KAT2A Antibody

What is KAT2A and what are its primary biological functions in cellular processes?

KAT2A belongs to the nuclear A-type histone acetyltransferase (HAT) family that directly impacts gene transcription through histone modifications. Research demonstrates that KAT2A plays critical roles in cell cycle regulation, DNA replication, and DNA repair, establishing it as a key player in maintaining genome stability . Additionally, KAT2A functions as a coactivator of the c-MYC oncogene protein, highlighting its relevance in cancer biology .

Recent studies have revealed that KAT2A can regulate cellular processes through both transcriptional and post-translational mechanisms. For example, in neural stem cells (NSCs), KAT2A can acetylate PAX6 protein, facilitating its ubiquitination-mediated degradation, which affects NSC proliferation and differentiation . This demonstrates KAT2A's versatility beyond its traditional role in histone modification.

How does KAT2A contribute to epigenetic regulation in different cell types?

KAT2A primarily contributes to epigenetic regulation through histone acetylation, particularly H3K9 acetylation (H3K9ac), which typically promotes gene expression. In macrophages, KAT2A facilitates glycolysis reprogramming by suppressing nuclear factor-erythroid 2-related factor 2 (NRF2) activity, supporting H3K9ac and limiting NRF2-mediated transcriptional repression of proinflammatory genes .

In colorectal cancer (CRC), KAT2A-dependent cells display higher gene expression levels and enriched H3K27ac marks at gene loci linked to enterocytic differentiation . The epigenetic regulation by KAT2A appears to be context-dependent, with its activity varying across different cellular environments and developmental stages.

What are the validated applications for KAT2A antibodies in research protocols?

Based on extensive validation studies, KAT2A antibodies have been successfully employed in multiple experimental applications with specific dilution recommendations:

It's important to note that certain KAT2A antibodies may not be suitable for IHC applications, highlighting the necessity of selecting appropriate antibody clones for specific experimental purposes .

How can researchers design robust co-immunoprecipitation experiments to study KAT2A protein interactions?

When designing co-immunoprecipitation experiments to study KAT2A interactions:

• Begin by selecting a validated KAT2A antibody with demonstrated specificity in immunoprecipitation applications.

• Include proper controls: IgG control from the same species as the KAT2A antibody and input controls to assess pull-down efficiency.

• Consider crosslinking procedures for transient interactions.

• Optimize lysis conditions to preserve protein-protein interactions while effectively disrupting cellular structures.

This methodology has been successfully utilized to demonstrate KAT2A interaction with PAX6 in neural stem cells. Researchers employed anti-PAX6 antibody for immunoprecipitation and detected both PAX6 and KAT2A in the precipitate by Western blot . Notably, the same approach demonstrated that KAT2A did not interact with SOX2 in neural stem cells, highlighting the specificity of the KAT2A-PAX6 interaction .

How does KAT2A influence neural stem cell differentiation and what experimental approaches best capture these effects?

KAT2A plays a crucial role in regulating neural stem cell (NSC) differentiation through post-translational modification of PAX6, a key transcription factor in neural development. Current research indicates that KAT2A inhibition results in:

• Accelerated NSC proliferation

• Delayed differentiation

• Potential apoptosis (context-dependent)

The effects of KAT2A on NSC differentiation appear to be mediated through a KAT2A/PAX6 axis that balances self-renewal and differentiation .

For experimental approaches, researchers have successfully employed:

• Electroporation-mediated knockdown of Kat2a in NSCs followed by differentiation induction

• EdU labeling to assess proliferation effects

• Morphological analysis of cell processes to evaluate differentiation status

• Western blotting to monitor PAX6 protein levels

After Kat2a knockdown, cells showed shorter processes after 24 hours of differentiation induction, suggesting inhibited differentiation. EdU labeling revealed increased proliferation, and Western blotting demonstrated elevated PAX6 levels .

What are the methodological considerations when analyzing KAT2A-mediated post-translational modifications in neural contexts?

When investigating KAT2A-mediated post-translational modifications in neural contexts, researchers should consider:

• Combined transcriptional and protein analysis: KAT2A inhibition does not affect Pax6 mRNA levels in NSCs but increases PAX6 protein levels, indicating regulation at the post-translational level rather than transcriptional control .

• Analysis of acetylation status: Use acetylation-specific antibodies or mass spectrometry to detect KAT2A-mediated acetylation of target proteins.

• Ubiquitination assessment: KAT2A facilitates ubiquitination-mediated degradation of PAX6. Research has identified ring finger protein 8 (RNF8) as the E3 ligase responsible for PAX6 ubiquitination, working in conjunction with KAT2A .

• Protein-protein interaction studies: Immunoprecipitation assays are essential to confirm direct interactions. For example, KAT2A has been shown to directly interact with PAX6 but not with SOX2 in NSCs .

• Comparative HAT expression analysis: When studying KAT2A specifically, compare with other histone acetyltransferases (HATs) like CREB-binding protein (CBP) and P300/CBP-associated factor (PCAF) to establish specificity, as these showed different expression patterns during NSC differentiation .

How can researchers determine KAT2A dependency in cancer cells and what molecular markers indicate this dependency?

To determine KAT2A dependency in cancer cells, researchers should employ a multi-omics approach:

• CRISPR-Cas9 screening: This methodology has been successfully used to identify KAT2A-dependent cancer cell populations. In colorectal cancer (CRC), this approach revealed that KAT2A dependency is not simply correlated with KAT2A expression levels .

• Genomic profiling: KAT2A dependency in CRC is associated with microsatellite stability and lower mutational burden. These genomic features can serve as potential predictive markers .

• Transcriptomic analysis: Gene expression profiling can identify cancer cells with increased molecular differentiation signatures, which correlate with KAT2A dependency in CRC .

• Epigenomic mapping: H3K27ac ChIP-seq analysis can reveal enrichment patterns at gene loci associated with cell differentiation states. In KAT2A-dependent CRC cells, enriched H3K27ac marks are observed at loci linked to enterocytic differentiation .

• Functional validation: CRISPR-interference-mediated KAT2A knockdown experiments in both cancer cell lines and patient-derived 3D spheroid cultures can confirm dependency by assessing effects on cell growth, viability, and differentiation marker expression .

Additionally, in diffuse large B-cell lymphoma (DLBCL), KAT2A has been identified as a potential biomarker related to immune infiltration and malignant pathways .

What experimental designs best evaluate the effects of KAT2A inhibition or depletion in cancer models?

For robust evaluation of KAT2A inhibition or depletion in cancer models, consider the following experimental design approaches:

• Comprehensive in vitro models:

  • Traditional 2D cell culture with established cancer cell lines

  • Patient-derived 3D spheroid cultures to better recapitulate tumor architecture and heterogeneity

  • Assessment of cell proliferation using CCK-8 or similar assays at multiple time points (24, 48, and 72 hours)

• Gene expression analysis:

  • qRT-PCR for targeted gene expression studies using validated primers

  • RNA-seq for global transcriptomic changes

  • Focus on proliferation-associated, stem cell-associated, and differentiation marker genes

• In vivo models:

  • Patient-derived xenograft mouse models to assess effects in a physiological context

  • Monitor tumor growth, differentiation status, and molecular changes

• Mechanistic studies:

  • Pathway analysis to identify affected signaling networks

  • Epigenetic profiling to assess changes in histone modification patterns

  • Cell cycle and apoptosis analyses using flow cytometry

• Statistical robustness:

  • Conduct at least three independent experiments

  • Present data as mean ± standard deviation

  • Use appropriate statistical tests (t-tests, ANOVA, or Kruskal-Wallis) with significance threshold of p < 0.05

Research has shown that loss of KAT2A leads to decreased cell growth and viability both in vitro and in vivo, downregulation of proliferation and stem cell-associated genes, and induction of differentiation markers in colorectal cancer models .

How does KAT2A regulate inflammatory pathways in macrophages and what techniques best capture these mechanisms?

KAT2A plays a crucial role in regulating inflammatory pathways in macrophages through multiple mechanisms:

• Transcriptional regulation of inflammatory genes: KAT2A supports the transcription of proinflammatory genes such as Il1b and Nlrp3. Both pharmacological inhibition and siRNA silencing of KAT2A have been shown to suppress innate stimuli-triggered proinflammatory gene transcription .

• NLRP3 inflammasome activation: KAT2A is required for proper activation of the NLRP3 inflammasome, a critical component of the innate immune response .

• Metabolic reprogramming: KAT2A facilitates macrophage glycolysis reprogramming by suppressing nuclear factor-erythroid 2-related factor 2 (NRF2) activity and downstream antioxidant molecules .

• Epigenetic regulation: KAT2A supports histone 3 lysine 9 acetylation (H3K9ac) and limits NRF2-mediated transcriptional repression of proinflammatory genes .

To effectively study these mechanisms, researchers should employ:

• Gene expression analysis (qPCR, RNA-seq) to track inflammatory gene expression

• Western blotting to assess protein levels of inflammasome components

• Metabolic flux analysis to measure glycolytic activity

• ChIP-seq to map H3K9ac patterns at inflammatory gene loci

• In vivo models of inflammatory disease (such as rheumatoid arthritis)

• Inflammasome activation assays measuring IL-1β secretion

What are the optimal experimental designs to study KAT2A's role in immune microenvironments of tumors?

To effectively study KAT2A's role in tumor immune microenvironments, researchers should consider:

• Immune cell profiling in KAT2A-stratified tumors:

  • Apply the ESTIMATE algorithm to calculate stromal, immune, and estimated scores based on RNA expression levels

  • Assess immunosuppressive, immune cytolytic, and antigen-processing effects in tumors stratified by KAT2A expression or activity

• Development of a histone acetylation scoring system:

  • Establish a HAscore model for analyzing histone acetylation patterns in tumor samples

  • Use this model to correlate acetylation patterns with immune infiltration profiles

• Functional validation experiments:

  • KAT2A knockdown in cancer cell lines followed by co-culture with immune cells

  • Analysis of changes in immune cell recruitment, activation, and function

• Clinical correlation studies:

  • Stratify patient samples into high and low KAT2A expression groups

  • Compare immune cell infiltration patterns between groups

  • Correlate findings with patient outcomes

Research has demonstrated that patients with low histone acetylation scores (HAscore) have distinct tumor immune microenvironments and poorer prognoses in diffuse large B-cell lymphoma (DLBCL) . Additionally, KAT2A has been identified as a potential biomarker related to immune infiltration and malignant pathways in DLBCL .

How can researchers validate KAT2A antibody specificity for conclusive experimental results?

Validating KAT2A antibody specificity is crucial for obtaining reliable experimental results. A comprehensive validation approach should include:

• Knockout/knockdown controls:

  • Test antibody in KAT2A knockout/knockdown models (CRISPR-Cas9, siRNA, or shRNA)

  • Absence or significant reduction of signal confirms specificity

  • Publications have documented successful use of KAT2A antibody in knockdown validation experiments

• Multiple antibody concordance:

  • Use at least two different KAT2A antibodies targeting distinct epitopes

  • Consistent results between antibodies increase confidence in specificity

• Western blot validation:

  • Confirm detection of a single band at the expected molecular weight of 94 kDa

  • Test across multiple cell lines (HeLa, MCF-7, HSC-T6, NIH/3T3, SKOV-3) to ensure consistent detection

• Immunoprecipitation followed by mass spectrometry:

  • Perform IP with the KAT2A antibody

  • Analyze precipitated proteins by mass spectrometry to confirm KAT2A identity and assess off-target binding

• Peptide competition assay:

  • Pre-incubate antibody with excess immunizing peptide

  • Signal elimination confirms epitope-specific binding

• Cross-reactivity assessment:

  • Test antibody against closely related proteins (e.g., other HAT family members like PCAF)

  • Absence of cross-reactivity increases confidence in specificity

What experimental considerations are critical when using KAT2A antibodies for multi-protein complex analysis?

When using KAT2A antibodies to study multi-protein complexes:

• Optimization of lysis conditions:

  • Different lysis buffers may preserve or disrupt specific protein-protein interactions

  • For nuclear protein complexes, consider nuclear extraction protocols rather than whole-cell lysis

  • Gentle lysis conditions (low detergent concentrations) help maintain protein complexes

• Crosslinking considerations:

  • For transient or weak interactions, chemical crosslinking (formaldehyde, DSS, or BS3) may be necessary

  • Optimize crosslinking time and concentration to avoid artifactual associations

• Sequential immunoprecipitation:

  • For specific sub-complexes, consider tandem IP approaches

  • First IP with KAT2A antibody followed by elution and second IP with antibody against suspected interaction partner

• Mass spectrometry-based approaches:

  • Label-free quantitative proteomics can identify specific vs. non-specific interactors

  • SILAC or TMT labeling offers quantitative comparison between conditions

  • Consider proximity-dependent biotinylation (BioID or TurboID) as complementary approaches

• Functional validation of interactions:

  • Confirm biological relevance of identified interactions through functional assays

  • For example, the KAT2A-PAX6 interaction was validated by demonstrating KAT2A-mediated acetylation of PAX6 and its effect on PAX6 protein stability

• Control experiments:

  • Include IgG control from the same species as the KAT2A antibody

  • Include reverse IP (IP with antibody against suspected interaction partner)

  • Test interaction in both endogenous context and with exogenously expressed proteins

This approach has successfully revealed that KAT2A interacts with PAX6 but not with SOX2 in neural stem cells, demonstrating the specificity of protein interaction studies when properly controlled .

How can researchers design experiments to explore KAT2A's therapeutic potential in inflammatory diseases?

Based on recent findings of KAT2A's role in inflammatory pathways, researchers can design experiments to assess its therapeutic potential using:

• Pharmacological inhibition studies:

  • Evaluate existing KAT2A inhibitors in inflammatory disease models

  • Monitor inflammation markers, disease progression, and clinical outcomes

  • Research has demonstrated that pharmacological inhibition of KAT2A suppresses proinflammatory gene transcription and impairs NLRP3 inflammasome activation both in vivo and in vitro

• Genetic modulation approaches:

  • Conditional knockout models in specific cell types (e.g., macrophages)

  • Inducible systems to control timing of KAT2A depletion

  • siRNA silencing of KAT2A has been shown to effectively suppress inflammatory responses

• Disease model selection:

  • Rheumatoid arthritis models (as suggested by existing research)

  • Other inflammatory conditions with NLRP3 inflammasome involvement

  • Patient-derived samples for ex vivo experiments

• Mechanism-focused experiments:

  • Measure effects on NRF2 activity and downstream antioxidant molecules

  • Assess glycolytic reprogramming in inflammatory cells

  • Examine H3K9ac patterns at proinflammatory gene loci

• Combination therapy evaluation:

  • Test KAT2A inhibition alongside existing anti-inflammatory agents

  • Assess potential synergistic effects or reduced side effect profiles

What experimental approaches can identify biomarkers for KAT2A dependency in different cancer types?

To identify biomarkers of KAT2A dependency across cancer types, researchers should implement:

• Multi-omics screening approaches:

  • Integrate CRISPR-Cas9 screening data with genomics, transcriptomics, and global acetylation patterns

  • This approach has successfully identified molecular markers indicative of KAT2A dependency in colorectal cancer

• Stratification criteria development:

  • Assess microsatellite stability status and mutational burden as potential biomarkers

  • Evaluate molecular differentiation signatures as indicators of KAT2A dependency

  • Research has shown that KAT2A dependency in colorectal cancer is associated with these features, independent of KAT2A expression levels

• Epigenetic profiling:

  • Map H3K27ac and H3K9ac patterns across cancer types

  • Correlate enrichment patterns with KAT2A dependency

  • KAT2A-dependent colorectal cancer cells display higher gene expression levels and enriched H3K27ac marks at gene loci linked to enterocytic differentiation

• Functional validation experiments:

  • Test KAT2A inhibition or depletion effects in patient-derived models

  • Correlate response with molecular and genetic features

  • Establish predictive algorithms for response likelihood

• Clinical correlation studies:

  • Develop a histone acetylation score (HAscore) model for tumor samples

  • Correlate scores with clinical outcomes and treatment responses

  • This approach has demonstrated utility in diffuse large B-cell lymphoma, where patients with low HAscores have poorer prognoses

These combined approaches can potentially identify patients most likely to benefit from KAT2A-targeted therapies across different cancer types.