HIST1H3A (Ab-10) Antibody

What is HIST1H3A and why is it important in epigenetic research?

HIST1H3A encodes histone H3.1, one of the canonical histone H3 variants essential for nucleosome structure. Two molecules of each of the four core histones (H2A, H2B, H3, and H4) form an octamer, around which approximately 146 bp of DNA is wrapped in repeating units called nucleosomes . Histone H3.1 is expressed during S-phase, distinguishing it from the constitutively expressed H3.3 variant . As a core component of chromatin, HIST1H3A is crucial for understanding DNA packaging, gene expression regulation, and epigenetic modifications that influence cellular identity and function.

What applications are HIST1H3A antibodies typically used for?

HIST1H3A antibodies are primarily used in Western Blot (WB), Immunohistochemistry (IHC), and ELISA applications . For Western Blot applications, recommended dilutions typically range from 1:5000 to 1:50000, while for IHC applications, dilutions of 1:500 to 1:2000 are commonly used . These antibodies are valuable tools for studying histone modifications, chromatin dynamics, and epigenetic regulation in various experimental contexts. Researchers should optimize antibody dilutions for each specific application and sample type to achieve optimal signal-to-noise ratios.

How can I verify the specificity of my HIST1H3A antibody?

Antibody specificity can be verified through multiple approaches:

• Positive controls: Use cell lines known to express HIST1H3A, such as LNCaP, HEK-293, HeLa, Jurkat, HSC-T6, or NIH/3T3 cells .

• Western blot analysis: Look for a band at approximately 15 kDa, which is the observed molecular weight of histone H3 .

• Comparison across species: Test antibody reactivity across species if your research involves cross-species comparisons, as some antibodies show cross-reactivity with human, mouse, rat, chicken, zebrafish, and wheat samples .

• Peptide competition assay: Pre-incubate the antibody with the immunogen peptide to confirm binding specificity.

• Knockout/knockdown validation: Compare signals between wild-type and HIST1H3A-depleted samples.

What is the difference between histone H3.1 (HIST1H3A) and other H3 variants?

The major histone H3 variants include H3.1, H3.2, and H3.3, which differ in their expression patterns and functions:

• H3.1 and H3.2 (canonical): Expressed only during S-phase of the cell cycle .

• H3.3 (replacement): Expressed constitutively throughout the cell cycle .

• Tissue-specific variants: Some variants are expressed in a tissue-restricted fashion (H3.5, H3.X, H3.Y) .

• CENP-A: Deposited only at centromeres .

A unique feature of H3.1 compared to other H3 variants is that it contains an oxidizable cysteine residue at position 96 (Cys96), which makes it susceptible to redox regulation . This feature is absent in H3.2 and H3.3, making H3.1 more sensitive to oxidation compared to these variants .

How does histone H3.1 function as a redox sensor in cancer cells?

Recent research has revealed that histone H3.1 serves as a chromatin-embedded redox sensor that responds to mitochondrial H₂O₂ in cancer cells . The mechanism involves:

• Cysteine oxidation: H3.1 contains Cys96, which can be oxidized by H₂O₂ to form sulfenic acid (Cys-SOH) .

• H3.1 depletion: Oxidation promotes the exchange of H3.1 for the variant H3.3 .

• Chromatin remodeling: This exchange leads to chromatin decompaction and increased accessibility at promoter regions .

• Gene activation: The resulting chromatin changes activate plasticity genes associated with epithelial-to-mesenchymal transition (EMT) .

Experimental evidence supporting this model includes:

• Treatment with H₂O₂ leads to DCP-Bio1 adduct formation with H3.1 but not with H3.2 or H3.3, indicating specific oxidation of H3.1 .

• Expression of oxidation-resistant H3.1(C96S) mutant prevents H₂O₂-induced histone exchange and EMT gene activation .

• ChIP-seq experiments demonstrate significant loss of H3.1/H3.2 histones near transcription start sites after nH₂O₂ induction, mirrored by increased H3.3 deposition .

These findings suggest that targeting H3.1 oxidation could potentially inhibit cancer cell plasticity and metastasis.

What are the effects of histone H3 mutations in cancer and how can they be studied using antibodies?

Histone H3 mutations have been identified in various cancers and exert dominant effects on chromatin regulation . Two major classes of H3 mutations with significant impacts include:

• "K to M" mutations: Lysine residues normally subject to methylation or acetylation are mutated to methionine (e.g., H3K27M) .

• Glycine 34 mutations: Mutations at G34 impact the modification of the nearby K36 residue .

The H3K27M mutation, for example, exerts dominant effects by:

• Stabilizing the binding of PRC2 to the mutant histone

• Sequestering the methyltransferase

• Preventing further deposition of H3K27me

• Leading to a global decrease in H3K27me3

Researchers can study these mutations using:

• Co-immunoprecipitation (Co-IP): To detect enhanced co-purification of mutant H3 with PRC2 complexes .

• ChIP-seq: To assess genome-wide changes in histone variant distribution and modifications .

• Specific antibodies: That recognize either the mutant histones or their modification states.

• Transmission electron microscopy: To visualize changes in chromatin structure .

How do H1 histones interact with H3 modifications and what methodologies can detect these relationships?

H1 linker histones play crucial roles in regulating H3 modifications and chromatin structure . Research has shown that:

• H1 depletion leads to decreased H3K27 methylation and increased H3K36 methylation .

• H1 promotes PRC2-mediated H3K27 methylation in vitro .

• H1 inhibits NSD2-mediated H3K36 methylation .

• These effects are mediated by H1's promotion of physical compaction of chromatin substrate .

Methodologies to study H1-H3 interactions include:

• Quantitative mass spectrometry: To detect global changes in histone modifications upon H1 depletion .

• Immunoblotting: To compare specific histone modifications across different tissues .

• ChIP-seq: To map genome-wide distribution of H3K27me3 and H3K36me2 in wild-type versus H1-depleted cells .

• In vitro reconstitution assays: To directly test the effects of H1 on histone-modifying enzymes like PRC2 and NSD2 .

What methodological considerations are important when designing ChIP experiments with HIST1H3A antibodies?

When designing Chromatin Immunoprecipitation (ChIP) experiments with HIST1H3A antibodies, consider the following methodological aspects:

• Fixation conditions: Optimize crosslinking time and formaldehyde concentration to preserve protein-DNA interactions without overextending crosslinks.

• Sonication parameters: Adjust sonication conditions to generate DNA fragments of 200-500 bp for optimal resolution.

• Antibody selection: Choose antibodies that specifically recognize the C-terminal region of H3.1 to distinguish it from other H3 variants .

• Controls:

  • Include input DNA control (non-immunoprecipitated)

  • Use IgG control to account for non-specific binding

  • Consider using H3.1(C96S) mutant as a control for redox-related studies

• Validation: Verify ChIP efficiency using qPCR of known H3.1-enriched regions before proceeding to sequencing.

• Bioinformatic analysis: Apply appropriate normalization methods to account for differences in antibody efficiency and H3.1 abundance.

ChIP-qPCR experiments have demonstrated that after oxidative stress, wild-type H3.1-FLAG shows decreased association with promoter regions of key EMT genes (SOX9, fibronectin, ZEB1), whereas oxidation-resistant H3.1(C96S)-FLAG maintains stable promoter occupancy .

How can I optimize antigen retrieval for HIST1H3A antibody in IHC applications?

Antigen retrieval is critical for successful IHC staining with HIST1H3A antibodies. Based on experimental data:

• Primary recommendation: Use TE buffer at pH 9.0 for antigen retrieval .

• Alternative method: Citrate buffer at pH 6.0 may also be used if TE buffer doesn't provide optimal results .

• Protocol optimization:

  • Heat-induced epitope retrieval (HIER) is generally preferred over enzymatic methods

  • Optimize heating time (typically 10-20 minutes)

  • Ensure consistent temperature throughout the tissue section

  • Allow gradual cooling to room temperature after heating

• Tissue-specific considerations: Mouse testis tissue has been validated for positive IHC detection , but optimization may be required for other tissue types.

• Blocking optimization: Increase blocking time or blocking agent concentration if high background is observed.

Testing multiple antigen retrieval conditions in parallel using the same tissue sample can help identify optimal conditions for your specific experimental setup.

What factors contribute to variability in Western blot results with HIST1H3A antibodies?

Several factors can affect the reproducibility and quality of Western blot results when using HIST1H3A antibodies:

• Sample preparation:

  • Histone extraction methods (acid extraction vs. whole cell lysates)

  • Presence of protein phosphatases and deacetylase inhibitors

  • Storage conditions of samples

• Gel electrophoresis:

  • Gel percentage (15-18% gels recommended for histones)

  • Running conditions (voltage and time)

  • Transfer efficiency (wet transfer typically preferred for small proteins)

• Antibody factors:

  • Antibody dilution (recommended range: 1:5000-1:50000)

  • Incubation time and temperature

  • Secondary antibody selection and dilution

• Detection method:

  • Chemiluminescence vs. fluorescence detection

  • Exposure time optimization

• Histone modifications:

  • Post-translational modifications can affect antibody recognition

  • Oxidation state of Cys96 in H3.1 can influence antibody binding

To minimize variability, include positive controls from validated cell lines such as LNCaP, HEK-293, HeLa, Jurkat, HSC-T6, or NIH/3T3 , and maintain consistent sample preparation and experimental conditions across experiments.

How can I distinguish between different histone H3 variants in my experiments?

Distinguishing between highly similar histone H3 variants presents a significant challenge. Consider these methodological approaches:

• Variant-specific antibodies:

  • Use antibodies that target unique regions or residues specific to each variant

  • Validate antibody specificity using recombinant H3 variants or tagged constructs

• Mass spectrometry:

  • Use MS/MS to detect variant-specific peptides

  • Analyze post-translational modification patterns characteristic of each variant

• Genetic approaches:

  • Express tagged variants (FLAG, HA) to distinguish from endogenous proteins

  • Use site-directed mutagenesis to create variant-specific mutations (e.g., H3.1(C96S))

• ChIP-seq analysis:

  • Compare binding patterns of different H3 variants across the genome

  • Analyze enrichment at specific genomic features (promoters, enhancers, etc.)

• Biochemical properties:

  • Exploit differential sensitivity to oxidation (H3.1 is more sensitive than H3.2/H3.3)

  • Use DCP-Bio1 labeling to detect oxidized Cys96 in H3.1

Research has shown that H3.1 and H3.3 have distinct genomic distributions, with H3.3 being enriched at transcriptionally active regions and associated with chromatin decompaction .

How can HIST1H3A antibodies be used to study cancer progression and metastasis?

Recent studies have revealed that histone H3.1 oxidation and subsequent replacement by H3.3 plays a critical role in cancer progression . Researchers can leverage HIST1H3A antibodies to:

• Track EMT progression:

  • Monitor H3.1 occupancy at EMT gene promoters (SOX9, fibronectin, ZEB1) using ChIP-qPCR

  • Assess correlation between H3.1 displacement and EMT marker expression

• Study redox-sensitive chromatin changes:

  • Detect oxidized H3.1 using specific antibodies or chemical probes (DCP-Bio1)

  • Compare oxidation patterns between normal and cancer cells

• Investigate therapeutic vulnerabilities:

  • Identify cancer subtypes with altered H3.1/H3.3 ratios

  • Screen for compounds that target H3.1 oxidation or exchange mechanisms

  • Evaluate combination therapies targeting both histone dynamics and oncogenic signaling

• Monitor chromatin structure:

  • Analyze heterochromatin-to-euchromatin transitions during cancer progression

  • Correlate nuclear enlargement with H3.1 replacement by H3.3

Experimental data shows that H3.1 oxidation at Cys96 promotes its replacement by H3.3, leading to increased accessibility of EMT gene promoters and upregulation of EMT markers like SOX9 and ZEB1 .

What methodological approaches can detect the interaction between histone H3 mutations and PRC2 complex activity?

Histone H3 mutations, particularly H3K27M, affect the activity of the Polycomb Repressive Complex 2 (PRC2) . To study these interactions, researchers can employ:

• Co-immunoprecipitation (Co-IP):

  • Pull down PRC2 components (EZH2, SUZ12, EED) and detect association with mutant H3

  • Data shows enhanced co-purification of H3K27M with PRC2 compared to wild-type H3

• Binding affinity measurements:

  • Structural analysis of the interaction between EZH2's SET domain and H3K27M peptides

  • Studies demonstrate 16-fold higher affinity of EZH2 for H3K27M peptide compared to wild-type

• Enzyme kinetics assays:

  • Measure PRC2 methyltransferase activity in the presence of wild-type vs. mutant H3

  • Determine inhibitory mechanisms (competitive, non-competitive, uncompetitive)

• ChIP-seq analysis:

  • Map genome-wide distribution of H3K27me3 in cells expressing wild-type vs. mutant H3

  • Identify loci with altered PRC2 recruitment or activity

• Functional genomics:

  • Employ CRISPR screens to identify synthetic lethal interactions with H3 mutations

  • Test combination therapies targeting both the mutation and compensatory pathways

These approaches have revealed that H3K27M mutation exerts dominant effects by stabilizing PRC2 binding, sequestering the methyltransferase, and preventing further deposition of H3K27me, leading to a global decrease in H3K27me3 .

Recommended dilutions for HIST1H3A antibody applications

Data sourced from product information sheet

Validated cell lines and tissues for HIST1H3A antibody testing

Data sourced from product validation information

Comparison of histone H3 variant properties

Table compiled from multiple research sources