ATXN7L3 Antibody

What is the biological function of ATXN7L3 in transcriptional regulation?

ATXN7L3 functions as a critical component of the SAGA deubiquitination module (DUBm), where it participates in a subcomplex that specifically deubiquitinates both histones H2A and H2B . Within the SAGA complex, ATXN7L3 is required to recruit USP22 and ENY2, playing an essential role in chromatin regulation and gene expression . The SAGA complex is recruited to specific gene promoters by activators such as MYC, where it facilitates transcription through histone modification . Recent evidence indicates that ATXN7L3 serves as a coactivator for estrogen receptor α (ERα)-mediated transactivation in hepatocellular carcinoma cells, thereby enhancing SMAD7 transcription .

How does ATXN7L3 influence H2B ubiquitination levels?

ATXN7L3 plays a crucial role in regulating H2B monoubiquitination (H2Bub1) levels at lysine 120. Studies using shRNA-mediated depletion of ATXN7L3 have shown that its loss leads to a substantial increase (approximately 5-6 fold) in global H2Bub1 levels . Interestingly, while ATXN7L3 and ENY2 depletion significantly increases H2Bub1, the depletion of USP22 (the catalytic subunit) has a milder effect, suggesting that the adapter proteins have a greater impact on H2B deubiquitination than the catalytic subunit itself . This indicates that ATXN7L3 may coordinate multiple deubiquitinases or influence H2Bub1 levels through additional mechanisms beyond direct catalytic activity.

What is the cellular and subcellular localization of ATXN7L3?

ATXN7L3 is predominantly localized in the nucleus, consistent with its role in transcriptional regulation and chromatin modification . The protein contains nuclear localization signals that facilitate its transport into the nucleus where it performs its functions as part of the SAGA complex. Immunofluorescence studies using anti-ATXN7L3 antibodies typically show nuclear staining patterns, often with enrichment in areas of active transcription. When designing experiments with ATXN7L3 antibodies, proper nuclear extraction protocols are essential to effectively detect and analyze this protein.

What critical factors should be considered when selecting an ATXN7L3 antibody for specific research applications?

When selecting an ATXN7L3 antibody, researchers should consider:

• Application compatibility: Verify that the antibody has been validated for your specific application (Western blot, ChIP, immunofluorescence, etc.)

• Epitope recognition: Choose antibodies targeting well-conserved regions if working across species, or species-specific epitopes when specificity is paramount

• Published validation: Review literature for successful applications with the antibody

• Clonality: Polyclonal antibodies may provide higher sensitivity but potentially lower specificity compared to monoclonals

• Controls: Ensure appropriate positive and negative controls are available for validation

• Lot-to-lot consistency: Consider antibodies with demonstrated reproducibility between production lots

Commercial antibodies like the Polyclonal Antibody (PA5-103624) have been validated to detect endogenous levels of total ATXN7L3 , making them suitable for various applications.

What strategies can be employed to validate ATXN7L3 antibody specificity?

Comprehensive validation of ATXN7L3 antibodies should include:

• Knockout/knockdown controls: Testing on ATXN7L3 knockout cell lines or cells treated with siRNAs targeting different sequences of ATXN7L3, as demonstrated in studies where three different siRNAs led to obvious reductions in ATXN7L3 mRNA and protein expression

• Western blot analysis: Confirming detection of a single band at the expected molecular weight of approximately 38.65 kDa

• Cross-reactivity assessment: Testing on tissues known to express or not express ATXN7L3

• Immunoprecipitation followed by mass spectrometry: Confirming that the antibody captures ATXN7L3 and its known interacting partners

• Comparing multiple antibodies: Using different antibodies targeting distinct epitopes of ATXN7L3 to confirm specificity

• Immunohistochemical validation: Testing expression patterns across tissues with known ATXN7L3 expression profiles

How should researchers interpret inconsistent results when using ATXN7L3 antibodies across different experimental systems?

When encountering inconsistent results:

• Consider cell type-specific variations: ATXN7L3 regulates different subsets of genes in different cellular environments. For example, differential gene expression analysis between ATXN7L3 knockout mESCs and MEFs showed minimal overlap in affected genes, indicating context-dependent functions

• Evaluate expression levels of interacting partners: The function of ATXN7L3 depends on its interactions with other proteins like ENY2, whose depletion significantly reduces ATXN7L3 protein levels

• Assess experimental conditions: Nuclear extraction efficiency, buffer composition, and fixation methods can significantly impact detection

• Consider post-translational modifications: ATXN7L3 function may be regulated by modifications affecting antibody recognition

• Verify antibody performance: Different lots or storage conditions may affect antibody performance

What are the optimal conditions for Western blot analysis of ATXN7L3?

For optimal Western blot detection of ATXN7L3:

• Sample preparation: Use nuclear extraction protocols with protease inhibitors to preserve ATXN7L3 integrity

• Protein amount: Load 20-50 μg of nuclear extract per lane

• Gel percentage: Use 10-12% SDS-PAGE gels to properly resolve the 38.65 kDa ATXN7L3 protein

• Transfer conditions: Semi-dry transfer at 15-20V for 30-45 minutes or wet transfer at 100V for 60-90 minutes

• Blocking: 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

• Primary antibody: Anti-ATXN7L3 (such as Bethyl A302-800A) at 1:1000-1:2000 dilution overnight at 4°C

• Controls: Include ATXN7L3 knockdown samples as negative controls

• Detection: Enhanced chemiluminescence with appropriate exposure times to avoid saturation

Studies have successfully used these conditions to detect changes in ATXN7L3 expression and correlate them with alterations in H2Bub1 levels .

How should researchers design ChIP experiments to study ATXN7L3 binding to chromatin?

For effective ChIP experiments targeting ATXN7L3:

• Crosslinking: Use 1% formaldehyde for 10 minutes at room temperature to preserve protein-DNA interactions

• Sonication: Optimize fragmentation to generate 200-500 bp chromatin fragments

• Antibody selection: Use ChIP-validated ATXN7L3 antibodies (typically 2-5 μg per IP)

• Controls:

  • Input chromatin (non-immunoprecipitated)

  • IgG control (non-specific antibody)

  • Positive control regions (known ATXN7L3 binding sites like SMAD7 promoter regions)

  • Negative control regions (non-bound regions)

• Analysis: qPCR primers targeting regions of interest or ChIP-seq for genome-wide binding profiles

• Data interpretation: Compare binding patterns with H2Bub1 levels and gene expression data

Research has shown that ATXN7L3 is recruited to the promoter regions of genes like SMAD7, where it regulates histone H2B ubiquitination levels to enhance transcription .

What experimental approaches are most effective for studying ATXN7L3-mediated regulation of gene expression?

To comprehensively investigate ATXN7L3's role in gene regulation:

• RNA-seq after ATXN7L3 manipulation: Differential gene expression analysis between wild-type and ATXN7L3-depleted cells has revealed significant numbers of up- and down-regulated genes (e.g., 1116 upregulated and 810 downregulated transcripts in ATXN7L3 knockout mESCs)

• ChIP-seq for ATXN7L3 and H2Bub1: Map genome-wide binding patterns and correlate with ubiquitination status

• Reporter assays: Using promoter constructs of ATXN7L3 target genes to quantify transcriptional impact

• Co-immunoprecipitation: Identify interacting transcription factors and cofactors

• CRISPR/Cas9-mediated genomic editing: Create precise mutations in ATXN7L3 or its binding sites

• Cell type-specific analysis: Compare ATXN7L3 function across different cellular contexts, as studies have shown that ATXN7L3 regulates different gene sets in different cell types

How can researchers investigate the interaction between ATXN7L3 and other components of the SAGA complex?

To study ATXN7L3's interactions with SAGA components:

• Sequential ChIP (ChIP-reChIP): Determine co-occupancy of ATXN7L3 with other SAGA components at specific genomic loci

• Proximity ligation assay (PLA): Visualize protein-protein interactions in situ

• Co-immunoprecipitation experiments: Using ATXN7L3 antibodies to pull down associated proteins

• Mass spectrometry: Identify the complete interactome of ATXN7L3

• Genetic interaction studies: Perform double knockdowns/knockouts of ATXN7L3 and other SAGA components

• Structure-function analysis: Create domain deletion mutants to map interaction regions

Research has demonstrated that ENY2 depletion leads to significant reduction of ATXN7L3 protein levels, suggesting interdependence among SAGA components .

What approaches should be used to resolve contradictory data regarding ATXN7L3 function?

When confronting contradictory findings:

• Cell type considerations: ATXN7L3 regulates different subsets of genes in different cellular environments. For example, comparisons between ATXN7L3 knockout mESCs and MEFs showed very few transcripts similarly affected in both systems

• Temporal dynamics: Consider the timing of analyses after ATXN7L3 manipulation

• Compensatory mechanisms: Look for upregulation of related deubiquitinases

• Technical validation: Use multiple techniques to validate observations

• Genetic background effects: Consider how genetic background might influence ATXN7L3 function

• Comprehensive gene ontology analysis: GO analyses of genes regulated by ATXN7L3 in different cell types have revealed distinct functional categories, suggesting context-dependent roles

How should researchers interpret changes in histone modification patterns after ATXN7L3 manipulation?

For proper interpretation of histone modification changes:

• Direct vs. indirect effects: Determine whether changes in histone modifications are directly caused by ATXN7L3 loss or are secondary effects

• Global vs. local changes: Assess whether modifications are altered genome-wide or at specific loci

• Temporal dynamics: Monitor changes over time to distinguish primary from secondary effects

• Correlation with gene expression: Analyze how histone modification changes correlate with transcriptional outcomes

• Compensation by other deubiquitinases: Consider potential redundant mechanisms

• Cross-talk between modifications: Examine how changes in H2Bub1 affect other histone modifications

Studies have shown that depletion of ATXN7L3 leads to a 5-6 fold increase in global H2Bub1 levels and also affects H2Aub1 levels to a lesser extent .

How can ATXN7L3 antibodies be used to investigate its role in hepatocellular carcinoma?

For HCC-focused research:

• Expression analysis: Immunohistochemical staining of HCC tissue arrays revealed that ATXN7L3 is expressed at lower levels in HCC samples compared to normal liver tissues

• Prognostic correlations: Lower expression of ATXN7L3 positively correlates with poor clinical outcomes in HCC patients

• Functional studies: ATXN7L3 knockdown experiments showed decreased SMAD7 expression, while overexpression increased it, demonstrating a role in gene regulation

• In vivo tumor models: Xenograft experiments have shown that ATXN7L3 participates in suppression of tumor growth

• Mechanism investigation: ChIP experiments confirmed that ATXN7L3 is recruited to promoter regions of genes like SMAD7 to regulate their expression

What methodological approaches are recommended for studying the relationship between ATXN7L3 and SMAD7 in cancer contexts?

To investigate the ATXN7L3-SMAD7 axis:

• Expression correlation analysis: Studies have demonstrated a strong positive correlation between ATXN7L3 and SMAD7 mRNA levels in HCC samples using TCGA database analysis

• Knockdown validation: siRNAs targeting different sequences of ATXN7L3 led to significant reductions in both ATXN7L3 and SMAD7 mRNA and protein levels

• Overexpression studies: Ectopic expression of ATXN7L3 increased SMAD7 expression

• Chromatin immunoprecipitation: ATXN7L3 is recruited to the promoter regions of SMAD7 gene, regulating histone H2B ubiquitination levels to enhance transcription

• Mechanistic investigations: ATXN7L3 functions as a coactivator for ERα-mediated transactivation in HCC cells, contributing to enhanced SMAD7 transcription

• Clinical correlation: Expression of ATXN7L3 negatively correlates with poor clinical outcomes in HCC patients

What experimental design considerations are important when studying cell adhesion gene regulation by ATXN7L3?

For investigating ATXN7L3's role in cell adhesion:

• Gene expression profiling: RNA-seq analysis of ATXN7L3 knockout MEFs showed significant downregulation of genes in the "Cell adhesion" GO category, including cadherins, catenins, collagens, and other adhesion molecules

• Cytoskeletal analysis: Fluorescence imaging of actin cytoskeletal proteins in ATXN7L3 knockout MEFs revealed massively reduced abundance of F-actin filaments and β-actin compared to wild-type cells

• Functional assays: Assess cell adhesion, migration, and invasion properties after ATXN7L3 manipulation

• Rescue experiments: Re-express ATXN7L3 in knockout cells to confirm specificity of observed phenotypes

• Chromatin analysis: Determine whether ATXN7L3 directly regulates adhesion gene promoters through ChIP experiments

• Signaling pathway investigation: Examine how ATXN7L3 loss affects signaling pathways governing cell adhesion

What are the key factors in optimizing immunofluorescence protocols for ATXN7L3 detection?

For successful immunofluorescence:

• Fixation: 4% paraformaldehyde for 15 minutes at room temperature preserves nuclear structure

• Permeabilization: 0.2% Triton X-100 for 10 minutes enables antibody access to nuclear proteins

• Blocking: 3-5% BSA or normal serum for 1 hour reduces background

• Primary antibody: Anti-ATXN7L3 at 1:100-1:500 dilution, incubated overnight at 4°C

• Controls:

  • ATXN7L3 knockdown cells as negative control

  • Co-staining with known nuclear markers

• Nuclear counterstain: DAPI or Hoechst to visualize nuclei

• Mounting: Use anti-fade mounting medium to preserve fluorescence

• Confocal microscopy: Required for detailed nuclear localization analysis

How should researchers analyze ATXN7L3-dependent changes in cellular phenotypes?

For comprehensive phenotypic analysis:

• Cell proliferation: ATXN7L3 participates in suppression of tumor growth in vitro and in vivo, as demonstrated in colony formation, cell growth curve, and xenograft tumor experiments

• Gene expression profiling: RNA-seq analysis of ATXN7L3 knockout cells revealed significant transcriptional changes affecting multiple biological processes

• GO term enrichment: Analysis of differentially expressed genes in ATXN7L3 knockout mESCs revealed enrichment of categories linked to regulation of transcription and cell differentiation in down-regulated genes, and "Metabolic processes" and "Cell adhesion" in up-regulated genes

• Cell morphology: ATXN7L3 knockout MEFs display unusual morphology, likely due to disruption of the actin cytoskeleton

• Functional assays: Design experiments specific to the biological processes identified in GO analysis

• Temporal analysis: Monitor phenotypic changes over time to distinguish primary from secondary effects

What strategies can resolve technical challenges in measuring H2B ubiquitination changes following ATXN7L3 manipulation?

To accurately measure H2Bub1 changes:

• Antibody selection: Use highly specific antibodies targeting H2Bub1 at K120, such as Cell Signaling Technology #5546

• Controls: Include samples with known H2Bub1 status (e.g., RNF20/40 knockdown cells as negative controls)

• Extraction protocols: Use specialized histone extraction methods to preserve modifications

• Quantification: Perform densitometric analysis normalized to total H2B levels

• ChIP-seq approach: Map genome-wide H2Bub1 distribution before and after ATXN7L3 manipulation

• Comparison with other SAGA components: Compare the effects of ATXN7L3 depletion with those of USP22, ENY2, and ATXN7 depletion

Research has shown that depletion of ATXN7L3 or ENY2 leads to a major increase (5-6 fold) in H2Bub1 levels, while USP22 or ATXN7 depletion results in a mild reduction, highlighting the complex regulation of this modification .

What novel approaches could advance our understanding of ATXN7L3's regulatory mechanisms?

Emerging approaches include:

• Single-cell technologies: Apply scRNA-seq and scATAC-seq to understand cell-to-cell variability in ATXN7L3 function

• CRISPR screens: Perform genome-wide CRISPR screens in ATXN7L3 wild-type versus knockout backgrounds to identify synthetic interactions

• Cryo-EM structural analysis: Determine the three-dimensional structure of ATXN7L3 within the SAGA complex

• Live-cell imaging: Use fluorescently tagged ATXN7L3 to track its dynamics during transcriptional activation

• Computational modeling: Develop predictive models of ATXN7L3-dependent gene regulation

• Multi-omics integration: Combine transcriptomics, proteomics, and epigenomics to build comprehensive models of ATXN7L3 function

How can ATXN7L3 research be translated into potential therapeutic applications?

Translational research directions:

• Biomarker development: ATXN7L3 expression correlates with clinical outcomes in HCC patients, suggesting potential as a prognostic biomarker

• Drug target identification: Screen for compounds that modulate ATXN7L3 activity or its interactions

• Synthetic lethality: Identify contexts where ATXN7L3 loss creates therapeutic vulnerabilities

• Gene therapy approaches: Restore ATXN7L3 expression in cancers where it is downregulated

• Combination therapies: Target ATXN7L3-regulated pathways in conjunction with standard treatments

• Patient stratification: Use ATXN7L3 expression patterns to guide personalized treatment approaches

Experimental evidence shows that ATXN7L3 participates in suppression of tumor growth, and its lower expression in HCC samples correlates with poor clinical outcomes, suggesting therapeutic relevance .

How should researchers interpret contradictory findings about ATXN7L3's role in different cellular contexts?

Strategies for resolving contextual differences:

• Consider cell type specificity: RNA-seq analyses have shown that ATXN7L3 regulates different gene sets in different cell types, with minimal overlap between affected genes in mESCs versus MEFs

• Examine interacting partners: The function of ATXN7L3 depends on its interaction with other proteins, which may vary across cell types

• Assess differentiation state: ATXN7L3's role may differ in stem cells versus differentiated cells

• Consider compensatory mechanisms: Other deubiquitinases may compensate for ATXN7L3 loss in specific contexts

• Evaluate experimental conditions: Different growth conditions or stress levels may affect ATXN7L3 function

• Temporal dynamics: Consider the timing of analyses after ATXN7L3 manipulation

What are the most common technical issues with ATXN7L3 antibodies and their solutions?

Troubleshooting guide for ATXN7L3 antibodies:

• Weak signal in Western blot:

  • Increase protein loading (40-50 μg nuclear extract)

  • Optimize antibody concentration

  • Use enhanced chemiluminescence detection systems

  • Ensure proper nuclear extraction

• High background in immunohistochemistry:

  • Increase blocking time and concentration

  • Reduce primary antibody concentration

  • Use biotin-free detection systems

  • Include appropriate controls

• Poor reproducibility in ChIP:

  • Optimize chromatin fragmentation

  • Increase antibody amount or incubation time

  • Optimize washing conditions

  • Verify antibody lot consistency

• Cross-reactivity issues:

  • Validate with knockout/knockdown controls

  • Use monoclonal antibodies for higher specificity

  • Pre-absorb antibody with recombinant protein

• Nuclear extraction efficiency:

  • Use specialized nuclear extraction kits

  • Verify extraction efficiency with nuclear markers

  • Include protease inhibitors to prevent degradation