Di-Methyl-Histone H3 (Lys4) Monoclonal Antibody

What is the biological significance of H3K4 dimethylation?

H3K4 dimethylation is an epigenetic modification associated with transcriptional activation. Methylation events that weaken the binding between histone tails and DNA lead to increased transcription by making DNA more accessible to transcription factor proteins and RNA polymerase . H3K4me2 levels are highest in the 5'-end of transcriptionally active genes . When examining the genome-wide distribution of histone modifications, H3K4me2 shows a distinct pattern from mono-methylation (H3K4me1), which is enriched at enhancer elements, and tri-methylation (H3K4me3), which is concentrated at active promoters .

How does H3K4 methylation status relate to other histone modifications?

H3K4 methylation works in concert with other histone modifications to regulate chromatin structure and gene expression. Research has demonstrated an inverse relationship between H3K4 methylation and histone deacetylation. Genomic regions with high H3K4 methylation show almost no overlap with regions deacetylated by histone deacetylase (HDAC) enzymes like Rpd3 and Hda1 . Additionally, H3K4 methylation can stimulate histone acetylation through recruitment of histone acetyltransferase complexes like NuA4 and SAGA . This interplay creates a dynamic equilibrium that maintains appropriate levels of histone acetylation in transcribed coding sequences.

What enzymes regulate H3K4 methylation status?

H3K4 methylation is dynamically controlled by "writer" lysine methyltransferases (KMTs) that deposit methyl groups and "eraser" lysine demethylases (KDMs) that remove them . In yeast, the Set1 protein is responsible for all detectable H3K4 methylation . The enzymatic regulation of H3K4 methylation is highly conserved from yeast to humans, with multiple KMTs and KDMs showing specificity for different methylation states (mono-, di-, or tri-methylation) . This dynamic regulation allows for rapid changes in chromatin structure in response to cellular signals.

How do I select the appropriate H3K4me2 antibody for my specific application?

When selecting an H3K4me2 antibody, consider these key factors:

• Specificity: Choose antibodies validated to distinguish between different methylation states of H3K4 (mono-, di-, or tri-methylation) and that don't cross-react with other histone modifications. For example, the C64G9 Rabbit mAb shows weak cross-reactivity with H3K4me1 but does not cross-react with non-methylated or tri-methylated H3K4, nor with methylated H3K9, H3K27, H3K36, or H4K20 .

• Application compatibility: Verify that the antibody has been validated for your specific application (Western blot, ChIP, immunofluorescence, etc.). Search results show that many commercial H3K4me2 antibodies have been validated for multiple applications including ChIP, Western blot, immunocytochemistry, and immunohistochemistry .

• Species reactivity: Confirm that the antibody recognizes your species of interest. Many H3K4me2 antibodies react with human, mouse, rat, and non-human primate samples .

• Clone type: Consider whether a monoclonal or polyclonal antibody is more suitable for your application.

What validation experiments should I perform before using an H3K4me2 antibody?

Before using an H3K4me2 antibody in critical experiments, perform these validation steps:

• Specificity testing: Use peptide dot blots or Western blots with recombinant histones containing different methylation states to confirm antibody specificity .

• Positive control testing: Include a positive control sample known to contain H3K4me2, such as acid extracts from HeLa cells .

• Negative control testing: Include appropriate negative controls such as samples treated with demethylase enzymes or cells with genetic knockdown of H3K4 methyltransferases (e.g., Set1 knockout cells) .

• Dilution optimization: Test different antibody dilutions to determine optimal concentration for your specific application and sample type. Guidelines from search results suggest 1:1000 for Western blotting, 1:50 for immunoprecipitation, 1:750-1:3000 for IHC, and 1:400-1:1600 for immunofluorescence .

What is the optimal protocol for ChIP experiments using H3K4me2 antibodies?

For optimal ChIP experiments with H3K4me2 antibodies:

• Sample preparation: Fix cells with 1% formaldehyde for 10 minutes at room temperature to crosslink proteins to DNA.

• Chromatin fragmentation: Sonicate chromatin to generate fragments of 200-500 bp, which is optimal for resolution in ChIP experiments.

• Antibody amount: Use approximately 1-2 μg of H3K4me2 antibody per ChIP reaction with 25 μg of chromatin .

• Controls: Include appropriate controls such as:

  • Input sample (non-immunoprecipitated chromatin)

  • IgG negative control

  • Positive control regions known to be enriched for H3K4me2

  • Negative control regions known to lack H3K4me2, such as telomeric regions

• Analysis: Analyze immunoprecipitated DNA by qPCR, microarray (ChIP-chip), or sequencing (ChIP-seq).

Example ChIP-seq data from yeast studies showed that H3K4me2 is enriched in the coding regions of transcriptionally active genes but depleted in heterochromatic regions like telomeres, silent mating-type loci, and rDNA regions .

How do I analyze H3K4me2 patterns using acid-urea gel electrophoresis?

Acid-urea (AU) gel electrophoresis is valuable for separating histones with different acetylation states, which can be combined with Western blotting to analyze H3K4me2 modifications:

• Sample preparation: Extract histones using acid extraction (e.g., with 0.2M H₂SO₄) followed by TCA precipitation and acetone washing.

• Gel preparation: Prepare 15% acrylamide gels containing 5% acetic acid and 6M urea.

• Electrophoresis: Run samples at constant voltage (e.g., 200V) for 3-4 hours in 5% acetic acid running buffer.

• Western blotting: Transfer proteins to PVDF membrane and probe with H3K4me2 antibody.

• Analysis: The resulting ladder of bands (levels 0-3) corresponds to different acetylation states of H3K4me2 histones, with each acetylation reducing the positive charge by one and causing the histone to migrate more slowly .

This technique effectively revealed that TSA treatment in Dictyostelium caused a rapid accumulation of acetylation preferentially on H3K4me3-modified histones, shifting the major bands from levels 1 and 2 to levels 2 and 3 within 1 hour of treatment .

What are the best approaches for visualizing H3K4me2 distribution in situ?

For optimal visualization of H3K4me2 distribution in cells:

• Sample preparation:

  • For immunocytochemistry: Fix cells with 4% paraformaldehyde (10 min) or 100% methanol (5 min) .

  • For immunohistochemistry: Use formalin-fixed, paraffin-embedded tissue sections .

• Antibody dilution: Use H3K4me2 antibody at a dilution of 1:400-1:1600 for immunofluorescence applications .

• Detection method: Use fluorophore-conjugated secondary antibodies for immunofluorescence or HRP-conjugated antibodies for chromogenic detection.

• Counter-staining: Co-stain with DAPI to visualize DNA/chromatin distribution, which helps analyze the relationship between H3K4me2 and chromatin condensation states.

• Imaging: For high-resolution analysis, use confocal microscopy with maximum intensity projection of multiple z-sections .

Immunofluorescence studies have shown that H3K4me2 appears in several hundred small nuclear foci that do not colocalize with condensed regions of chromatin. Notably, perinuclear and perinucleolar heterochromatin typically do not stain with H3K4me2 antibodies .

How do I interpret the relationship between H3K4me2 and transcriptional activity?

When interpreting H3K4me2 ChIP data in relation to transcriptional activity:

• Genome location analysis: H3K4me2 is typically enriched in the 5' regions of transcriptionally active genes but shows a distribution pattern distinct from both H3K4me1 (enhancers) and H3K4me3 (promoters) .

• Correlation with gene expression: Compare H3K4me2 ChIP-seq data with RNA-seq or microarray expression data to establish correlations between H3K4me2 levels and transcriptional activity.

• Integration with other histone marks: Analyze H3K4me2 in conjunction with other activating marks (H3K9ac, H3K14ac, H3K36me3) and repressive marks (H3K9me3, H3K27me3) for a comprehensive view of chromatin state .

• Spatial distribution analysis: Examine the distribution pattern along genes - H3K4me2 is generally depleted in heterochromatic regions like telomeres and enriched in coding regions of active genes .

Studies in yeast have demonstrated that regions with high levels of H3K4 methylation show minimal overlap with regions deacetylated by histone deacetylases like Rpd3 and Hda1, supporting a model where H3K4 methylation facilitates transcription by preventing deacetylation of coding regions .

How can I distinguish between specific antibody binding and background in H3K4me2 ChIP experiments?

To distinguish specific H3K4me2 signal from background:

• Control regions: Compare enrichment at known positive regions (active gene bodies) versus negative regions (heterochromatin, telomeres) .

• Peak shape analysis: True H3K4me2 enrichment typically shows a characteristic distribution pattern along genes, while non-specific binding produces random peaks.

• Statistical analysis: Use appropriate statistical methods (e.g., MACS, SICER) to call significant peaks above background.

• Controls comparison: Compare your H3K4me2 ChIP data with:

  • Input control

  • IgG control

  • H3 occupancy control (to normalize for nucleosome density)

• Validation: Validate key findings using independent methods such as targeted ChIP-qPCR.

In the studies by Santos-Rosa et al., they evaluated the specificity of H3K4me2 enrichment by comparing it to telomere-proximal regions, which showed significantly lower Cy5:Cy3 ratios than the global average, indicating hypomethylation at H3K4 in those heterochromatic regions .

How can I use H3K4me2 antibodies to study the relationship between histone methylation and acetylation?

To investigate the relationship between H3K4 methylation and histone acetylation:

• Sequential ChIP (Re-ChIP): Perform initial ChIP with H3K4me2 antibody followed by a second IP with antibodies against specific acetylation marks (H3K9ac, H3K14ac, H4K5ac, etc.).

• Inhibitor studies: Treat cells with histone deacetylase inhibitors (e.g., TSA) and analyze changes in H3K4me2 distribution using ChIP-seq or acid-urea gel electrophoresis .

• Genetic models: Use cells lacking methyltransferases (e.g., Set1Δ) or histone deacetylases (e.g., Rpd3Δ, Hda1Δ) to study the interdependence of these modifications .

• Biochemical assays: Perform in vitro assays with recombinant or purified native complexes to determine how H3K4 methylation affects the activity of histone acetyltransferases or deacetylases.

Huang et al. demonstrated that in Dictyostelium, H3K4me3-directed H3 acetylation is mediated by Sgf29, a subunit of the SAGA acetyltransferase complex that specifically recognizes H3K4me3 via a tandem tudor domain . This system provides a valuable model for studying the interplay between methylation and acetylation.

What approaches can I use to study the dynamics of H3K4 methylation during cellular processes?

To study H3K4 methylation dynamics:

• Time-course experiments: Perform ChIP-seq at different time points during cellular processes like differentiation, cell cycle progression, or response to stimuli.

• Live-cell imaging: Use engineered antibody fragments or methylation-specific reader domains fused to fluorescent proteins for real-time tracking of H3K4 methylation.

• Pulse-chase approaches: Use inducible systems to express tagged histones and track newly deposited versus pre-existing methylated histones.

• Single-cell analysis: Combine H3K4me2 antibodies with single-cell technologies to examine cell-to-cell variation.

• Pharmacological inhibition: Use methyltransferase or demethylase inhibitors to perturb the dynamic equilibrium of methylation.

In their study on the effects of HDAC inhibitors, Huang et al. used a time-course approach to demonstrate that TSA treatment leads to rapid accumulation of acetylation on H3K4me3-modified histones within 1 hour, revealing the dynamic nature of this histone modification interplay .

How can I use H3K4me2 antibodies to study the role of histone methylation in disease models?

For studying H3K4me2 in disease contexts:

• Disease tissue analysis: Compare H3K4me2 patterns in normal versus diseased tissues using ChIP-seq or immunohistochemistry with validated H3K4me2 antibodies .

• Patient-derived models: Use patient-derived cell lines or xenografts to study disease-specific H3K4me2 patterns.

• Drug response studies: Examine how epigenetic drugs (e.g., HDAC inhibitors like TSA) affect H3K4me2 distribution in normal versus disease models .

• Genetic manipulation: Create disease models with mutations in H3K4 methyltransferases or demethylases to study causality.

• Correlation studies: Correlate H3K4me2 patterns with disease progression, prognosis, or treatment response.

The research by Huang et al. demonstrated that cells lacking H3K4me3 due to disruption of the Set1 methyltransferase or mutations in endogenous H3 genes showed resistance to TSA-induced developmental inhibition . This suggests that H3K4 methylation levels could serve as biomarkers for sensitivity to HDAC inhibitors, which are approved for clinical use against certain cancers.

What are common problems with H3K4me2 ChIP experiments and how can I resolve them?

Here are common problems and solutions for H3K4me2 ChIP experiments:

For optimal results, verify that your antibody has been specifically validated for ChIP applications, as many commercial antibodies are specifically tested and optimized for this purpose .

How do I troubleshoot western blots using H3K4me2 antibodies?

For troubleshooting western blots with H3K4me2 antibodies:

When performing western blots for H3K4me2, use calf thymus histone preparations or acid extracts from HeLa cells as positive controls . The expected molecular weight for histone H3 is approximately 15-17 kDa .

What factors affect the specificity of H3K4me2 antibodies?

Key factors affecting H3K4me2 antibody specificity include:

• Antibody type: Monoclonal antibodies generally offer higher specificity than polyclonal antibodies. For example, the C64G9 Rabbit mAb shows minimal cross-reactivity with other methylation states .

• Epitope recognition: Some antibodies may recognize the surrounding sequence context in addition to the H3K4me2 modification.

• Cross-reactivity with other methylation states: Many H3K4me2 antibodies show some degree of cross-reactivity with H3K4me1, so validation against different methylation states is critical .

• Buffer conditions: Salt concentration, pH, and detergent types can affect antibody binding specificity.

• Sample preparation: Fixation methods, extraction procedures, and protein denaturation conditions can influence epitope accessibility and recognition.

When selecting an H3K4me2 antibody, examine the validation data for cross-reactivity testing against other histone modifications. For example, RevMAb RM135 is reported to have no cross-reactivity with H3K4me1, H3K4me3, or other methylations in histone H3 , while Cell Signaling's C64G9 shows weak cross-reactivity with H3K4me1 but not with non-methylated or tri-methylated H3K4 .