KDM5A Antibody

What is KDM5A and what are its primary biological functions?

KDM5A (also known as JARID1A or RBP2) is a histone demethylase that specifically removes methyl groups from lysine 4 of histone H3 (H3K4). It has specificity for trimethylated and dimethylated H3K4 but does not demethylate monomethylated H3K4 . KDM5A does not demethylate other histone marks such as H3K9, H3K27, H3K36, H3K79, or H4K20 .

Functionally, KDM5A plays diverse roles in cellular processes, including:

• Transcriptional regulation, particularly in stimulating nuclear receptor-mediated transcription

• Repression of genes involved in antigen processing and presentation pathways

• Regulation of HOX protein transcription during cell differentiation

• Transcriptional repression of cytokines such as CXCL12

• Cell cycle regulation through interaction with the Retinoblastoma (pRb) protein

• Maintenance of neural progenitor cells (NPCs) in an undifferentiated state by repressing astrocytogenesis

• Contribution to replication stress response and DNA damage tolerance mechanisms

How does KDM5A expression vary across different tissue types and disease states?

KDM5A exhibits variable expression patterns across tissues and is frequently dysregulated in pathological conditions. In cancer biology, KDM5A is often overexpressed compared to normal adjacent tissues. For example, studies have demonstrated significantly higher KDM5A expression in osteosarcoma tissues compared to adjacent normal tissue . This overexpression correlates with enhanced tumor cell proliferation and inhibition of apoptosis .

In neural development, KDM5A shows an interesting expression pattern where mRNA levels increase during differentiation of neural progenitor cells into astrocytes, but paradoxically, the protein levels decrease during this transition . This suggests sophisticated post-transcriptional regulation of KDM5A during developmental processes.

KDM5A is also implicated in various cancer types as a mediator of drug tolerance and treatment resistance. Together with KDM5B, it contributes to replication stress responses that may help cancer cells survive therapeutic interventions .

What are the key structural domains of KDM5A that antibodies typically target?

KDM5A is a large protein of approximately 192.1 kilodaltons . Antibodies against KDM5A can be raised against different domains, but many commercial antibodies are developed against the full-length recombinant human KDM5A protein . When selecting a KDM5A antibody, researchers should consider which domain they wish to target based on their experimental question.

Key structural domains that may be targeted include:

• The JmjC catalytic domain responsible for demethylase activity

• The ARID DNA-binding domain

• The PHD finger domains that recognize specific histone modifications

• C-terminal regions that interact with binding partners such as pRb

Understanding the epitope specificity of a KDM5A antibody is crucial for experimental design, as some domains may be masked in certain protein complexes or affected by post-translational modifications.

What are the most common experimental applications for KDM5A antibodies?

KDM5A antibodies are employed in a variety of experimental techniques to investigate its function and regulation. Based on the literature, the most common applications include:

• Western Blot (WB): For detecting and quantifying KDM5A protein expression levels in cell or tissue lysates . This technique is particularly useful for comparing KDM5A protein levels between different experimental conditions or cell types.

• Chromatin Immunoprecipitation (ChIP): For identifying genomic regions bound by KDM5A. ChIP followed by sequencing (ChIP-seq) has been used to map KDM5A binding sites genome-wide, revealing its preferential binding to transcription start sites (TSS), especially among highly expressed genes .

• Immunoprecipitation (IP): For studying protein-protein interactions involving KDM5A, such as its association with PCNA and Chk1 at replication forks .

• Immunocytochemistry/Immunofluorescence (ICC/IF): For visualizing the subcellular localization of KDM5A or studying its co-localization with other proteins .

• Flow Cytometry (FCM): For analyzing KDM5A expression in specific cell populations .

When designing experiments, researchers should consider the validation status of their chosen antibody for specific applications, as performance can vary significantly between different experimental contexts.

How should I validate a KDM5A antibody for my specific application?

Proper validation of KDM5A antibodies is essential for generating reliable research data. A comprehensive validation approach should include:

• Specificity testing: This can be accomplished by comparing signal in KDM5A knockout or knockdown cells versus wild-type cells. For example, ChIP-seq experiments using KDM5A antibodies in cells with conditional KDM5A alleles (Kdm5a^f/f) versus deleted alleles (Kdm5a^-/-) showed no significant enrichment in the knockout cells, confirming antibody specificity .

• Positive and negative controls: Include known KDM5A-expressing cells/tissues as positive controls and KDM5A-negative samples as negative controls.

• Application-specific validation:

  • For WB: Confirm the observed band appears at the expected molecular weight (~192 kDa) and disappears with KDM5A knockdown

  • For ChIP: Verify enrichment at known KDM5A target genes

  • For ICC/IF: Confirm nuclear localization pattern and verify knockdown reduces signal

• Cross-validation with multiple antibodies: When possible, use antibodies from different sources or raised against different epitopes to confirm results.

• Peptide competition: Pre-incubation of the antibody with its specific immunogen peptide should abolish specific binding.

For critical experiments, it is advisable to use antibodies validated through multiple methodologies and, ideally, those that have been cited in peer-reviewed publications for your specific application.

What are the optimal sample preparation methods for KDM5A detection?

Effective detection of KDM5A requires careful consideration of sample preparation methods:

• For Western Blotting:

  • Use nuclear extraction protocols, as KDM5A is primarily a nuclear protein

  • Include protease inhibitors to prevent degradation

  • Consider adding phosphatase inhibitors if investigating phosphorylation states

  • For optimal resolution of this large protein (~192 kDa), use low percentage (6-8%) SDS-PAGE gels

  • Transfer proteins to membranes using longer transfer times or specialized protocols for high molecular weight proteins

• For ChIP assays:

  • Crosslinking conditions are critical; standard 1% formaldehyde for 10 minutes at room temperature works for most applications

  • Sonication conditions should be optimized to achieve chromatin fragments of 200-500 bp

  • Use buffer conditions that preserve protein-DNA interactions while minimizing non-specific binding

• For Immunofluorescence:

  • Fixation method affects epitope accessibility; try both paraformaldehyde and methanol fixation

  • Permeabilization steps are essential for nuclear protein detection

  • Consider antigen retrieval methods if working with tissue sections

  • Nuclear counterstains like DAPI help confirm nuclear localization

• For Flow Cytometry:

  • Ensure complete permeabilization to allow antibody access to nuclear proteins

  • Consider fixation that preserves both cell surface markers and nuclear proteins

  • Optimize antibody concentration to achieve good signal-to-noise ratio

Preserving post-translational modifications may require specialized buffers containing specific inhibitors relevant to the modification of interest (e.g., deacetylase inhibitors, phosphatase inhibitors).

How can I utilize KDM5A antibodies to study its role in cancer biology?

KDM5A has emerged as a significant player in cancer biology, and antibodies against this protein can be instrumental in investigating its roles through several sophisticated approaches:

• Tumor microenvironment studies: KDM5A has been shown to impair immune cell infiltration into tumors by silencing genes involved in antigen processing and presentation pathways . Researchers can use KDM5A antibodies for multiplex immunofluorescence to simultaneously visualize KDM5A expression and immune cell markers in tumor sections.

• ChIP-seq analysis in tumor samples: This approach can identify genes directly regulated by KDM5A in cancer cells. Studies have shown that KDM5A preferentially binds to transcription start sites, especially among highly expressed genes . Differential binding analysis between normal and tumor tissues can reveal cancer-specific KDM5A targets.

• Protein complex analysis: Co-immunoprecipitation using KDM5A antibodies followed by mass spectrometry can identify novel interaction partners in cancer cells. For example, KDM5A has been found to associate with PCNA and Chk1 at replication forks .

• Response to therapy monitoring: KDM5A expression changes in response to treatment can be monitored using antibodies in western blot, IHC, or flow cytometry analyses. KDM5A and KDM5B have been implicated in drug tolerance mechanisms in cancer cells .

• Chromatin landscape mapping: Combining KDM5A ChIP-seq with histone modification ChIP-seq (particularly H3K4me3/me2) can provide insights into how KDM5A reshapes the epigenetic landscape in cancer cells.

For translational research, correlative studies between KDM5A expression patterns (detected by IHC) and clinical outcomes can help establish its potential as a prognostic biomarker.

What approaches can resolve contradictions in KDM5A functional data from different experimental systems?

Researchers frequently encounter contradictory data regarding KDM5A function across different experimental systems. Several methodological approaches can help resolve these contradictions:

• Cell type-specific context analysis: KDM5A may exhibit different functions in different cell types. For example, while KDM5A represses astrocytogenesis in neural progenitor cells , it promotes proliferation in osteosarcoma cells . Systematically comparing KDM5A binding patterns via ChIP-seq across different cell types can reveal cell type-specific targets.

• Functional domain dissection: Using antibodies that recognize specific domains of KDM5A, researchers can determine whether contradictory functions are mediated by different protein domains. Structure-function studies using domain-specific mutants can complement antibody-based approaches.

• Temporal dynamics investigation: KDM5A function may vary temporally during cellular processes. For instance, despite higher Kdm5a mRNA expression during astrocyte differentiation, KDM5A protein levels decrease , suggesting complex regulatory mechanisms. Time-course studies with KDM5A antibodies can capture these dynamics.

• Protein complex characterization: KDM5A functions as part of different protein complexes. IP-mass spectrometry under different experimental conditions can identify context-specific interaction partners that might explain functional differences.

• Post-translational modification profiling: KDM5A function may be modulated by PTMs. Combining general KDM5A antibodies with modification-specific antibodies can reveal how PTMs change across experimental systems.

• Integrated multi-omics approach: Combining ChIP-seq, RNA-seq, and proteomics data can provide a comprehensive view of KDM5A function. For example, KDM5A knockout in osteosarcoma cells affected interferon, EMT, IL6/JAK/STAT3, and TNF-α/NF-κB pathways , demonstrating its multifaceted roles.

These approaches can help reconcile seemingly contradictory functions by placing KDM5A activities within specific biological contexts.

How can I design experiments to distinguish between demethylase-dependent and -independent functions of KDM5A?

KDM5A has both enzymatic (demethylase-dependent) and non-enzymatic (demethylase-independent) functions. Designing experiments to distinguish between these requires careful consideration:

• Catalytic dead mutants: Compare the effects of wild-type KDM5A versus catalytically inactive mutants (e.g., mutations in the JmjC domain) in rescue experiments. Use KDM5A antibodies to confirm equal expression levels of both constructs.

• Histone mark correlation studies: Combine KDM5A ChIP-seq with H3K4me3/me2 ChIP-seq to identify regions where KDM5A binding correlates with reduced H3K4 methylation (demethylase-dependent) versus regions where KDM5A binds without affecting H3K4 methylation (potentially demethylase-independent).

• Small molecule inhibitors: Compare the effects of KDM5A knockdown with selective KDM5A inhibitors. Differences may indicate demethylase-independent functions. Studies have shown that KDM5A inhibitors demonstrated limited effectiveness at suppressing cancer cell viability as single agents in vitro, despite effectively blocking enzymatic activity .

• Protein-protein interaction mapping: Use KDM5A antibodies for co-IP experiments to identify interaction partners. Correlate these with functional outcomes to determine which interactions depend on catalytic activity.

• Domain-specific antibodies: Use antibodies recognizing different KDM5A domains to determine which regions are involved in specific functions through ChIP-seq or IP studies.

• Direct comparison with other KDM5 family members: Compare KDM5A with other family members that share demethylase activity but have different non-catalytic domains. Studies have shown that KDM5A and KDM5B both contribute to replication stress response , suggesting possible functional redundancy in some contexts.

• Measuring global versus local H3K4me3/me2 levels: Use immunoblotting to assess global changes in H3K4 methylation versus ChIP-qPCR at specific loci to distinguish between genome-wide and locus-specific demethylase functions.

These approaches can help delineate the multifaceted roles of KDM5A beyond its canonical histone demethylase activity.

What are the most common technical challenges when using KDM5A antibodies in ChIP experiments?

Chromatin immunoprecipitation with KDM5A antibodies presents several technical challenges that researchers should anticipate:

• Antibody specificity and quality: The success of ChIP experiments heavily depends on antibody quality. Researchers should validate KDM5A antibodies in knockout or knockdown systems. Studies have demonstrated that ChIP-seq experiments using KDM5A antibodies in Kdm5a^-/- cells showed no significant enrichment, confirming antibody specificity .

• Chromatin preparation: KDM5A binds to active promoters, particularly at transcription start sites of highly expressed genes . Optimal chromatin fragmentation is crucial - fragments should be 200-500bp for standard ChIP-seq. Over-sonication can destroy epitopes, while under-sonication results in poor resolution and non-specific binding.

• Low signal-to-noise ratio: KDM5A binding may not be as strong as some transcription factors, resulting in lower enrichment. Optimizing crosslinking conditions (formaldehyde concentration, time, temperature) can improve signal. Using multiple antibodies recognizing different epitopes can provide validation.

• Dynamic binding patterns: KDM5A binding can be dynamic and context-dependent. For example, E2F4 influences KDM5A recruitment to certain sites . Time-course experiments or specific cell state synchronization may be necessary to capture relevant binding events.

• Buffer composition: Salt concentration and detergent levels in wash buffers significantly affect specificity. Titrate these components carefully to balance between maintaining specific interactions and reducing background.

• Technical controls: Include input chromatin, IgG controls, and positive control regions (known KDM5A binding sites) in each experiment. If possible, include biological controls such as KDM5A-depleted cells.

• Data analysis challenges: KDM5A binding patterns can be complex, with both sharp peaks at promoters and broader domains. Peak-calling algorithms should be selected and parameterized appropriately for the expected binding pattern.

To overcome these challenges, researchers often need to test multiple antibodies, optimize chromatin preparation protocols specifically for KDM5A, and carefully validate results with orthogonal approaches.

How should I interpret changes in KDM5A expression versus changes in its genomic localization?

Distinguishing between changes in KDM5A expression levels and alterations in its genomic localization is crucial for understanding its biological functions:

Understanding this distinction is particularly important when interpreting KDM5A function during cellular transitions, such as differentiation or malignant transformation.

What experimental controls are essential when studying KDM5A's role in gene regulation?

Rigorous experimental controls are crucial for accurately interpreting KDM5A's role in gene regulation:

• Genetic controls:

  • KDM5A knockdown/knockout: Essential for demonstrating KDM5A-dependent effects. Studies showed that KDM5A knockdown increased GFAP expression in neural progenitor cells, confirming its role in repressing astrocytogenesis .

  • Rescue experiments: Reintroduction of wild-type KDM5A should reverse phenotypes observed in knockout/knockdown cells.

  • Catalytic mutants: Comparing wild-type with catalytically inactive KDM5A distinguishes between demethylase-dependent and -independent functions.

• Target specificity controls:

  • Off-target effect assessment: Use multiple siRNAs/shRNAs targeting different regions of KDM5A.

  • Family member specificity: Determine whether other KDM5 family members (KDM5B, KDM5C, KDM5D) compensate for KDM5A loss. Studies have shown functional overlap between KDM5A and KDM5B in replication stress response .

• Mechanistic controls:

  • Histone modification analysis: Monitor H3K4me3/me2 levels at KDM5A target genes to confirm demethylase activity.

  • Direct binding verification: Use ChIP-qPCR to verify KDM5A binding at regulated genes.

  • Promoter-reporter assays: Studies demonstrated that co-transfection of cells with an HA-tagged KDM5A and a luciferase vector containing the Gfap promoter sequence significantly reduced luciferase activity compared to controls .

• Biological context controls:

  • Cell-type specificity: Test KDM5A function across multiple cell types to determine context-specific effects.

  • Differentiation stage comparison: KDM5A function may vary during differentiation or cell state transitions.

  • Stimulation time course: Include appropriate time points when studying dynamic responses.

• Technical controls:

  • Antibody validation: Confirm antibody specificity using KDM5A-depleted samples.

  • Multiple methodologies: Corroborate findings using different techniques (e.g., RNA-seq, RT-qPCR, Western blot).

  • Appropriate statistical analysis: Include sufficient biological replicates and appropriate statistical tests.

Implementing these controls ensures that observed effects can be confidently attributed to KDM5A's specific activities in gene regulation.

How can KDM5A antibodies be utilized to study its role in immune regulation?

KDM5A has emerging roles in immune regulation, and antibodies can be powerful tools to investigate these functions:

• Tumor immune microenvironment analysis: KDM5A has been shown to impair epithelial ovarian cancer infiltration by immune cells and inhibit antitumor immune responses by silencing genes involved in antigen processing and presentation pathways . Multiplex immunohistochemistry using KDM5A antibodies combined with immune cell markers can characterize its expression in relation to tumor-infiltrating lymphocytes.

• Mechanistic studies of antigen presentation: KDM5A inhibition restored the expression of genes involved in the antigen-presentation pathway and promoted antitumor immune responses mediated by CD8+ T cells in vivo . Co-ChIP experiments can identify where KDM5A co-localizes with other transcriptional regulators at promoters of antigen presentation genes like HLA-A and HLA-B.

• KDM5A-immune cell correlation studies: A negative correlation exists between expression of KDM5A and genes involved in antigen processing and presentation in most cancer types . Researchers can use KDM5A antibodies for immunoblotting or IHC to correlate its expression with immune infiltration markers across patient samples.

• Therapeutic response monitoring: KDM5A inhibition reduced tumor burden and improved survival of tumor-bearing mice in a CD8+ T cell-dependent manner . KDM5A antibodies can monitor changes in its expression during immunotherapy to determine potential resistance mechanisms.

• NK cell activation studies: KDM5A is necessary for natural killer cell activation by associating with p50 to suppress the suppressor of cytokine signaling 1, which inhibits STAT4 phosphorylation and nuclear localization . Proximity ligation assays using KDM5A antibodies can visualize its interaction with p50 in NK cells.

• Immune checkpoint regulation: Investigate potential connections between KDM5A and immune checkpoint molecules using ChIP-seq and co-immunoprecipitation approaches with specific antibodies.

These applications can illuminate KDM5A's dual role as both a regulator of cancer cell intrinsic properties and as a modulator of anti-tumor immunity.

What are the current challenges in developing specific inhibitors of KDM5A function?

Developing specific inhibitors of KDM5A presents several significant challenges that researchers are actively addressing:

• Selectivity within KDM5 family: KDM5A belongs to a family that includes KDM5B, KDM5C, and KDM5D, all of which share a highly conserved catalytic domain. Achieving inhibitors that selectively target KDM5A requires identifying unique structural features. Antibody epitope mapping studies can help identify KDM5A-specific regions that might be targeted.

• Catalytic versus non-catalytic functions: Studies have shown that while current KDM5A inhibitors effectively block enzymatic activity, they demonstrate limited effectiveness at suppressing cancer cell viability as single agents in vitro . This suggests important non-catalytic functions of KDM5A that would not be affected by catalytic inhibitors. Antibody-based studies identifying KDM5A protein complexes can reveal non-catalytic functions as potential therapeutic targets.

• Context-dependent function: KDM5A functions differently across cell types and biological contexts. For example, it represses astrocytogenesis in neural progenitor cells while promoting proliferation in osteosarcoma cells . This context dependency complicates inhibitor development and necessitates tissue-specific testing of candidate compounds.

• Redundancy with other demethylases: Functional overlap between KDM5A and other family members, particularly KDM5B in replication stress response , means that selective KDM5A inhibition might be compensated for by other demethylases. Combination targeting strategies may be necessary.

• Biomarker development: Identifying patients who would benefit from KDM5A inhibition requires reliable biomarkers. Immunohistochemistry protocols using validated KDM5A antibodies need to be standardized for potential clinical applications.

• Delivery to nuclear targets: As a nuclear protein, KDM5A presents drug delivery challenges. Antibody-drug conjugates or proteolysis-targeting chimeras (PROTACs) that degrade KDM5A may offer alternative approaches to direct catalytic inhibition.

Addressing these challenges requires multidisciplinary approaches combining structural biology, medicinal chemistry, and biological validation with highly specific KDM5A antibodies to characterize inhibitor effects on both enzymatic and non-enzymatic functions.

How can I apply KDM5A antibodies in studies of therapy resistance mechanisms?

KDM5A has been implicated in therapy resistance across multiple cancer types, making it an important target for understanding and overcoming treatment failure:

• Expression dynamics during treatment: Monitor KDM5A expression changes before, during, and after therapy using immunoblotting or immunohistochemistry with validated antibodies. KDM5A and KDM5B are overexpressed in many cancers and have been involved in drug tolerance mechanisms .

• Drug-tolerant persister cell identification: Use KDM5A antibodies in combination with other markers to identify and isolate drug-tolerant persister cells via flow cytometry or immunofluorescence. These cells can then be characterized for molecular features driving resistance.

• Chromatin dynamics assessment: Perform ChIP-seq with KDM5A antibodies in sensitive versus resistant cells to identify altered binding patterns that may contribute to resistance. KDM5A, together with KDM5B, contributes to replication stress response and tolerance , which may protect cancer cells from DNA-damaging therapies.

• Pathway modulation studies: KDM5A knockdown in osteosarcoma suppressed cell proliferation and induced apoptosis . Combine KDM5A antibodies with pathway-specific markers (e.g., cell cycle regulators like P27 and Cyclin D1) to understand how KDM5A modulation affects therapeutic response pathways.

• Combination therapy rational design: KDM5A inhibition restored antigen presentation and promoted CD8+ T cell-mediated antitumor immune responses . Use KDM5A antibodies to monitor the effects of combining KDM5A inhibitors with immunotherapies or conventional treatments.

• Replication stress response analysis: KDM5A is enriched at ongoing replication forks and associates with both PCNA and Chk1 . Proximity ligation assays with KDM5A antibodies can visualize these interactions during therapy.

• Translational biomarker development: Correlate KDM5A protein levels or localization patterns (detected by immunohistochemistry) with clinical response to identify potential predictive biomarkers for therapy selection.

These approaches can reveal KDM5A-dependent mechanisms of therapy resistance and provide rationale for combination strategies targeting KDM5A alongside standard treatments.

What are the key considerations when planning KDM5A antibody-based experiments?

Successful implementation of KDM5A antibody-based experiments requires careful consideration of multiple factors to ensure reliable and interpretable results. Researchers should prioritize antibody validation, recognizing that KDM5A functions are context-dependent and often involve complex protein interactions. The experimental design should include appropriate controls, particularly genetic controls such as KDM5A knockdown/knockout samples, to definitively attribute observed effects to KDM5A .

Technical aspects require special attention, including optimized sample preparation methods appropriate for nuclear proteins, and technique-specific considerations such as chromatin fragmentation for ChIP or transfer conditions for Western blotting of this large protein. Researchers should also consider KDM5A's multiple functions—both enzymatic and non-enzymatic—when interpreting results, and utilize complementary approaches to distinguish between these roles .

For translational research applications, particularly in cancer biology or immune regulation, it is essential to understand KDM5A's context-specific functions and potential redundancy with other KDM5 family members. This understanding will inform more effective therapeutic targeting strategies and biomarker development efforts .

How is the field of KDM5A research likely to evolve in the coming years?

The field of KDM5A research is poised for significant advances in several key areas. First, the development of more selective KDM5A inhibitors will likely progress beyond targeting purely catalytic functions to address non-enzymatic activities through innovative approaches like protein degraders or interaction disruptors . These advances will be informed by deeper structural understanding of KDM5A complexes and domain-specific functions.

The role of KDM5A in immune regulation represents an exciting frontier, with emerging evidence suggesting its importance in modulating antitumor immunity through regulation of antigen presentation pathways . This intersection between epigenetic regulation and immunology will likely yield novel immunotherapeutic strategies targeting the KDM5A axis.

Single-cell technologies combined with KDM5A antibodies will provide unprecedented resolution of its function in heterogeneous cell populations and during developmental transitions. This approach will be particularly valuable for understanding KDM5A's context-dependent roles, such as its function in maintaining neural progenitor cells in an undifferentiated state .

The translational impact of KDM5A research will expand through the development of biomarker strategies using validated antibodies in clinical specimens. These biomarkers may help stratify patients for targeted therapies and monitor treatment responses, particularly in cancers where KDM5A contributes to therapy resistance mechanisms .