ZMYM3 Antibody

What is ZMYM3 and why is it significant in research?

ZMYM3 (Zinc finger MYM-type protein 3, also known as ZNF261, DXS6673E, or KIAA0385) is a chromatin-binding protein with significant roles in DNA damage response pathways. It functions as a critical factor in modulating BRCA1 functions within chromatin to maintain genome integrity . ZMYM3 is a 1370-amino-acid protein containing 10 tandem zinc finger domains along with a domain of unknown function (DUF3504) . The protein plays essential roles in:

• DNA repair through homologous recombination (HR) mechanisms

• Cell morphology and cytoskeletal organization regulation

• Spermatogenesis in males, with knockout studies showing arrest at the metaphase of the first meiotic division

Research interest in ZMYM3 has increased due to its implications in genome stability pathways and potential connections to disease mechanisms when dysregulated.

Which applications are most suitable for ZMYM3 antibodies in research settings?

Based on validated research applications, ZMYM3 antibodies are most suitable for:

• Western blotting (WB): Detecting ZMYM3 protein in nuclear extracts, with expected band sizes around 152 kDa for the primary isoform , though multiple isoforms have been observed at approximately 200 kDa and 95 kDa

• Immunohistochemistry on paraffin-embedded sections (IHC-P): Visualizing ZMYM3 distribution in tissue sections

• Immunoprecipitation (IP): Particularly useful for studying protein-protein interactions, such as the association between ZMYM3 and LSD1

• Immunofluorescence: For examining subcellular localization patterns, which vary between isoforms (nuclear localization for the larger isoform, both nuclear and cytoplasmic distribution for the smaller isoform)

When selecting an antibody for these applications, researchers should verify specificity for the target region of interest, as alternative splicing may generate multiple ZMYM3 isoforms with differing functional domains.

How should researchers validate ZMYM3 antibody specificity for experimental use?

Proper validation of ZMYM3 antibodies requires a multi-faceted approach:

• Knockout/knockdown controls: Compare antibody signals between wild-type cells and ZMYM3 knockout or knockdown models. Complete loss of signal in knockout samples provides strong evidence of specificity

• Western blot analysis: Verify band patterns match predicted molecular weights, acknowledging that post-translational modifications often result in migration patterns different from theoretical weights. For ZMYM3, expect bands at approximately 152 kDa theoretical weight, though observed bands of ~200 kDa and 95 kDa have been reported due to post-translational modifications

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide (synthetic peptide within Human ZMYM3 aa 250-300 for some commercial antibodies) before application in your experimental system. Signal elimination confirms epitope-specific binding

• Subcellular fractionation correlation: Confirm that detected signals align with expected subcellular distribution patterns. For ZMYM3, the larger isoform (~200 kDa) should be predominantly nuclear, while the smaller isoform (~95 kDa) should be detected in both nuclear and cytoplasmic fractions

• Cross-reference multiple antibodies: Where possible, compare results using different antibodies targeting distinct ZMYM3 epitopes to confirm consistent patterns

What are the optimal conditions for detecting ZMYM3 in chromatin fractions?

Detecting ZMYM3 in chromatin fractions requires careful optimization due to its tight association with both histone and DNA components of nucleosomes. The following protocol has been validated in research settings:

Chromatin Fractionation Protocol for ZMYM3 Detection:

• Cell preparation: Harvest approximately 1-5×10⁶ cells and wash twice with ice-cold PBS

• Cytoplasmic extraction:

  • Resuspend cell pellet in 200 μl cytoplasmic extraction buffer (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl₂, 0.34 M sucrose, 10% glycerol, 1 mM DTT, protease inhibitors)

  • Add Triton X-100 to 0.1% final concentration

  • Incubate on ice for 8 minutes

  • Centrifuge at 1,300 × g for 5 minutes at 4°C

  • Collect supernatant as cytoplasmic fraction

• Nuclear extraction:

  • Wash nuclear pellet once with cytoplasmic extraction buffer

  • Resuspend in 200 μl nuclear extraction buffer (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, protease inhibitors)

  • Incubate on ice for 30 minutes with occasional vortexing

  • Centrifuge at 1,700 × g for 5 minutes at 4°C

  • Collect supernatant as nuclear soluble fraction

• Chromatin extraction:

  • Resuspend pellet in 200 μl chromatin extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM DTT, benzonase 250 U/ml, protease inhibitors)

  • Incubate at room temperature for 15 minutes with rotation

  • Centrifuge at 16,000 × g for 10 minutes

  • Collect supernatant as chromatin-bound fraction

• Western blot detection:

  • Load 10-20 μg of protein per lane

  • For ZMYM3 detection, use 0.1 μg/ml antibody concentration

  • Allow extended exposure time (up to 15 minutes) for optimal detection of the chromatin-bound fraction

Research has demonstrated that endogenous ZMYM3 is exclusively present in the chromatin fraction, making this extraction protocol particularly important for successful detection .

How can researchers optimize ChIP-seq protocols for studying ZMYM3-chromatin interactions?

ChIP-seq for ZMYM3 requires special considerations due to its bivalent interactions with both histone and DNA components of nucleosomes. The following optimized protocol incorporates techniques from published ZMYM3 ChIP-seq studies:

• Cell preparation:

  • Culture 15-20 million cells per ChIP experiment

  • For DNA damage studies, treat cells with appropriate DNA damaging agents before crosslinking (e.g., 1 μM camptothecin for 1 hour)

• Crosslinking and chromatin preparation:

  • Double crosslinking approach is recommended: first with 2 mM disuccinimidyl glutarate (DSG) for 45 minutes, followed by 1% formaldehyde for 10 minutes at room temperature

  • Quench with 0.125 M glycine for 5 minutes

  • Wash cells twice with ice-cold PBS

  • Lyse cells and isolate nuclei using SDS lysis buffer

  • Sonicate chromatin to 200-500 bp fragments

• Immunoprecipitation optimization:

  • Pre-clear chromatin with protein A/G beads

  • Use 5-10 μg of validated ZMYM3 antibody per ChIP reaction

  • Include appropriate controls: IgG negative control and H3K4me3 positive control

  • Incubate overnight at 4°C with rotation

• Epitope tagging approach:

  • For challenging epitopes, consider the CETCh-seq approach (CRISPR epitope tagging ChIP-seq) to introduce a 3X FLAG tag at the endogenous ZMYM3 locus

  • This approach allows use of highly specific anti-FLAG antibodies for immunoprecipitation

• Bioinformatic analysis:

  • Use specialized tools such as SPP for peak calling

  • Apply Irreproducible Discovery Rate (IDR) methodology to ensure reproducibility

  • For differential binding analysis, implement the R package csaw v.1.28.0

  • Focus analysis on regions containing DNA damage response elements and known BRCA1 binding sites

This optimized protocol accounts for ZMYM3's unique chromatin binding properties through double crosslinking and considers the alternative CETCh-seq approach for challenging epitopes.

How should researchers interpret multiple ZMYM3 bands in Western blot analysis?

Multiple ZMYM3 bands in Western blots reflect biologically significant isoforms rather than non-specific binding. Proper interpretation requires understanding the following key points:

• Expected band patterns:

  • Theoretical molecular weight: 152 kDa for the canonical isoform

  • Observed bands: ~200 kDa and ~95 kDa in multiple tissues and cell types

  • The size discrepancy between predicted and observed weights is attributed to post-translational modifications

• Isoform-specific subcellular distribution:

  • The larger ~200 kDa isoform localizes predominantly to the nucleus

  • The smaller ~95 kDa isoform is present in both cytoplasmic and nuclear fractions

• Alternative splicing considerations:

  • Multiple verified and predicted alternatively spliced ZMYM3 mRNAs have been reported

  • Different isoforms may have distinct functional roles

• Troubleshooting inconsistent band patterns:

  Issue	Potential Cause	Solution
  Missing larger (200 kDa) band	Insufficient nuclear extraction	Ensure complete nuclear lysis; use appropriate extraction buffers with nuclease treatment
  Missing smaller (95 kDa) band	Exposure time too short	Increase exposure time up to 15 minutes
  Additional unexpected bands	Cross-reactivity with related proteins	Perform peptide competition assay to identify specific bands
  Smeared bands	Protein degradation	Add fresh protease inhibitors; maintain cold temperatures throughout extraction

• Functional interpretation:

  • Different isoforms may have distinct roles in cellular processes

  • When possible, correlate specific bands with functional outcomes in your experimental system

When reporting ZMYM3 Western blot results, clearly specify which isoform(s) you are detecting and include molecular weight markers to facilitate interpretation by other researchers.

What are the most effective cell models for studying ZMYM3 functions in DNA damage response?

Selecting appropriate cell models is critical for investigating ZMYM3's role in DNA damage response pathways. Based on published research, the following models offer distinct advantages:

• HeLa cells:

  • Widely used for ZMYM3 Western blot detection in nuclear extracts

  • Well-characterized DNA damage response pathways

  • Readily transfectable for overexpression or knockdown studies

  • Appropriate for initial characterization studies

• U2OS cells:

  • Excellent model for DNA repair studies

  • Form distinct DNA damage foci that are easily visualized

  • Compatible with laser micro-irradiation techniques to create localized DNA damage

  • Suitable for studying ZMYM3 recruitment to DNA damage sites

• HEK293T cells:

  • Used in published studies demonstrating ZMYM3's role in DNA damage repair

  • High transfection efficiency for genetic manipulation

  • Suitable for protein-protein interaction studies

• Spermatogonial stem cells (SSCs):

  • Physiologically relevant for studying ZMYM3 in reproductive contexts

  • Express both ZMYM3 isoforms with distinct subcellular localization patterns

  • Allow investigation of ZMYM3 interaction with LSD1 and regulation by growth factors

  • More challenging to culture but provide insights into tissue-specific functions

• ZMYM3 knockout models:

  • Essential for determining protein specificity and function

  • CRISPR-Cas9 generated knockouts show clear phenotypes:

    • Genome instability

    • PARP inhibitor sensitivity

    • Impaired DNA repair by homologous recombination

    • Arrested spermatogenesis in mouse models

For comprehensive characterization, a multi-model approach is recommended, starting with established cell lines for mechanistic studies and progressing to more specialized models for context-specific functions.

How can researchers effectively analyze ZMYM3's interaction with BRCA1-A complex components?

Analyzing the interaction between ZMYM3 and the BRCA1-A complex requires specialized approaches due to the dynamic nature of these interactions, particularly in the context of DNA damage. The following methodological approach is recommended:

• Co-immunoprecipitation (Co-IP) studies:

  • Prepare nuclear extracts from cells with and without DNA damage induction

  • Use anti-ZMYM3 antibody for immunoprecipitation, followed by Western blotting for BRCA1-A components (ABRA1, RAP80)

  • Alternatively, perform reverse Co-IP using antibodies against BRCA1-A components

  • Include appropriate controls: IgG negative control and input samples

• Proximity ligation assay (PLA):

  • This technique allows visualization of protein-protein interactions in situ

  • Fix cells with 4% paraformaldehyde after appropriate treatments

  • Perform PLA using antibody pairs targeting ZMYM3 and individual BRCA1-A components

  • Quantify PLA signals in different cellular compartments and in response to DNA damage

• Sequential ChIP (ChIP-reChIP):

  • Perform first round of ChIP using ZMYM3 antibody

  • Elute complexes and perform second round using antibodies against BRCA1-A components

  • This approach identifies genomic regions where both proteins co-localize

• FRET-based interaction analysis:

  • Generate fluorescently tagged ZMYM3 and BRCA1-A components

  • Measure FRET efficiency before and after DNA damage induction

  • This approach provides spatiotemporal information about interaction dynamics

• Deletion mutant analysis:

  • Generate deletion constructs of ZMYM3 based on domain organization

  • Focus on the DNA-binding domain (amino acids 300-330) and chromatin-interacting regions

  • Perform Co-IP experiments with each mutant to map interaction domains

  • Complement with functional assays to correlate binding with biological activities

This multi-method approach provides comprehensive characterization of the ZMYM3-BRCA1-A interaction, which is critical for understanding ZMYM3's role in facilitating BRCA1 localization to damaged chromatin and promoting DNA repair by homologous recombination .

What experimental controls are essential when studying ZMYM3 recruitment to DNA damage sites?

Rigorous experimental controls are critical when investigating ZMYM3 recruitment to DNA damage sites to ensure valid and reproducible results. The following controls should be incorporated:

• Antibody specificity controls:

  • ZMYM3 knockout or knockdown cells as negative controls

  • Peptide competition assays to confirm antibody specificity

  • Secondary antibody-only controls to assess background staining

• DNA damage induction controls:

  • Untreated control cells to establish baseline ZMYM3 distribution

  • Positive control proteins with established damage recruitment patterns (e.g., γH2AX, 53BP1)

  • Time course analysis to capture transient recruitment dynamics

  • Different DNA damage agents to distinguish damage-type specificity:

    • Camptothecin for replication-dependent DSBs

    • Ionizing radiation for direct DSB induction

    • UV for nucleotide excision repair pathways

• Domain-specific recruitment controls:

  • ZMYM3 deletion mutants lacking key functional domains:

    • Deletion of DNA-binding domain (Δ300-330)

    • Deletion of both DNA-binding and chromatin-interacting regions (Δ270-330)

    • These mutants should show reduced recruitment to damage sites

• Cell cycle controls:

  • Synchronize cells in different cell cycle phases

  • Co-stain with cell cycle markers (e.g., PCNA for S-phase)

  • ZMYM3 recruitment may vary depending on cell cycle stage due to the availability of homologous recombination repair pathways

• Quantification controls:

  • Establish clear criteria for positive recruitment (e.g., >1.5-fold enrichment at damage sites)

  • Perform quantification on sufficient numbers of cells (minimum 50-100 cells per condition)

  • Conduct statistical analysis to determine significance of recruitment

  • Use objective image analysis software to avoid confirmation bias

By implementing these controls, researchers can confidently assess ZMYM3 recruitment to DNA damage sites and make valid comparisons between experimental conditions, thereby advancing understanding of ZMYM3's role in the DNA damage response.

How should researchers reconcile differences in ZMYM3 expression patterns across tissue types?

ZMYM3 exhibits variable expression patterns across different tissues, which can lead to seemingly contradictory results. To properly interpret and reconcile these differences, researchers should consider the following methodological approach:

• Tissue-specific expression profiling:

  • RNA-seq and qRT-PCR data show ZMYM3 is most abundant in gonads and brain, but is ubiquitously expressed across multiple organs

  • Systematically quantify both mRNA and protein levels across tissues using standardized methods

  • Create a reference expression atlas as baseline for comparative studies

• Isoform-specific analysis:

  • Different tissues may preferentially express specific ZMYM3 isoforms

  • Use isoform-specific primers for RT-PCR analysis

  • For Western blotting, use antibodies that can detect multiple isoforms and carefully document all observed bands

• Developmental timing considerations:

  • ZMYM3 expression varies during development and differentiation

  • In testicular cells, expression is documented in spermatogonia and early spermatocytes but not in late spermatocytes or spermatids

  • Include age-matched controls when comparing across studies

• Regulatory influences:

  • ZMYM3 is down-regulated by GDNF and up-regulated by retinoic acid in spermatogonial stem cells

  • Similar regulatory mechanisms may exist in other tissues

  • Document treatment conditions that might affect expression levels

• Reconciliation strategy for contradictory results:

  Observation	Possible Explanation	Reconciliation Approach
  Different band patterns on Western blots	Tissue-specific isoform expression	Use multiple antibodies targeting different epitopes; include tissue-specific positive controls
  Varying subcellular localization	Cell type-specific regulation of nuclear import/export	Perform careful subcellular fractionation; use consistent cell fixation methods
  Discrepancies between mRNA and protein levels	Post-transcriptional regulation	Measure both parameters in the same samples; investigate miRNA regulation
  Inconsistent immunostaining patterns	Epitope masking in certain contexts	Test multiple antibodies and fixation protocols; include appropriate controls

By systematically addressing these factors, researchers can develop a more comprehensive understanding of ZMYM3 biology that accounts for tissue-specific variations while establishing core functional principles.

What are the potential pitfalls when interpreting ZMYM3 knockout phenotypes in different experimental systems?

Interpreting ZMYM3 knockout phenotypes requires careful consideration of several factors that could lead to misinterpretation. Researchers should be aware of these potential pitfalls and implement appropriate controls:

• Sex-specific effects:

  • ZMYM3 is X-linked, leading to different phenotypic manifestations in males versus females

  • Male ZMYM3 knockout mice show complete infertility due to meiotic arrest

  • Female phenotypes may be influenced by X-chromosome inactivation patterns

  • Always analyze and report sex-specific outcomes separately

• Compensatory mechanisms:

  • Acute versus chronic loss of ZMYM3 may yield different phenotypes

  • Consider using inducible knockout systems to distinguish between direct effects and adaptive responses

  • Examine expression changes in related family members (other ZMYM proteins) that might compensate for ZMYM3 loss

• Cell type-specific functions:

  • ZMYM3 knockout in spermatogonial stem cells shows distinct phenotypes compared to somatic cells

  • In meiotic cells, knockout leads to metaphase arrest and elevated BUB3-positive spermatocytes

  • In other cell types, DNA damage repair defects predominate

  • Always validate findings across multiple cell types relevant to your research question

• Methodological considerations:

  • Complete versus partial knockouts may yield different phenotypes

  • Verify knockout efficiency at both mRNA and protein levels using isoform-specific detection methods

  • For CRISPR-Cas9 generated knockouts, check for potential off-target effects

• Interpretation framework for complex phenotypes:

  Observed Phenotype	Potential Misinterpretation	Recommended Controls
  Growth defects	Direct effect of ZMYM3 loss	Check for DNA damage accumulation and cell cycle checkpoint activation
  Apoptosis	Non-specific cellular stress	Assess specific pathways (e.g., spindle assembly checkpoint activation in meiotic cells)
  Altered gene expression	Direct transcriptional effect	Distinguish between direct targets and secondary effects through ChIP-seq analysis
  Epistatic interactions	Simple linear pathway	Test multiple genetic backgrounds and perform careful epistasis analysis

• Molecular mechanism validation:

  • Correlate phenotypes with specific molecular functions of ZMYM3

  • For DNA repair defects, assess specifically homologous recombination efficiency

  • For meiotic phenotypes, examine spindle assembly and chromosome segregation

  • Rescue experiments with wild-type and mutant ZMYM3 are essential for causality determination

By addressing these considerations, researchers can develop more accurate interpretations of ZMYM3 knockout phenotypes across different experimental systems.

How can contradictory findings about ZMYM3's role in epigenetic regulation be resolved through experimental design?

Resolving contradictory findings regarding ZMYM3's role in epigenetic regulation requires carefully designed experiments that address specific aspects of this complex function. The following methodological approach can help reconcile apparently contradictory results:

By implementing this comprehensive approach, researchers can develop a more nuanced understanding of ZMYM3's epigenetic functions that accounts for context-specific effects and resolves apparent contradictions in the literature.

What emerging technologies hold promise for advancing understanding of ZMYM3 function in chromatin dynamics?

Several cutting-edge technologies are particularly well-suited for investigating ZMYM3's complex roles in chromatin dynamics and DNA damage response. Researchers should consider these approaches to advance beyond current knowledge limitations:

• Proximity labeling technologies:

  • BioID or TurboID fusion with ZMYM3 to identify proximal interacting proteins in living cells

  • APEX2-based proximity labeling for temporal resolution of interactions following DNA damage

  • These approaches can capture transient interactions that might be missed by traditional Co-IP methods

• Advanced microscopy techniques:

  • Super-resolution microscopy (STORM, PALM, SIM) to visualize ZMYM3 localization at the nanoscale

  • Live-cell imaging with lattice light-sheet microscopy to track ZMYM3 dynamics during DNA damage responses

  • Single-molecule tracking to analyze ZMYM3 mobility on chromatin before and after DNA damage

• Genomic technologies:

  • CUT&Tag for improved mapping of ZMYM3 genomic binding sites with lower background

  • HiChIP to connect ZMYM3 binding with 3D chromatin structure

  • Single-cell technologies to address cellular heterogeneity:

    • scRNA-seq to identify cell state-specific transcriptional effects

    • scATAC-seq to link ZMYM3 to chromatin accessibility changes at single-cell resolution

• CRISPR-based screening approaches:

  • CRISPR activation/inhibition screens targeting ZMYM3-regulated genes

  • CRISPR tiling screens across the ZMYM3 locus to identify functional regulatory elements

  • Base editing to introduce specific point mutations for structure-function analysis

• Structural biology advances:

  • Cryo-EM studies of ZMYM3 in complex with nucleosomes and DNA repair machinery

  • Hydrogen-deuterium exchange mass spectrometry to map protein-protein interaction surfaces

  • AlphaFold2-based structural predictions to guide targeted mutagenesis of key domains

• Multi-omics integration platforms:

  Technology	Application to ZMYM3 Research	Expected Advancement
  Spatial transcriptomics	Map ZMYM3-dependent gene expression in tissue context	Connect molecular mechanisms to physiological roles
  Proteomics with PTM analysis	Identify ZMYM3 modifications and their regulatory role	Understand activation/inactivation mechanisms
  Metabolomics integration	Link ZMYM3 function to cellular metabolic states	Discover unexpected connections to cellular physiology
  4D Nucleome approaches	Connect ZMYM3 to chromatin architecture changes over time	Understand higher-order regulatory functions

These emerging technologies promise to resolve current knowledge gaps regarding ZMYM3's precise molecular mechanisms, tissue-specific functions, and potential roles in disease processes, guiding more targeted therapeutic interventions for conditions involving dysregulated ZMYM3.

How can researchers design experiments to elucidate the functional significance of different ZMYM3 isoforms?

Designing experiments to distinguish and characterize the functions of different ZMYM3 isoforms requires a systematic approach that combines molecular, cellular, and genomic techniques. The following experimental design strategy addresses this complex question:

• Comprehensive isoform identification and characterization:

  • Perform long-read RNA sequencing (Oxford Nanopore or PacBio) to identify all ZMYM3 transcript isoforms

  • Compare isoform expression across tissues, with focus on brain and testis where ZMYM3 is highly expressed

  • Develop isoform-specific RT-PCR assays for quantitative analysis

  • Create a reference map of protein domains present in each isoform

• Isoform-specific antibody development and validation:

  • Design peptide antigens unique to each major isoform

  • Generate and validate isoform-specific antibodies

  • Confirm specificity using knockout models and overexpression systems

  • These tools are essential for distinguishing the ~200 kDa and ~95 kDa isoforms observed in Western blots

• Isoform-specific knockout/knockin strategies:

  • Design CRISPR-Cas9 editing strategies that target specific exons unique to individual isoforms

  • Create cell lines expressing only one isoform at a time

  • Generate knockin models with epitope-tagged versions of each isoform for tracking

• Comparative functional studies:

  Functional Aspect	Experimental Approach	Expected Outcome
  Subcellular localization	Live-cell imaging with isoform-specific fluorescent tags	Confirm differential localization patterns (nuclear for 200 kDa; nuclear/cytoplasmic for 95 kDa)
  Protein interactions	IP-MS with isoform-specific antibodies	Identify isoform-specific interaction partners (e.g., LSD1 interacts with larger isoform)
  Chromatin binding	ChIP-seq with isoform-specific antibodies	Map genomic binding sites unique to each isoform
  DNA damage response	Laser microirradiation followed by isoform tracking	Determine which isoform(s) are recruited to DNA damage sites
  Gene regulation	RNA-seq in isoform-specific rescue models	Identify genes regulated by specific isoforms

• Domain swap experiments:

  • Create chimeric constructs that exchange domains between isoforms

  • Test these constructs in rescue experiments with ZMYM3 knockout cells

  • This approach can identify which domains confer isoform-specific functions

• Regulation of isoform expression:

  • Investigate mechanisms controlling alternative splicing of ZMYM3

  • Test effects of growth factors (GDNF, retinoic acid) on isoform ratios

  • Examine isoform expression changes during differentiation and development

• Physiological significance assessment:

  • Analyze isoform expression in response to cellular stresses (DNA damage, oxidative stress)

  • Examine isoform ratios in relevant disease models

  • Correlate isoform expression with functional outcomes in tissue-specific contexts

This comprehensive experimental approach will provide crucial insights into the distinct biological roles of ZMYM3 isoforms, potentially explaining the diverse functions attributed to this protein across different cellular contexts.