasis through several mechanisms revealed through knockout studies:

Cardiac-Specific DNMT3A Functions:

• Contractile Protein Regulation:

  • DNMT3A knockout in cardiomyocytes leads to altered gene expression of contractile proteins

  • Higher atrial gene expression is observed

  • Lower MYH7/MYH6 ratio correlates with different contraction kinetics

• Metabolic Regulation:

  • DNMT3A deficiency causes aberrant activation of peroxisome proliferator-activated receptor gamma (PPARγ)

  • This activation is associated with accumulation of lipid vacuoles within cardiomyocytes

• Hypoxia Response:

  • DNMT3A knockout leads to hypoxia-inducible factor 1α (HIF1α) protein instability

  • This results in impaired glucose metabolism and lower glycolytic enzyme expression

  • DNMT3A-deficient cardiac tissue shows increased sensitivity to metabolic stress conditions (serum withdrawal, restrictive feeding)

Recommended Research Approaches:

• Engineered Heart Tissue (EHT) Models:

  • Generate EHTs using CRISPR/Cas9-modified hiPSC-derived cardiomyocytes with DNMT3A mutations

  • Compare heterozygous (DNMT3A+/-) and homozygous (DNMT3A-/-) models to wild-type

  • Analyze functional parameters including contraction kinetics and force generation

• Comprehensive Methylome Analysis:

  • Perform whole-genome DNA methylation analysis of differentiated cardiomyocytes

  • Compare methylation patterns across developmental stages and stress conditions

  • Correlate methylation changes with gene expression alterations

• Metabolic Stress Testing:

  • Subject wild-type and DNMT3A-deficient cardiac models to controlled stress conditions

  • Measure parameters such as:

    • Glucose utilization rates

    • Lipid accumulation (using Oil Red O staining)

    • Mitochondrial function

    • Cellular viability under restricted nutrient conditions

• Comparative Tissue Analysis:

  • Investigate DNMT3A function across different cardiac cell types (cardiomyocytes, fibroblasts, endothelial cells)

  • Compare cardiac-specific effects to those in other tissues to identify unique regulatory networks

These approaches can help elucidate the tissue-specific roles of DNMT3A in cardiac function and potentially identify new therapeutic targets for cardiac diseases.

What are the most common technical challenges when using DNMT3A antibodies, and how can researchers overcome them?

Researchers frequently encounter several technical challenges when working with DNMT3A antibodies. Here are the most common issues and recommended solutions:

Challenge 1: Non-specific Banding Patterns in Western Blots

• Cause: Cross-reactivity with DNMT3B or other methyltransferases due to conserved domains

• Solutions:

  • Optimize antibody dilution (start with 0.1 μg/mL for monoclonal antibodies like MAB63151)

  • Use reducing conditions with Immunoblot Buffer Group 1

  • Include positive controls (HeLa, HEK293, U2OS) and negative controls (Daudi) in parallel lanes

  • Consider using monoclonal antibodies (MAB6315, MAB63151) for higher specificity in applications where cross-reactivity is problematic

Challenge 2: Weak Nuclear Signal in Immunofluorescence

• Cause: Insufficient nuclear permeabilization or epitope masking

• Solutions:

  • Optimize fixation methods (immersion fixation works well for DNMT3A detection)

  • Extend antibody incubation time to 3 hours at room temperature

  • Increase antibody concentration to 10-15 μg/mL

  • Use antigen retrieval techniques for formalin-fixed samples

  • Counter-stain with DAPI to confirm nuclear localization

Challenge 3: Variant-Specific Detection

• Cause: Different DNMT3A variants (especially the 100kDa DNMT3A2) may show variable detection

• Solutions:

  • Select antibodies that target conserved regions (e.g., Ala353-Lys486) for detecting all variants

  • For variant-specific detection, choose antibodies targeting unique regions

  • Verify variant expression patterns using RT-PCR before antibody-based detection

  • Consider transcript-specific expression patterns when interpreting results

Challenge 4: Poor Signal in Tissues with Low DNMT3A Expression

• Cause: DNMT3A expression varies across tissues, with low levels in some adult tissues

• Solutions:

  • Use signal amplification systems (HRP-polymer detection, tyramide signal amplification)

  • Increase tissue section thickness for IHC applications

  • Consider more sensitive detection methods like RNAscope for transcript detection in low-expressing tissues

How can researchers optimize DNMT3A antibody applications for detecting specific mutations associated with leukemia?

Detecting DNMT3A mutations, particularly the clinically significant R882 mutations found in leukemia, requires specialized approaches:

Mutation-Specific Detection Strategies:

• Mutation-Specific Antibodies:

  • When available, use antibodies specifically raised against common mutant epitopes (R882H, R882C)

  • Validate specificity using cell lines with known mutation status

  • Employ paired antibodies (targeting both wild-type and mutant epitopes) for comparative analysis

• Indirect Detection Approaches:

  • Functional Readouts: Monitor downstream effects of DNMT3A mutations:

    • DNA damage markers (increased in DNMT3A-mutant cells under replication stress)

    • Cell cycle checkpoint activation markers (persistent in mutants)

    • PARP1 recruitment (impaired in mutants)

  • Methylation Signatures: Assess characteristic methylation patterns associated with DNMT3A mutations:

    • Hypomethylation at specific genomic regions

    • Correlation with transcript 2 expression patterns in AML

• Optimization Protocols:

  • For Western Blot Detection:

    • Use cell fractionation to enrich nuclear proteins

    • Load higher protein amounts (50-100μg) when detecting mutant forms

    • Optimize gel percentage (7-8% acrylamide) for better resolution of the 120-130kDa band

  • For Immunofluorescence:

    • Increase antibody concentration to 15μg/mL for detection in leukemia samples

    • Use paired staining with DNA damage markers (γH2AX) to identify cells with mutant DNMT3A phenotype

    • Consider co-staining with cell cycle markers to identify cells in S-phase

• Validation Approaches:

  • Use isogenic cell lines with and without DNMT3A mutations as controls

  • Compare antibody results with genetic testing methods (sequencing)

  • Correlate antibody signals with functional assays (cytarabine sensitivity)

These optimized approaches can help researchers better characterize DNMT3A mutations in leukemia samples and potentially identify patients who might benefit from targeted therapeutic strategies.

What considerations are important when using DNMT3A antibodies across different species for comparative studies?

Cross-species applications of DNMT3A antibodies require careful consideration of several factors to ensure valid comparative analyses:

Species Reactivity Profiles:

Sequence Conservation Considerations:

• Epitope Mapping:

  • The region Ala353-Lys486 of human DNMT3A used for generating several antibodies shows high conservation across mammals

  • Verify sequence homology of the target epitope across species of interest

  • Higher conservation increases likelihood of cross-reactivity

• Isoform Variations:

  • Expression patterns of DNMT3A variants may differ between species

  • The 100kDa DNMT3A2 variant is conserved in mammals but may show species-specific regulation

  • Verify which isoforms are expressed in the tissue of interest across species

Validation Recommendations:

• Positive Control Tissues/Cells:

  • For mouse studies: NIH-3T3 cells show reliable DNMT3A detection

  • For human studies: HeLa, HEK293, U2OS, and OVCAR-3 cell lines

  • Include species-matched positive controls in all experiments

• Antibody Dilution Optimization:

  • Perform separate titration experiments for each species

  • Starting recommendations:

    • Western blot: 0.1 μg/mL for mouse and human samples

    • Immunofluorescence: 10-15 μg/mL for human samples, may require adjustment for other species

• Species-Specific Technical Modifications:

  • Adjust fixation protocols based on species (tissue composition differences)

  • Consider species-specific blocking reagents to reduce background

  • Optimize antigen retrieval methods for each species

• Performance Verification:

  • Confirm antibody specificity in each species using genetic models (knockouts where available)

  • Verify expected molecular weight (approximately 120kDa in humans and mice)

  • Confirm expected subcellular localization (nuclear) across species

By carefully addressing these considerations, researchers can conduct valid cross-species comparisons of DNMT3A expression and function while avoiding artifacts due to species-specific technical variations.

How can DNMT3A antibodies be used to investigate the relationship between DNA methylation and genome stability in cancer?

DNMT3A's dual role in epigenetic regulation and genome stability makes antibody-based investigations particularly valuable for understanding cancer mechanisms:

Research Applications:

• DNA Damage Response Analysis:

  • Use DNMT3A antibodies in combination with DNA damage markers (γH2AX, 53BP1) to investigate co-localization at sites of damage

  • Compare wild-type and mutant DNMT3A recruitment patterns following treatment with replication stress inducers like cytarabine

  • Analyze temporal dynamics of DNMT3A localization during DNA damage response

• Replication Fork Stability Assessment:

  • Combine DNMT3A immunoprecipitation with chromatin components to identify interactions at replication forks

  • Investigate DNMT3A association with replication machinery proteins (PCNA, MCM complex)

  • Compare fork protection mechanisms between cells with wild-type and mutant DNMT3A

• Chromatin Structure Analysis:

  • Use DNMT3A antibodies for ChIP-seq to map genome-wide binding patterns in relation to chromatin states

  • Correlate DNMT3A binding with regions of genomic instability in cancer cells

  • Investigate differential associations between DNMT3A variants and heterochromatin vs. euchromatin regions

Methodological Approaches:

• Sequential ChIP (Re-ChIP):

  • First immunoprecipitate with DNMT3A antibodies (MAB6315)

  • Follow with second immunoprecipitation using antibodies against:

    • DNA repair factors (PARP1, RAD51)

    • Replication proteins (PCNA)

    • Chromatin modifiers (SETDB1)

  • This approach can identify genomic regions where DNMT3A co-localizes with specific functional partners

• DNMT3A Interactome Analysis in Response to Replication Stress:

  • Immunoprecipitate DNMT3A complexes before and after treatment with replication stress inducers

  • Identify differential protein interactions in sensitive vs. resistant cell lines

  • Correlate with patient outcomes in leukemia and other cancers

• Live-Cell Imaging:

  • Generate fluorescently tagged DNMT3A constructs (wild-type and mutant)

  • Track recruitment dynamics to sites of induced DNA damage

  • Compare mobilization rates between DNMT3A variants

These approaches can help elucidate how DNMT3A mutations contribute to genomic instability in cancer and potentially identify novel therapeutic strategies targeting the intersection of epigenetic regulation and genome maintenance.

What emerging applications of DNMT3A antibodies could advance our understanding of epigenetic regulation in tissue-specific development and disease?

Several emerging applications of DNMT3A antibodies show promise for advancing our understanding of epigenetic regulation in development and disease:

Single-Cell Applications:

• Single-Cell CUT&Tag/CUT&RUN:

  • Adapting DNMT3A antibodies for single-cell chromatin profiling techniques

  • Mapping heterogeneity in DNMT3A binding patterns across individual cells within tissues

  • Identifying rare cell populations with unique DNMT3A-associated epigenetic states

• Spatial Transcriptomics Integration:

  • Combining DNMT3A immunohistochemistry with spatial transcriptomics

  • Correlating DNMT3A protein localization with gene expression patterns in tissue contexts

  • Mapping the spatial relationship between DNMT3A activity and tissue architecture in development and disease

3D Chromatin Organization:

• HiChIP/PLAC-seq with DNMT3A Antibodies:

  • Investigating how DNMT3A influences 3D genome organization

  • Mapping the relationship between DNMT3A binding, DNA methylation, and chromatin looping

  • Comparing 3D epigenomic landscapes in wild-type versus DNMT3A-mutant contexts

• Liquid-Liquid Phase Separation (LLPS) Studies:

  • Investigating DNMT3A's potential role in forming membraneless organelles through LLPS

  • Using super-resolution microscopy with DNMT3A antibodies to visualize potential condensate formation

  • Analyzing how DNMT3A mutations affect phase separation properties and subsequent epigenetic regulation

Tissue-Specific Applications:

• Cardiovascular Research:

  • Using DNMT3A antibodies to study its role in cardiac stress responses

  • Investigating DNMT3A-mediated regulation of metabolism in cardiac tissue

  • Exploring potential cardioprotective interventions targeting DNMT3A pathways

• Developmental Biology:

  • Tracking DNMT3A variant expression during lineage specification

  • Correlating DNMT3A binding patterns with developmental milestones

  • Investigating the interplay between DNMT3A variants in orchestrating tissue-specific differentiation programs

• Cancer Therapeutic Development:

  • Screening for small molecules that selectively target mutant DNMT3A

  • Developing combination therapies exploiting synthetic lethality with DNMT3A mutations

  • Using DNMT3A antibodies to monitor treatment responses in replication stress-targeting therapies

These emerging applications could significantly advance our understanding of how DNMT3A contributes to normal development and disease pathogenesis while potentially identifying novel therapeutic approaches for conditions involving DNMT3A dysregulation.

How might advanced microscopy techniques combined with DNMT3A antibodies reveal new insights into its nuclear organization and function?

Advanced microscopy techniques paired with DNMT3A antibodies offer powerful approaches to investigate the protein's nuclear organization and function at unprecedented resolution:

Super-Resolution Microscopy Applications:

• Stimulated Emission Depletion (STED) Microscopy:

  • Reveals nano-scale organization of DNMT3A within the nucleus

  • Can distinguish between heterochromatin and euchromatin associations of different DNMT3A variants

  • Enables visualization of DNMT3A clustering patterns in normal versus disease states

  • Implementation:

    • Use directly labeled primary antibodies (MAB6315, AF6315) for optimal resolution

    • Combine with chromatin markers to map precise nuclear subdomains

• Structured Illumination Microscopy (SIM):

  • Provides ~100nm resolution for detailed nuclear architecture analysis

  • Enables multi-color imaging to visualize DNMT3A alongside interaction partners

  • Implementation:

    • Compare nuclear distribution patterns between wild-type and mutant DNMT3A

    • Analyze co-localization with replication fork markers during S-phase

• Single-Molecule Localization Microscopy (STORM/PALM):

  • Achieves molecular-scale resolution (~20nm) to visualize individual DNMT3A molecules

  • Can detect molecular clustering and stoichiometry changes

  • Implementation:

    • Map precise changes in DNMT3A organization during DNA damage response

    • Quantify molecular density at specific nuclear structures

Live-Cell Imaging Approaches:

• FRAP (Fluorescence Recovery After Photobleaching):

  • Measures DNMT3A mobility and binding dynamics in living cells

  • Can detect changes in protein-chromatin interactions

  • Implementation:

    • Compare mobility of wild-type versus mutant DNMT3A

    • Analyze how replication stress affects DNMT3A dynamics

• Single-Particle Tracking:

  • Tracks individual DNMT3A molecules to characterize diffusion properties

  • Reveals transient interactions with chromatin and other nuclear components

  • Implementation:

    • Compare diffusion coefficients across nuclear compartments

    • Identify potential phase-separated domains containing DNMT3A

Correlative Microscopy:

• CLEM (Correlative Light and Electron Microscopy):

  • Combines fluorescence imaging of DNMT3A with ultrastructural context

  • Reveals relationship between DNMT3A localization and nuclear ultrastructure

  • Implementation:

    • Correlate DNMT3A clusters with specific nuclear bodies or chromatin states

    • Analyze structural changes in DNMT3A-associated chromatin in disease models

• Lattice Light-Sheet Microscopy:

  • Enables long-term 3D imaging of DNMT3A dynamics with minimal phototoxicity

  • Captures rapid nuclear reorganization events during cellular responses

  • Implementation:

    • Track DNMT3A redistribution during cell cycle progression

    • Visualize dynamic responses to DNA damage or replication stress

These advanced microscopy approaches can provide unprecedented insights into DNMT3A's nuclear organization, revealing how its spatial distribution and dynamics contribute to its diverse functions in epigenetic regulation, DNA damage response, and cellular differentiation.