HIST1H3A (Ab-9) Antibody

What is HIST1H3A (Ab-9) Antibody and what epigenetic mark does it specifically recognize?

The HIST1H3A (Ab-9) Antibody is a rabbit polyclonal antibody designed to recognize the histone H3 protein specifically at lysine 9 (K9) . This antibody targets a peptide sequence around lysine 9 of human Histone H3.1 . Histone H3 lysine 9 is a critical residue that can undergo methylation at different levels (mono-, di-, and tri-methylation), each associated with distinct biological functions . These modifications play essential roles in heterochromatin formation, gene silencing, and transcriptional repression .

Through validation studies, this antibody has been shown to specifically recognize its target epitope with minimal cross-reactivity. Immunoblot studies have demonstrated that the antibody reacts with H3 purified from wild-type yeast but not from isogenic strains where lysine 37 is replaced by alanine (H3K37A) .

What are the validated applications for the HIST1H3A (Ab-9) Antibody?

The HIST1H3A (Ab-9) Antibody has been validated for multiple experimental applications:

The antibody has been verified in Western blotting to detect a band of approximately 16 kDa, which corresponds to the predicted molecular weight of histone H3.1 . In ChIP applications, it has been used successfully to investigate chromatin modifications and protein-DNA interactions in various cellular contexts .

How should samples be prepared for optimal results with this antibody?

For optimal results with the HIST1H3A (Ab-9) Antibody, sample preparation depends on the specific application:

For Western Blotting:

• Use acid extraction methods for isolating histones from cell lysates. This is critical as histones are tightly bound to DNA .

• Examples from published studies show successful detection in HEK293, A375, HepG2 cell acid extracts, as well as in mouse tissue lysates (liver, brain, kidney) .

• Use histone acid extracts (0.2M H₂SO₄ or similar) followed by TCA precipitation for optimal enrichment of histones.

For ChIP Applications:

• Crosslink cells with 1% formaldehyde for 10 minutes at room temperature.

• Sonicate chromatin to fragments of 200-500 bp.

• Pre-clear lysates with protein A/G beads before immunoprecipitation.

• Include appropriate controls (IgG, input samples) .

For Immunohistochemistry/Immunofluorescence:

• For paraffin-embedded tissues, use antigen retrieval methods (typically heat-induced epitope retrieval in citrate buffer pH 6.0).

• For cell lines, fixation with 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100 has shown good results .

What species reactivity has been confirmed for the HIST1H3A (Ab-9) Antibody?

The HIST1H3A (Ab-9) Antibody has been confirmed to react with:

• Human samples (primary reactivity)

• Mouse samples (validated in multiple tissues)

• Rat samples (in some product variants)

The broad cross-reactivity stems from the high conservation of histone H3 sequences across species. Published studies have demonstrated that this antibody detects H3K9 methylation in a wide variety of mammalian cell lines . Researchers should note that while the antibody has been validated primarily for human samples, the high conservation of histone proteins makes it suitable for use in model organisms.

What are the storage and handling recommendations for maintaining antibody activity?

For optimal performance and longevity of the HIST1H3A (Ab-9) Antibody:

• Store at -20°C or -80°C for long-term storage .

• Avoid repeated freeze/thaw cycles by preparing small aliquots before freezing .

• The antibody is typically provided in a buffer containing 50% glycerol with preservatives such as 0.03% Proclin 300 in PBS (pH 7.4) .

• Working dilutions should be prepared fresh before use.

• The antibody remains stable for at least 12 months from the date of receipt when stored properly .

• For daily use, antibody aliquots can be kept at 4°C for up to two weeks .

How does H3K9 methylation status change during cell cycle progression and differentiation?

H3K9 methylation undergoes dynamic changes during various cellular processes:

Cell Cycle Regulation:

• H3K9 methylation patterns show specific temporal dynamics during the cell cycle, with changes in methylation status correlating with RNA polymerase II recruitment and release .

• Studies have shown relatively low constitutive levels of H3K9 methylation that are erased upon gene activation and restored during post-induction transcriptional repression .

• The remethylation of H3K9 strongly correlates with RNA polymerase II release from chromatin, suggesting a role in transcriptional termination .

Differentiation Processes:

• During cellular differentiation, changes in H3K9 methylation help establish cell-type-specific gene expression patterns.

• Research indicates that H3K9 methylation can generate a time window during which transcription is permitted, adding an additional regulatory level to transcriptional activation of tightly controlled inducible genes .

• The dynamics of H3K9 methylation can vary between different cell types. For example, in dendritic cells (DCs), certain inflammatory genes show demethylation of H3K9 upon activation, while monocytes do not exhibit this response despite similar basal levels of H3K9 methylation .

What experimental controls are essential when using this antibody in ChIP experiments?

When performing ChIP experiments with the HIST1H3A (Ab-9) Antibody, several critical controls should be included:

Negative Controls:

• IgG Control: Include a ChIP reaction with non-specific IgG from the same species as the HIST1H3A antibody to determine background signal.

• Peptide Competition: Pre-incubate the antibody with excess peptide containing the target epitope to validate specificity.

• Genetic Controls: When possible, use cell lines with mutations or deletions in the target histone residue (e.g., H3K9A mutants) as negative controls .

Positive Controls:

• Input DNA: Include a sample of chromatin before immunoprecipitation (typically 5-10% of starting material).

• Known Target Regions: Amplify regions known to be enriched for H3K9 methylation, such as heterochromatic regions or silenced genes .

• Sequential ChIP: For advanced validation, perform sequential ChIP with another antibody targeting a mark known to co-occur with H3K9 methylation.

Quantification Controls:

• Serial dilutions of input can be used to create a standard curve for quantitative assessment of H3K9 methylation density at specific genomic loci .

• Comparison with heterochromatic regions like the Xist gene can provide relative quantification of methylation levels .

How can researchers validate the specificity of this antibody in their experimental systems?

Validating antibody specificity is crucial for reliable interpretation of results. For the HIST1H3A (Ab-9) Antibody, consider these validation approaches:

Biochemical Validation:

• Peptide Competition Assays: Pre-incubate the antibody with excess peptide containing the H3K9 epitope before using it in your assay. A specific antibody will show reduced or no signal.

• Western Blot Analysis: Compare the signal from wild-type cells with that from cells where the target modification is reduced (e.g., through treatment with methyltransferase inhibitors or genetic knockdown of relevant enzymes like SetDB1, G9a, or Suv39H1) .

Genetic Validation:

• Use cells with mutations in H3K9 (if available) or knockdowns of enzymes responsible for H3K9 methylation (SetDB1, G9a, Suv39H1) .

• Compare results from HIST1H3A (Ab-9) Antibody with those from other validated H3K9 antibodies targeting the same modification.

Functional Validation:

• Correlate antibody signals with functional states known to be associated with H3K9 methylation, such as heterochromatin regions or transcriptionally repressed genes .

• For ChIP experiments, verify enrichment at known heterochromatic regions (e.g., satellite repeats, telomeric and centromeric regions) .

What methodological considerations are important when studying the relationship between H3K9 methylation and heterochromatin formation?

When investigating H3K9 methylation in heterochromatin formation, consider these methodological aspects:

Distinguishing Different Methylation States:

• H3K9 can be mono-, di-, or tri-methylated, each with distinct functional implications. Ensure your antibody specifically recognizes the methylation state of interest .

• H3K9me1/me2 are typically associated with euchromatic gene regulation, while H3K9me3 is enriched at pericentric heterochromatin .

Spatial and Temporal Resolution:

• Consider using techniques that provide high spatial resolution, such as ChIP-seq, to map the genome-wide distribution of H3K9 methylation .

• For temporal dynamics, synchronized cell populations or time-course experiments may be necessary to capture changes in methylation patterns during processes like replication or transcriptional activation .

Protein Interactions:

• Investigate interactions with known heterochromatin components such as HP1 proteins, which specifically bind to methylated H3K9 .

• Co-immunoprecipitation experiments can reveal interactions between H3K9 methyltransferases (Suv39H1, G9a) and other chromatin-associated factors .

Cellular Context:

• Different cell types may show distinct patterns of H3K9 methylation. Consider using multiple cell types or tissues in your analysis .

• Replication timing analysis can provide insights into the relationship between H3K9 methylation and late-replicating heterochromatic domains .

How does H3K9 methylation interact with other histone modifications and what are the best approaches to study these interactions?

H3K9 methylation exists within a complex network of histone modifications. To study these interactions:

Co-occurrence Analysis:

• Sequential ChIP (Re-ChIP) can determine if different modifications co-occur on the same nucleosomes.

• ChIP-seq for multiple histone marks followed by correlation analysis can reveal genome-wide patterns of co-occurrence or mutual exclusivity.

Functional Interactions:

• H3K9 methylation has been shown to inhibit histone acetylation by p300, suggesting a direct functional antagonism between these modifications .

• Studies have shown that H3K9 methylation correlates inversely with H3K4 methylation across large chromosomal regions .

Enzymatic Crosstalk:

• Investigate how enzymes responsible for different modifications interact. For example, Suv39h HMTases require mono-methylated H3K9 (created by other enzymes like SetDB1) as a preferred substrate .

• Knockdown studies of specific enzymes can reveal hierarchical relationships between modifications.

Technological Approaches:

• Mass spectrometry-based approaches (like Mod Spec) can identify combinations of modifications on the same histone tail .

• Genome editing of specific lysine residues can help determine the interdependence of different modifications.

• Single-molecule techniques can provide insights into the dynamics and stoichiometry of modification complexes .

How can researchers interpret discrepancies between HIST1H3A (Ab-9) antibody signals and other histone modification markers?

When encountering discrepancies between H3K9 methylation signals and other histone marks, consider these analytical approaches:

Technical Considerations:

• Antibody Cross-Reactivity: HIST1H3A (Ab-9) Antibody specificity should be extensively validated. Some antibodies may recognize multiple methylation states or be affected by neighboring modifications .

• Epitope Masking: Certain protein interactions or adjacent modifications may mask the epitope recognized by the antibody, leading to false-negative results.

• Fixation Artifacts: Different fixation methods can affect epitope availability, especially in immunostaining applications.

Biological Interpretations:

• Heterogeneity within Cell Populations: Discrepancies may reflect cellular heterogeneity, with subpopulations displaying different modification patterns.

• Dynamic Regulation: Temporal differences in the establishment of various modifications may lead to apparent discrepancies in steady-state analyses .

• Nucleosome-Specific Patterns: H3K9 methylation might occur selectively on specific nucleosomes within a region, creating a mosaic pattern that differs from other modifications .

Resolution Strategies:

• Single-Cell Approaches: Techniques like single-cell ChIP-seq or mass cytometry can resolve population heterogeneity.

• Time-Course Experiments: Analyzing modifications at multiple time points can reveal sequential establishment of different marks.

• Quantitative Analysis: Compare the relative densities of different modifications rather than simple presence/absence .

• Alternative Techniques: Validate findings using orthogonal methods such as mass spectrometry-based histone analysis.

What are the latest findings regarding the role of H3K9 methylation in co-translational and post-translational histone modification pathways?

Recent research has revealed intricate mechanisms of H3K9 methylation establishment:

Co-translational Methylation:

• Groundbreaking studies have demonstrated that H3K9 is mono- and dimethylated while histone H3 is still bound to the ribosome, indicating that this modification occurs co-translationally .

• The methyltransferase SetDB1 has been identified as the enzyme that associates with ribosomes and catalyzes H3K9me1 and H3K9me2 during translation .

• This co-translational modification represents a previously unrecognized mechanism for establishing epigenetic marks before chromatin assembly.

Modification Pathways:

• SetDB1 exists in two distinct complexes: one associated with ribosomes for co-translational methylation and another nuclear complex with CAF-1 and HP1α for post-translational modification .

• This dual-localization system ensures sufficient H3K9 methylation for proper heterochromatin establishment and maintenance .

• The co-translational methylation may serve as a priming modification that marks histones for subsequent incorporation into specific chromatin domains.

Functional Implications:

• The early establishment of H3K9 methylation during histone synthesis may be critical for maintaining epigenetic memory through cell division.

• This mechanism provides insights into how cells ensure that a sufficient population of histones features H3K9me1, priming them for further modifications that enable heterochromatin formation .

• Understanding these pathways opens new therapeutic avenues for targeting SetDB1 in cancers with perturbed heterochromatin regions .

How does the density of H3K9 methylation differ between euchromatic and heterochromatic regions, and what methodologies best capture these differences?

The density and distribution of H3K9 methylation varies significantly across the genome:

Density Comparisons:

• Research has estimated that H3K9 methylation at inflammatory genes in euchromatin is approximately eight-fold lower than at heterochromatic regions like the Xist gene .

• This density difference may result from either selective modification of specific nucleosomes or hemi-methylation of several nucleosomes in euchromatic regions .

• In heterochromatin, particularly at pericentric regions, H3K9me3 forms a more uniform and dense pattern established by Suv39h HMTases .

Methodological Considerations:

• ChIP-seq with spike-in controls provides a quantitative measure of methylation density across different genomic regions.

• Sequential ChIP can determine whether multiple H3K9 methylation marks co-occur on the same nucleosome.

• High-affinity monoclonal antibodies may be required for precise quantification of methylation density differences .

• Single-molecule techniques can reveal the stoichiometry of modified nucleosomes within specific regions.

Analytical Challenges:

• The antibody's affinity can have non-linear effects on immunoprecipitation efficiency when detecting differences in modification density .

• Comparing relative enrichment across genomic regions requires normalization to account for differences in chromatin accessibility and nucleosome density.

• Integration with other data types (DNA methylation, chromatin accessibility) provides a more comprehensive view of heterochromatin structure.

What role does ORCA (Origin Recognition Complex-Associated) protein play in organizing H3K9 methylation, and how can researchers study this interaction?

ORCA represents a critical link between DNA replication and heterochromatin organization:

ORCA Function in Heterochromatin:

• ORCA preferentially localizes to heterochromatic regions in post-replicated cells and plays a key role in heterochromatin organization .

• It recognizes methylated H3K9 marks and interacts with repressive KMTs, including G9a/GLP and Suv39H1, in a chromatin context-dependent manner .

• Single-molecule pull-down assays have demonstrated that ORCA-ORC (Origin Recognition Complex) and multiple H3K9 KMTs exist in a single complex, with ORCA stabilizing the H3K9 KMT complex .

Experimental Approaches:

• Cells lacking ORCA show alterations in chromatin architecture, with significantly reduced H3K9 di- and tri-methylation at specific chromatin sites .

• ORCA depletion affects replication timing, preferentially at late-replicating regions, suggesting a connection between heterochromatin organization and DNA replication timing .

• ChIP-seq analysis following ORCA knockdown has revealed that approximately 18% of H3K9me3 peaks show highly significant decreases, particularly at satellite repeats and telomeric/centromeric regions .

Study Methods:

• Co-immunoprecipitation experiments can identify interactions between ORCA and various H3K9 methyltransferases.

• Peptide pull-down assays can quantify ORCA's binding affinity for different methylated H3K9 states .

• Single-molecule pull-down (SiMPull) techniques provide insights into the composition and stoichiometry of ORCA-containing complexes .

• ChIP-seq combined with replication timing analysis can reveal the functional consequences of disrupting ORCA-mediated heterochromatin organization .

What are the emerging therapeutic implications of targeting H3K9 methylation in diseases with heterochromatin dysregulation?

The critical role of H3K9 methylation in genome stability has significant therapeutic implications:

Disease Associations:

• Disruptions in H3K9 methylation patterns have been linked to various diseases, including cancer, where heterochromatin structure is frequently altered .

• Changes in the activity of H3K9 methyltransferases (SetDB1, G9a, Suv39H1/H2) have been observed in multiple pathological conditions.

• Heterochromatin instability due to altered H3K9 methylation can lead to genomic instability, a hallmark of cancer .

Therapeutic Strategies:

• Inhibitors targeting H3K9 methyltransferases represent a promising approach for diseases with heterochromatin perturbations .

• SetDB1, which catalyzes co-translational H3K9 methylation, represents a particularly interesting target given its role in priming histones for heterochromatin formation .

• Combination approaches targeting both the establishment and maintenance of H3K9 methylation might provide synergistic effects.

Research Approaches:

• Chemical probe development for specific H3K9 methyltransferases enables target validation in cellular and animal models.

• CRISPR-based screens can identify synthetic lethal interactions with H3K9 methylation machinery in different disease contexts.

• Patient-derived models can help identify biomarkers predictive of response to epigenetic therapies targeting H3K9 methylation.

• Functional genomics approaches can determine which genes are most affected by alterations in H3K9 methylation in specific disease states.