WIZ Antibody

What is WIZ protein and what are its primary cellular functions?

WIZ (Widely-interspaced zinc finger-containing protein, also known as ZNF803) is a zinc finger protein that functions primarily as a transcriptional regulator. Current research demonstrates that WIZ plays a critical role in linking EHMT1 and EHMT2 histone methyltransferases to the CTBP corepressor machinery. WIZ is particularly important for stabilizing EHMT1-EHMT2 heterodimer formation . Although WIZ has traditionally been characterized as a transcriptional repressor, more recent evidence suggests it may also function as a transcriptional activator in certain genomic contexts . WIZ has been found to bind active promoters and CTCF-binding sites, indicating its complex role in chromatin organization and gene expression regulation.

What experimental applications are WIZ antibodies validated for?

WIZ antibodies have been validated for multiple molecular and cellular applications, with specific performance characteristics depending on the antibody source and clone. The most commonly validated applications include:

Research has demonstrated antibody efficacy across multiple species, with human, mouse, and rat samples being the most thoroughly validated . It's essential to perform preliminary validation when applying these antibodies to less commonly tested species or novel applications.

How are WIZ protein isoforms detected using antibodies?

WIZ antibodies have detected multiple isoforms of this protein, with important implications for tissue-specific research. Studies have identified a ~100 kDa isoform that is widely expressed, as well as a larger ~160 kDa isoform that has been specifically observed in adult cerebellum . This size variation must be considered when analyzing Western blot results, as different antibodies may preferentially detect specific isoforms based on their epitope recognition sites. For example, ab92334 recognizes an epitope within amino acids 550-650 of human WIZ , while other antibodies may target alternative regions. When planning experiments, researchers should carefully examine the specific epitope targeted by their selected antibody and consider how this might affect detection of different WIZ isoforms in their experimental system.

How does WIZ interact with the G9a/GLP complex and what are the functional implications?

WIZ serves as a core structural component of the G9a/GLP histone methyltransferase complex. Co-immunoprecipitation studies have demonstrated that WIZ physically associates with ZNF644, G9a, and GLP, forming a stable multi-protein complex . The molecular architecture of this complex reveals that WIZ and ZNF644 interact indirectly, with their association being mediated by G9a and GLP. Research has shown that knockdown of either G9a or GLP impairs the interaction between WIZ and ZNF644, confirming the structural dependence of this complex .

Functionally, the WIZ-containing G9a/GLP complex regulates H3K9 methylation patterns across the genome. More specifically, WIZ appears to direct the complex to specific genomic loci through its DNA-binding capacity. ChIP-seq analysis has revealed that approximately 40% of G9a peaks, 45% of ZNF644 peaks, and 43% of WIZ peaks are distributed in gene promoter regions, suggesting coordinated action in transcriptional regulation . This genomic co-localization pattern provides strong evidence for WIZ's role in targeting the methyltransferase activity of the complex to specific regulatory elements.

What is the relationship between WIZ binding and CTCF sites in chromatin organization?

Analysis of WIZ ChIP-seq data has revealed a striking relationship between WIZ and CTCF binding patterns. Motif analysis identified a highly enriched sequence within WIZ binding sites (significance E-value of 2.3 × 10^-6903) that matches the CTCF-binding site consensus sequence with remarkable precision . This motif is present in approximately 70% of all WIZ binding peaks across the genome, suggesting a fundamental functional relationship between these two factors.

The extensive co-localization of WIZ and CTCF is visualized in the clustering patterns of their genomic distribution. When examining read density across ~40,000 WIZ ChIP-seq peaks, both WIZ and CTCF show highly correlated binding patterns . This genomic co-occupancy suggests that WIZ may cooperate with CTCF in establishing chromatin boundaries or coordinating long-range chromatin interactions. Researchers investigating chromatin architecture should consider examining both factors simultaneously to better understand their cooperative functions in genome organization.

How can WIZ degradation be induced, and what are the consequences for gene expression?

Recent research has identified molecular strategies to induce targeted degradation of WIZ protein as a potential therapeutic approach. Two molecular glue degraders, designated dWIZ-1 and dWIZ-2, have been developed that selectively target WIZ for proteasomal degradation . The second-generation compound, dWIZ-2, demonstrates improved pharmacokinetic properties making it suitable for both in vitro and in vivo applications.

The functional consequences of WIZ degradation have been particularly investigated in the context of hemoglobin regulation. Both genetic knockout of WIZ using CRISPR-Cas9 and pharmacological degradation using dWIZ compounds lead to pronounced de-repression of fetal hemoglobin (HbF) . Specifically:

• WIZ knockout using two independent sgRNAs elevated γ-globin mRNA, HbF protein levels, and the percentage of HbF+ cells in primary human erythroblasts

• dWIZ-2 treatment increased γ-globin mRNA, HbF+ cells, and HbF protein levels in sickle cell disease patient-derived erythroblasts

• In vivo treatment with dWIZ-2 in humanized mice resulted in dose-dependent WIZ degradation and increased HbF in human erythroblasts in the bone marrow

These findings establish WIZ as a previously unrecognized repressor of fetal hemoglobin and highlight potential therapeutic applications of targeting this transcription factor.

What validation controls are essential when using WIZ antibodies in research?

When using WIZ antibodies, comprehensive validation controls are critical to ensure experimental reliability. Based on established protocols in the literature, the following validation approaches are recommended:

• Genetic knockdown/knockout controls: Western blotting comparing WIZ+/+ and WIZ knockout/knockdown samples has been successfully used to confirm antibody specificity. For example, researchers validated NBP180586 antibody by comparing protein lysates from wild-type and homozygous mutant embryonic heads . This approach conclusively demonstrates that observed bands represent WIZ protein.

• Blocking peptide controls: For applications like Western blot, inclusion of a blocking peptide control lane confirms epitope specificity. This method has been effectively employed with antibodies like ab92334, where parallel samples with and without blocking peptide reveal which bands represent specific binding .

• Multiple antibody validation: Using multiple antibodies targeting different epitopes of WIZ provides stronger evidence of specificity. When testing a new WIZ antibody, comparing results with a previously validated antibody can increase confidence in the observations.

• ChIP-qPCR validation: For ChIP-seq applications, validation of selected peaks by ChIP-qPCR is essential. Studies have verified ChIP-seq results by examining 30 randomly picked loci that represent a broad range of fragment counts , confirming the reliability of genome-wide binding patterns.

• Negative controls: For immunoprecipitation experiments, parallel IgG control antibody samples are necessary to identify non-specific binding .

What are the optimal conditions for using WIZ antibodies in Western blot analysis?

Optimizing Western blot conditions for WIZ detection requires careful consideration of several parameters. Based on published protocols, the following conditions have proven effective:

• Sample preparation: RIPA buffer has been successfully used for tissue lysate preparation, particularly for cerebellum and other brain tissues . When analyzing cellular extracts, researchers should consider the anticipated WIZ isoform size (~100 kDa in most tissues, ~160 kDa in cerebellum) when selecting gel percentage.

• Protein loading: Optimal protein loading ranges from 25-50 μg total protein per lane, with 35 μg commonly used for brain tissue lysates . Higher loading amounts may be necessary for tissues with lower WIZ expression.

• Antibody concentration: Primary antibody concentrations of 1 μg/mL have been effective for antibodies like ab92334 and A05707 . Incubation overnight at 4°C typically yields optimal signal-to-noise ratios.

• Detection method: Chemiluminescence detection has been successfully employed, with ECL technique providing sufficient sensitivity for endogenous WIZ detection . For tissues with lower expression, enhanced sensitivity chemiluminescent substrates may be required.

• Expected band sizes: Researchers should anticipate detecting bands at approximately 178 kDa (predicted full-length size), with additional isoforms possible at ~100 kDa and ~160 kDa depending on the tissue being analyzed .

What strategies should be employed to optimize WIZ antibody performance in ChIP-seq experiments?

ChIP-seq experiments with WIZ antibodies require careful optimization to generate high-quality, reproducible data. Based on successful protocols in the literature, the following strategies are recommended:

• Tissue and sample pooling: Pooling of tissue samples has proven effective for WIZ ChIP-seq. Previous studies successfully used pooled samples from multiple cerebellums (two cerebellums per sample with two biological replicates) or pooled E13.5 brain tissue (from 5 individuals) . This approach helps overcome individual variation and ensures sufficient starting material.

• Sequencing depth: Generating at least 30 million reads per sample is recommended, with at least 24 million alignable reads after filtering out PCR duplicates . This depth ensures comprehensive coverage of WIZ binding sites across the genome.

• Peak identification parameters: Successful WIZ ChIP-seq analyses have employed peak calling with a stringent p-value threshold of ≤1 × 10^-20 when comparing ChIP samples to input controls . This threshold balances sensitivity and specificity.

• Data validation: ChIP-qPCR validation of selected peaks representing a range of enrichment levels should be performed to confirm the reliability of ChIP-seq results . This step is crucial for validating novel findings and establishing confidence in the dataset.

• Bioinformatic analysis: To identify WIZ binding motifs, MEME-ChIP program has been effectively used, revealing several motifs with high statistical significance . For analyzing WIZ binding relative to gene features, categorizing peaks into promoter regions (defined as 2kb up/downstream of TSS), intragenic, and intergenic regions provides valuable insights into binding patterns .

How should researchers interpret overlapping signals between WIZ and other chromatin factors?

Interpreting overlapping signals between WIZ and other chromatin factors requires sophisticated analytical approaches. Research has demonstrated substantial co-localization between WIZ binding sites and those of other factors, particularly CTCF and H3K4me3 . When analyzing such overlapping patterns, consider these interpretive frameworks:

• Quantitative correlation analysis: Calculate the correlation coefficient between binding intensity profiles of WIZ and other factors across co-bound regions. High correlation suggests cooperative binding or functional interaction.

• Categorical classification: Divide genomic regions into categories based on binding patterns: WIZ-only, factor X-only, and co-bound regions. This categorization facilitates the identification of context-specific functions.

• Motif enrichment analysis: For co-bound regions, determine if specific DNA motifs are enriched compared to regions bound by only one factor. Research has shown that ~70% of WIZ binding sites contain CTCF motifs , suggesting a functional relationship between these factors.

• Gene ontology analysis: Compare the functional annotations of genes associated with different binding patterns to identify biological processes specifically associated with co-bound regions versus uniquely bound sites.

• Integration with expression data: Correlate binding patterns with gene expression changes following perturbation of individual factors to distinguish between redundant, antagonistic, or synergistic relationships.

Venn diagram analysis of WIZ, CTCF, and H3K4me3 ChIP-seq peaks has revealed complex overlapping patterns that suggest both shared and unique functions . These relationships should be interpreted within the broader context of chromatin regulation to develop accurate mechanistic models.

What are common technical challenges when using WIZ antibodies and how can they be addressed?

Researchers working with WIZ antibodies may encounter several technical challenges that can be addressed through methodical troubleshooting:

• Multiple band detection in Western blot: The detection of multiple bands is common due to the presence of WIZ isoforms (~100 kDa and ~160 kDa) . To distinguish true isoforms from non-specific binding:

  • Compare with knockout/knockdown controls

  • Use blocking peptide competitions

  • Analyze tissue-specific expression patterns (the ~160 kDa isoform is enriched in cerebellum)

• Background signal in immunofluorescence: For applications like immunofluorescence where high antibody concentrations (20 μg/mL) are typically used , background signal can be problematic. This can be mitigated by:

  • Extending blocking steps (2 hours with 5% BSA)

  • Including additional wash steps with 0.1% Tween-20

  • Using fluorophore-conjugated secondary antibodies with minimal cross-reactivity

• ChIP efficiency variability: ChIP experiments with WIZ antibodies may show variability. To improve consistency:

  • Standardize chromatin fragmentation to 200-500 bp

  • Optimize antibody:chromatin ratios for each new antibody lot

  • Include spike-in controls with known enrichment patterns

• Cross-reactivity in multi-species studies: When comparing WIZ binding across species, potential cross-reactivity must be considered. The most thoroughly validated WIZ antibodies have confirmed reactivity with human, mouse, and rat samples . When extending to other species:

  • Perform preliminary validation with species-specific positive controls

  • Consider epitope conservation analysis before selecting antibodies

  • Include appropriate negative controls from the species of interest

How do different fixation methods affect WIZ antibody performance in immunohistochemistry?

The choice of fixation method significantly impacts WIZ antibody performance in immunohistochemistry applications. Based on established protocols and performance data:

• Formalin fixation and paraffin embedding (FFPE): This standard method has been validated for WIZ antibodies such as Boster's A05707, which is specifically recommended for IHC-P applications at a concentration of 2.5 μg/mL . FFPE tissues typically require antigen retrieval steps to unmask epitopes that may be crosslinked during fixation.

• Antigen retrieval methods: For WIZ detection in FFPE samples, heat-induced epitope retrieval (HIER) methods have proven most effective. The optimal pH for retrieval buffer may vary by antibody:

  • Citrate buffer (pH 6.0) provides good results for most WIZ antibodies

  • EDTA buffer (pH 9.0) may enhance signal for antibodies targeting certain epitopes

• Fresh frozen vs. FFPE: When comparing fresh frozen and FFPE samples, researchers should adjust antibody concentrations accordingly. Fresh frozen sections typically require lower antibody concentrations (approximately 50-60% of the concentration used for FFPE) due to better epitope preservation.

• Fixation duration considerations: Extended formalin fixation (>24 hours) may reduce WIZ antibody binding efficiency. If long fixation cannot be avoided, extended antigen retrieval times may be necessary to restore epitope accessibility.

• Alternative fixatives: For specialized applications where formalin fixation is problematic, methanol or acetone fixation has been used successfully for immunofluorescence detection of WIZ in cultured cells. These alternatives may preserve certain epitopes better than aldehyde-based fixatives.

What is the potential of targeting WIZ for therapeutic applications in hemoglobinopathies?

Recent research has uncovered promising therapeutic applications for targeting WIZ in hemoglobinopathies, particularly sickle cell disease (SCD). The development of molecular glue degraders specific to WIZ has opened new avenues for potential interventions:

• Mechanism of HbF induction: Targeted degradation of WIZ leads to de-repression of fetal hemoglobin (HbF), offering a potential therapeutic strategy for SCD . This approach addresses the disease's underlying pathophysiology by inducing expression of an alternative non-sickling hemoglobin form.

• In vivo validation: Studies in humanized mouse models have demonstrated that oral administration of dWIZ-2 (a WIZ degrader) results in dose-dependent WIZ degradation and corresponding increases in both total HbF and the proportion of HbF+ human erythroblasts in bone marrow . This provides proof-of-concept for the approach's efficacy in a complex biological system.

• Patient-derived cell validation: Experiments using sickle cell disease patient-derived erythroblasts showed that dWIZ-2 treatment increased γ-globin mRNA, HbF+ cells, and HbF protein levels . This validation in disease-relevant primary cells strengthens the translational potential of this approach.

• Advantages over existing approaches: Unlike genetic editing approaches for SCD, pharmacological degradation of WIZ offers a reversible, titratable intervention that doesn't require permanent genomic modification. This potentially addresses safety concerns associated with permanent genetic manipulations.

• Future development considerations: Several research questions remain to be addressed:

  • Long-term effects of WIZ degradation on other cellular processes

  • Potential for developing resistance mechanisms

  • Optimal dosing regimens to maintain therapeutic HbF levels

  • Potential combination approaches with other HbF-inducing agents

How can bioinformatic approaches enhance the analysis of WIZ ChIP-seq data?

Advanced bioinformatic approaches offer powerful methods for extracting meaningful insights from WIZ ChIP-seq data. Based on current research methodologies, the following analytical strategies are particularly valuable: