Tri-methyl-Histone H3(K9) Monoclonal Antibody

What is H3K9me3 and what is its biological significance?

H3K9me3 is a post-translational modification of histone H3 where the lysine at position 9 is trimethylated. This modification is primarily characterized as a repressive mark, serving as a hallmark of transcriptionally repressed chromatin and heterochromatin formation . It plays essential roles in:

• Ensuring transcriptional silencing at transposable elements, satellite repeats, and specific genes

• Maintaining genome stability

• Establishing constitutive heterochromatin

• Regulating cell identity through tissue-specific gene repression

H3K9me3 is deposited by specific histone methyltransferases (HMTs) including SUV39H1, SUV39H2, SETDB1, SETDB2, G9A, and GLP in mammals, which show considerable functional redundancy despite their distinct roles .

How do I select the appropriate H3K9me3 antibody for my experiments?

When selecting an H3K9me3 antibody, consider these critical factors:

For chromatin immunoprecipitation (ChIP), antibodies like EPR16601 and clone RM389 have been specifically validated and produce consistent results .

What are the optimal conditions for H3K9me3 ChIP experiments?

For successful H3K9me3 ChIP experiments:

• Chromatin preparation: Use fresh or properly stored samples with crosslinking typically performed with 1% formaldehyde for 10 minutes at room temperature.

• Antibody amount: For optimal results, use 5 μl of antibody and approximately 10 μg of chromatin (approximately 4 × 10^6 cells) per immunoprecipitation .

• Antibody incubation: Most protocols recommend incubating the primary antibody for 1 hour at room temperature in the dark in staining chambers, followed by washing cells 2x with 1x PBS for 4 minutes per wash on an orbital shaker .

• Controls: Include:

  • Input chromatin (10% of starting material)

  • Negative control with non-specific IgG

  • Positive control targeting a different histone mark (e.g., H3K4me3 for active chromatin)

• Validation: Validate ChIP efficiency with qPCR targeting known H3K9me3-enriched regions before proceeding to genome-wide analyses .

How can I verify the specificity of H3K9me3 antibodies?

To confirm antibody specificity:

• Peptide competition assays: Pre-incubate the antibody with H3K9me3 peptides to block specific binding. This should eliminate signal in your detection method .

• Cross-reactivity testing: Test against related modifications (H3K9me1, H3K9me2) to ensure specificity. For example, Western Blot analysis with recombinant histone H3.3 and acid extracts of cells should show specific bands for H3K9me3 .

• ELISA with designer nucleosomes: Use recombinant nucleosomes with defined modifications. The RevMAb RM389 antibody was validated using Designer Nucleosomes with H3 K4, 9, 27, 36 and H4 K20 in mono-, di-, and tri-methylated states .

• Multiple antibody comparison: Compare results from different H3K9me3 antibodies (e.g., from different suppliers or clones) to confirm consistent findings .

How do I interpret unexpected H3K9me3 enrichment at transcriptionally active regions?

While H3K9me3 is typically associated with repressed chromatin, several studies have reported its presence at transcriptionally active regions, creating apparent contradictions:

• Co-occurrence with active marks: H3K9me3 can co-occur with active histone marks like H3 acetyl K9 on the same or neighboring nucleosomes, particularly at switch regions in B cells undergoing class switch recombination .

• Sequential ChIP analysis: To determine if H3K9me3 and active marks (like H3K9ac) are present on the same DNA molecules, perform sequential ChIP (ChIP-reChIP). Studies have shown that some DNA fragments can be associated with both H3K9me3 and H3K9ac .

• Context-dependent functions: H3K9me3 may have different functions depending on genomic context and cell type. For example:

  • In B cells, H3K9me3 is associated with donor and recipient switch regions that undergo class switch recombination, contrasting with its typically repressive role

  • At certain loci, H3K9me3 may help maintain the boundaries between active and inactive chromatin regions

• Mutation analysis: DNA regions associated with H3K9me3 can still be targets for mutation by enzymes like AID, indicating accessibility despite the presence of this typically repressive mark .

What is the relationship between H3K9me3 and dominant-negative histone H3 K-to-M mutations?

H3K9 to methionine (H3K9M) mutations provide valuable insights into H3K9me3 function:

• Inhibitory mechanism: H3K9M acts as an orthosteric inhibitor of H3K9 methyltransferases like EHMT2 .

• Structural basis: The methionine side chain in H3K9M creates increased van der Waals forces compared to wild-type H3, resulting in higher affinity binding to methyltransferases .

• Sequestration model: H3K9M appears to sequester methyltransferases, preventing them from modifying wild-type H3. In yeast, H3K9M causes accumulation of the H3K9 methyltransferase Clr4 at heterochromatin nucleation centers .

• Heterochromatin spreading inhibition: Expressing H3K9M in yeast prevents the spread of H3K9me3 to regions surrounding heterochromatin nucleation centers, suggesting a complex mechanism beyond simple sequestration .

• Research applications: K-to-M mutations serve as valuable tools for studying histone methylation functions during development and homeostasis .

How do different histone modifications at H3K9 interact and what techniques can distinguish them?

The lysine 9 residue of histone H3 can undergo various modifications with distinct functions:

To distinguish these modifications:

• Western blot with specific antibodies: Verify modification-specific bands at approximately 17 kDa .

• ChIP-qPCR: Target known genomic regions enriched for specific modifications.

• Mass spectrometry: For unbiased quantification of different H3K9 modifications.

• Sequential ChIP: To determine co-occurrence of modifications on the same DNA molecules .

What is the temporal dynamic of H3K9me3 establishment during cellular differentiation and response to stimuli?

H3K9me3 establishment follows specific temporal patterns:

• During B cell activation: Time course ChIP experiments have shown that H3K9me3 at recipient switch regions becomes established between 24-48 hours after stimulation with appropriate cytokines and persists through 96 hours .

• Cell differentiation: H3K9 methyltransferases have both unique and redundant roles during tissue differentiation:

  • In haematopoiesis, muscle differentiation, and neurogenesis, H3K9me3 helps maintain tissue integrity by restricting transcription factor binding to lineage-specific promoters and enhancers

  • The timing of H3K9me3 establishment is critical for proper cell differentiation and identity maintenance

• Response to environmental stimuli: In models of chronic intermittent ethanol (CIE) exposure, changes in H3K9 modifications occur at specific gene promoters (e.g., NR2B gene), with decreased H3K9me3 observed alongside increased H3K9ac .

What are the cutting-edge applications of H3K9me3 antibodies in epigenetic research?

Recent innovations in H3K9me3 research include:

• ChIP-seq with low cell numbers: Optimized protocols allowing H3K9me3 profiling from limited biological samples.

• CUT&RUN and CUT&Tag: These techniques provide higher resolution mapping of H3K9me3 with lower background compared to traditional ChIP-seq.

• Single-cell epigenomics: Techniques like single-cell CUT&Tag allow mapping of H3K9me3 distribution across individual cells to study heterogeneity.

• Genomic engineering approaches: Using CRISPR-based systems to manipulate H3K9me3 at specific genomic loci.

• H3K9me3 reader domain tools: Engineered proteins containing H3K9me3-binding domains to target specific functions to H3K9me3-marked regions.

How can I differentiate between genuine H3K9me3 signal and antibody artifacts in my experiments?

To minimize artifacts and validate H3K9me3 signals:

• Multiple antibody approach: Use at least two different monoclonal antibodies (e.g., RM389 and EPR16601) targeting the same modification but with different epitopes .

• Spike-in controls: Add exogenous chromatin from a different species as an internal control for ChIP efficiency.

• Knockdown validation: Reduce the expression of H3K9 methyltransferases (SUV39H1/2, SETDB1) and verify the corresponding reduction in H3K9me3 signal.

• Domain structure analysis: Genuine H3K9me3 signals typically occur in broad domains rather than sharp peaks. Unusual peak patterns may indicate artifacts.

• Correlation with known H3K9me3 features: Validate by checking enrichment at expected regions like pericentromeric heterochromatin, telomeres, and repressed transposable elements.

What is the prognostic significance of H3K9me3 in disease states?

H3K9me3 has been implicated in various diseases:

• Cancer prognosis: H3K9me3 serves as a prognostic marker in some cancers. Altered levels correlate with clinical parameters:

  • H3K9ac shows a significant inverse correlation with tumor T-status (Rho = -0.149, p = 0.019)

  • Patients with FIGO I status had higher H3K9ac expression (median IRS of 8) compared to FIGO II or higher (median IRS of 4), with a negative Spearman's-rank correlation (Rho = -0.192, p = 0.016)

• Neurodegenerative diseases: Dysregulation of H3K9me3 has been associated with neurodegeneration through its effects on gene expression and genome stability.

• Aging: Loss of proper H3K9me3 distribution contributes to age-related cellular changes and genomic instability.

• Immunological disorders: Given its role in class switch recombination in B cells, H3K9me3 alterations may impact antibody production and immune function .

Understanding these connections provides potential therapeutic targets for epigenetic-based interventions.

What are common problems with H3K9me3 antibodies and how can they be resolved?