H3F3A (Ab-79) Antibody

What is H3F3A and why is it significant in cancer research?

H3F3A is a gene that encodes the histone variant H3.3, which plays crucial roles in chromatin dynamics and gene regulation. Its significance in cancer research stems from its demonstrated role in cancer progression, particularly in lung cancer. Studies have shown that H3F3A overexpression promotes lung cancer cell migration by activating metastasis-related genes . H3.3 functions as a transcriptional activator by occupying intronic regions that exhibit characteristics of regulatory DNA elements, thus modifying the chromatin landscape to influence gene expression . This makes H3F3A an important target for both basic cancer biology research and potential therapeutic development.

What specific epitope does the H3F3A (Ab-79) Antibody recognize?

The H3F3A (Ab-79) Antibody specifically recognizes the region around the Lysine 79 (K79) residue in human Histone H3.3. The immunogen used for developing this antibody consists of a peptide sequence derived from the region surrounding this lysine residue . This specificity is important because post-translational modifications at various histone residues can significantly alter chromatin structure and gene expression, making site-specific antibodies valuable tools for epigenetic research.

What experimental applications has the H3F3A (Ab-79) Antibody been validated for?

The H3F3A (Ab-79) Antibody has been validated for several experimental applications:

• Western Blot (WB): Recommended dilution range of 1:200-1:2000

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Chromatin Immunoprecipitation (ChIP)

This versatility makes it suitable for multiple research approaches, from protein expression analysis to investigations of DNA-protein interactions in chromatin contexts.

What are the optimal storage and handling conditions for preserving antibody activity?

For optimal preservation of antibody activity, the H3F3A (Ab-79) Antibody should be stored at -20°C or -80°C upon receipt. Researchers should avoid repeated freeze-thaw cycles as this can degrade antibody quality . The antibody is supplied in liquid form with a storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . These components help maintain antibody stability during storage. When working with the antibody, it's advisable to aliquot it to minimize freeze-thaw cycles and keep it on ice during experiments.

What positive controls are recommended for validating experimental results?

Based on validated Western blot data, multiple positive controls can be used when working with the H3F3A (Ab-79) Antibody:

• Human cell lines: HeLa, 293 (HEK293), HepG2, and K562 whole cell lysates

• Rat tissues: Brain and spleen tissue lysates

These positive controls have shown clear bands at the expected molecular weight of 16 kDa, which matches the predicted size of histone H3.3 . Including these controls in experiments helps validate antibody performance and ensures experimental reliability.

How should chromatin immunoprecipitation (ChIP) experiments be designed when using this antibody?

When designing ChIP experiments with the H3F3A (Ab-79) Antibody, researchers should follow protocols similar to those validated in previous studies. An effective approach includes:

• Cell preparation: Use approximately 4×10^6 cells (as demonstrated with HeLa cells in validation studies)

• Chromatin fragmentation: Treat with Micrococcal Nuclease followed by sonication to generate appropriately sized DNA fragments

• Immunoprecipitation: Use 5μg of the H3F3A antibody per ChIP reaction

• Controls: Include a normal rabbit IgG as a negative control

• Analysis: Quantify the resulting ChIP DNA using real-time PCR with appropriate primers

This methodology has been successfully employed to detect H3F3A binding at specific genomic regions, particularly for investigating its role in gene regulation.

How can the H3F3A (Ab-79) Antibody be used to investigate the role of H3.3 in cancer progression?

The H3F3A (Ab-79) Antibody can be instrumental in investigating H3.3's role in cancer progression through several advanced applications:

• ChIP followed by sequencing (ChIP-seq) to map genome-wide H3.3 occupancy in cancer cells, particularly at intronic regions where H3.3 has been shown to function as a transcriptional activator

• Combined ChIP with expression analysis to correlate H3.3 binding with changes in gene expression patterns in cancer models

• Immunofluorescence imaging to analyze subcellular localization and expression levels of H3.3 in different cancer cell lines or patient samples

• Western blot analysis to compare H3.3 expression levels between non-metastatic and metastatic cancer cell lines

Research has demonstrated that H3.3 specifically activates metastasis-related genes through its deposition at intronic regions. For example, H3.3 binds to a specific intronic region of GPR87, modifying the chromatin status and directly activating GPR87 transcription, which contributes to cancer progression .

What genomic regions does H3.3 predominantly occupy, and how can this be studied?

H3.3 predominantly occupies intronic (approximately 39%) and intergenic (approximately 56%) regions based on ChIP-seq analysis in cancer cells . This distribution differs significantly from canonical histones and suggests specialized regulatory functions.

To study these occupancy patterns:

• Perform ChIP-seq using the H3F3A (Ab-79) Antibody in relevant cell lines

• Analyze peak distribution with attention to intronic regions of metastasis-related genes

• Correlate H3.3 binding with chromatin accessibility markers

• Compare occupancy patterns between normal and cancer cells to identify cancer-specific binding sites

Research has shown that H3.3 binding regions often show characteristics of regulatory DNA elements, suggesting their importance in transcriptional control . This is particularly relevant when investigating how H3.3 regulates genes involved in cancer progression.

How should Western blot data be interpreted when using the H3F3A (Ab-79) Antibody?

When interpreting Western blot data using the H3F3A (Ab-79) Antibody:

• Expected band size: The predicted molecular weight of histone H3.3 is 16 kDa, which matches the observed band size in validated Western blots

• Specificity considerations: Since H3.3 shares high sequence homology with canonical H3 histones, it's important to verify that the antibody specifically recognizes the H3F3A-encoded variant. The specificity for the region around Lysine 79 helps ensure this distinction

• Expression correlation: When studying cancer samples, correlate H3.3 expression levels with phenotypic data, as higher H3F3A expression has been linked to cancer progression and poorer prognosis in lung cancer patients

• Loading controls: Use appropriate histone loading controls to normalize expression levels across samples

If unexpected bands appear, consider cross-reactivity with other histone variants or post-translational modifications that might alter antibody recognition or protein migration patterns.

What are common pitfalls in ChIP experiments with histone variant antibodies and how can they be avoided?

Common pitfalls in ChIP experiments with histone variant antibodies include:

• Cross-reactivity with canonical histones: Due to sequence similarity between H3.3 and canonical H3, validate antibody specificity using peptide competition assays

• Chromatin preparation issues: Insufficient fragmentation can lead to high background signal. Optimize Micrococcal Nuclease treatment and sonication conditions for each cell type

• Antibody concentration: Using too little antibody may result in poor enrichment, while excess antibody can increase non-specific binding. The validated protocol suggests 5μg per ChIP reaction for optimal results

• Appropriate controls: Always include a normal IgG control to account for non-specific binding, and consider including known positive and negative genomic regions for validation

• Quantification methods: For ChIP-qPCR, design primers for both expected binding sites and control regions to properly assess enrichment

How does H3F3A expression correlate with lung cancer prognosis?

H3F3A expression has been established as a significant prognostic marker for lung cancer patients, particularly those with early-stage disease. Analysis of public datasets (GSE13213 and GSE31210) revealed that:

• H3F3A expression was significantly higher in relapsed patient groups compared to non-relapsed patients:

  • All patients: P=1.1×10^-2 (GSE13213) and P=8.5×10^-7 (GSE31210)

  • Stage I patients: P=8.5×10^-3 (GSE13213) and P=2.0×10^-6 (GSE31210)

• Multivariate Cox proportional hazard analysis showed that H3F3A expression level was a better predictor of poor survival than other markers, including histological staging:

Table: Multivariate analysis of H3F3A expression and other markers in lung cancer patients (GSE13213 dataset)

These findings indicate that H3F3A overexpression strongly correlates with poorer clinical outcomes and could serve as a valuable prognostic biomarker, particularly for early-stage lung adenocarcinoma.

What molecular mechanisms link H3F3A overexpression to increased cancer cell invasion?

H3F3A overexpression promotes cancer cell invasion through several molecular mechanisms:

• Activation of invasion-related genes: H3F3A overexpression significantly increases the expression of metastasis-related genes, including MMP9, a well-established invasion effector (P=1.7×10^-3 for A549 and P=9.7×10^-5 for NCI-H23 cells)

• Regulation of collagen metabolic processes: Gene Ontology analysis of H3F3A-regulated genes identified "collagen metabolic process" as the top-ranked term among positively regulated genes, which includes several metastasis-related effectors like matrix metalloproteases

• Epithelial-Mesenchymal Transition (EMT): Gene Set Enrichment Analysis showed that genes positively regulated by H3F3A were significantly associated with "Epithelial Mesenchymal Transition" and "Metallopeptidase Activity" gene sets, which are closely involved in cancer metastasis

• Direct transcriptional activation: H3.3 directly activates specific target genes like GPR87 through deposition at intronic regions, where it modifies chromatin status to enhance transcription

Experimental validation demonstrated that H3F3A overexpression significantly increased lung cancer cell invasion (P=2.0×10^-5 for A549 and P=7.7×10^-6 for NCI-H23), while H3F3A knockdown significantly decreased invasion (P=1.0×10^-5 for A549 and P=1.7×10^-8 for NCI-H23) . Importantly, overexpression of canonical histone H3 genes (H3.1 and H3.2) did not significantly affect cancer cell invasion, highlighting the specific role of H3.3 in promoting this phenotype.

How might H3F3A-targeted therapies be developed based on current research findings?

Based on current understanding of H3F3A's role in cancer progression, several therapeutic approaches could be explored:

• GPR87 antagonists: Research has identified GPR87 as a direct downstream target of H3F3A, suggesting GPR87 antagonists could be effective in treating H3F3A-overexpressing cancers

• Epigenetic modulators: Since H3.3 functions by modifying chromatin status at specific genomic regions, drugs targeting the enzymes that regulate H3.3 deposition or its associated modifications could potentially disrupt its oncogenic functions

• Gene expression-based approaches: H3F3A and GPR87 expression levels, either alone or in combination, could serve as biomarkers for patient stratification in clinical trials, particularly for early-stage lung cancer patients

• Intronic regulation targeting: The finding that H3.3 functions primarily through binding to intronic regions suggests that disrupting this specific regulatory mechanism could represent a novel therapeutic strategy with potentially fewer off-target effects compared to general epigenetic inhibitors

Future research using the H3F3A (Ab-79) Antibody will be instrumental in validating these potential therapeutic targets and developing effective interventions for cancers characterized by H3F3A overexpression.