Tri-methyl-Histone H3(K79) Monoclonal Antibody

What is Tri-methyl-Histone H3(K79) and what is its biological significance?

Tri-methyl-Histone H3(K79) refers to histone H3 that has been trimethylated specifically at lysine 79. Histone H3 is a core component of nucleosomes, which wrap and compact DNA into chromatin structure. This compaction limits DNA accessibility to cellular machinery that requires DNA as a template for processes such as transcription, repair, and replication . H3K79 trimethylation (H3K79me3) represents a specific post-translational modification catalyzed by the methyltransferase Dot1 in mammalian cells .

The biological significance of H3K79me3 lies in its association with transcriptionally active genes. Unlike some histone modifications that repress transcription, H3K79me3 is generally considered an active mark . It contributes to the "histone code" - a complex set of post-translational modifications that regulate DNA accessibility and gene expression. Research has also implicated altered global H3K79 trimethylation in pathological processes, particularly in leukemogenesis in humans . This epigenetic mark is therefore important both in normal cellular function and in disease mechanisms.

How does the Tri-methyl-Histone H3(K79) Monoclonal Antibody differ from antibodies targeting other histone modifications?

The Tri-methyl-Histone H3(K79) Monoclonal Antibody is specifically engineered to recognize and bind to histone H3 only when it is trimethylated at lysine 79. This high specificity is a key distinguishing feature of this antibody compared to others targeting different histone modifications. According to peptide dotblot analyses, the antibody shows no cross-reactivity with non-modified lysine 79 (H3K79Ctrl), monomethylated lysine 79 (K79me1), or dimethylated lysine 79 (K79me2) .

Unlike antibodies for histone modifications such as H3K4me3 (associated with active promoters), H3K27me3 (associated with repressed genes), or acetylation marks like H3K14ac, the H3K79me3 antibody targets a modification that occurs within the globular domain of histone H3 rather than on the N-terminal tail . This is significant because most histone modifications occur on the N-terminal tails protruding from the nucleosome. The antibody's specificity is typically validated through multiple techniques including Western blot, where it identifies a single band corresponding to H3K79me3 in cell extracts like those from HeLa cells .

What techniques can be used to detect Tri-methyl-Histone H3(K79)?

Several established techniques can be employed to detect H3K79me3, each with specific applications in research:

• Western Blot (WB): The antibody can be used at concentrations of 0.2-1 μg/mL to detect H3K79me3 in protein extracts. This technique allows visualization of the specific band corresponding to trimethylated histone H3 at approximately 17 kDa . Western blotting is particularly useful for quantitative analysis of global H3K79me3 levels across different cell types or treatment conditions.

• Chromatin Immunoprecipitation (ChIP): This technique allows researchers to identify specific DNA sequences associated with H3K79me3-modified histones, helping to map the genomic distribution of this modification . ChIP is essential for understanding which genes are regulated by H3K79 trimethylation.

• Immunocytochemistry/Immunofluorescence (ICC/IF): These techniques enable visualization of H3K79me3 distribution within cellular compartments, providing insights into the spatial organization of this epigenetic mark .

• Dot Blot: This technique allows rapid screening of samples for the presence of H3K79me3 and can be used to test antibody specificity against various modified peptides .

• Fluorometric Quantification: Specialized kits like the EpiSeeker Histone H3 (tri-methyl K79) Quantification Kit use a capture-and-detect approach where strip wells are coated with anti-trimethyl H3-K79 antibody to measure global levels of this modification .

How does H3K79 trimethylation interact with other histone modifications in the context of gene regulation?

H3K79 trimethylation functions within a complex network of histone modifications that collectively regulate gene expression. The "histone code" hypothesis suggests that combinations of modifications create binding platforms for effector proteins that influence chromatin structure and function. H3K79me3 typically exists in regions with other active marks such as H3K4me3 and various acetylation marks . The coexistence of these modifications creates synergistic effects that reinforce the transcriptionally active state.

Recent research indicates that H3K79me3 has a unique relationship with H3K27me3, a repressive mark associated with Polycomb-mediated gene silencing. These marks tend to be mutually exclusive, suggesting antagonistic regulatory mechanisms . Additionally, the presence of H3K79me3 may prevent the binding of certain repressive complexes, thereby maintaining genes in an active state.

The methyltransferase Dot1, which deposits methyl groups at H3K79, lacks the SET domain common to most histone methyltransferases, suggesting a distinctive catalytic mechanism . This enzymatic uniqueness may explain the unusual location of K79 within the globular domain of H3 rather than on the N-terminal tail where most modifications occur. Understanding these interactions requires techniques that can simultaneously detect multiple modifications, such as sequential ChIP or mass spectrometry-based approaches.

What is the role of H3K79 trimethylation in disease pathogenesis and potential therapeutic interventions?

H3K79 trimethylation dysregulation has been implicated in several pathological processes, most notably in leukemogenesis. Altered activity of DOT1L, the enzyme responsible for H3K79 methylation, is associated with mixed lineage leukemia (MLL) rearrangements . In these cases, MLL fusion proteins recruit DOT1L to inappropriate genomic locations, leading to aberrant H3K79me3 patterns and consequently abnormal gene expression that drives leukemogenesis.

This understanding has prompted the development of DOT1L inhibitors as potential therapeutic agents for MLL-rearranged leukemias. Preclinical studies have shown that inhibition of DOT1L activity can selectively kill leukemia cells harboring MLL translocations while sparing normal cells. Beyond leukemia, emerging evidence suggests roles for H3K79me3 dysregulation in solid tumors, neurodegenerative disorders, and cardiovascular diseases.

Research methodologies to investigate these disease connections include:

• Genome-wide mapping of H3K79me3 in patient samples versus healthy controls using ChIP-seq

• Correlation analyses between H3K79me3 levels and disease progression markers

• In vivo studies using conditional Dot1l knockout or inhibition in disease models

• Combinatorial approaches targeting H3K79me3 alongside other epigenetic modifications

Understanding the mechanistic details of how H3K79me3 contributes to disease pathogenesis requires careful experimental design that accounts for cell-type specificity and temporal dynamics of this modification.

How does the chromatin environment affect the accessibility and functionality of H3K79me3?

The chromatin context significantly influences both the deposition and functional consequences of H3K79 trimethylation. Unlike most histone modifications that occur on the protruding N-terminal tails, K79 resides within the globular domain of histone H3, which is generally less accessible within the nucleosome structure . This position makes H3K79 methylation particularly sensitive to higher-order chromatin organization.

Several factors influence H3K79me3 accessibility and function:

• Nucleosome positioning: Well-positioned nucleosomes may restrict access of DOT1L to K79, thereby regulating methylation patterns.

• Chromatin remodeling complexes: ATP-dependent chromatin remodelers can alter nucleosome structure to expose K79 for modification or to reveal existing H3K79me3 to reader proteins.

• Histone variant incorporation: Variants such as H3.3 may influence the propensity for K79 methylation due to subtle structural differences.

• Pre-existing modifications: Other nearby histone modifications may either facilitate or inhibit DOT1L activity through allosteric effects on enzyme binding or activity.

Research approaches to study these relationships include in vitro reconstitution of nucleosomes with defined modification patterns, electron microscopy or crystallography to visualize structural changes, and proximity ligation assays to detect interactions between H3K79me3 and other chromatin components. Advanced techniques such as Micro-C or Hi-C can relate H3K79me3 distribution to three-dimensional genome organization.

What are the optimal protocols for using Tri-methyl-Histone H3(K79) Monoclonal Antibody in ChIP experiments?

Chromatin Immunoprecipitation (ChIP) with the Tri-methyl-Histone H3(K79) Monoclonal Antibody requires careful optimization to achieve high specificity and sensitivity. Here is a detailed methodological approach:

Sample Preparation:

• Cross-link protein-DNA complexes in cultured cells or tissue samples using 1% formaldehyde for 10 minutes at room temperature.

• Quench cross-linking with 125 mM glycine for 5 minutes.

• Wash cells with cold PBS and harvest by scraping or trypsinization.

• Lyse cells and isolate nuclei using appropriate buffers containing protease inhibitors.

• Sonicate chromatin to obtain fragments of 200-500 bp, verifying fragment size by agarose gel electrophoresis.

Immunoprecipitation:

• Pre-clear chromatin with protein A/G beads to reduce non-specific binding.

• Add Tri-methyl-Histone H3(K79) antibody at an optimized concentration (typically 2-5 μg per ChIP reaction) .

• Incubate overnight at 4°C with rotation.

• Add pre-blocked protein A/G beads and incubate for 2-3 hours.

• Wash stringently to remove non-specific interactions using increasingly stringent wash buffers.

• Elute protein-DNA complexes and reverse cross-links by heating at 65°C overnight.

• Treat with RNase A and Proteinase K to remove RNA and proteins.

• Purify DNA using column-based methods.

Quality Control:

• Include appropriate controls: input chromatin (5-10%), no-antibody control, and IP with IgG.

• Validate enrichment by qPCR using primers for known H3K79me3-enriched regions versus depleted regions.

• For genome-wide analysis, proceed to library preparation for ChIP-seq.

This methodology ensures specific enrichment of H3K79me3-associated genomic regions that can be further analyzed to understand the distribution pattern of this modification across the genome.

What quantification methods provide the most accurate measurement of global H3K79me3 levels?

Accurate quantification of global H3K79me3 levels is essential for comparative studies across different cell types, conditions, or disease states. Several methods offer complementary approaches to achieve reliable measurements:

Fluorometric Assays:
The EpiSeeker Histone H3 (tri-methyl K79) Quantification Kit employs a sensitive fluorometric approach . In this assay:

• Strip wells coated with anti-trimethyl H3-K79 antibody capture the modified histone.

• A labeled detection antibody binds to the captured proteins.

• A fluorescent development reagent generates a signal proportional to H3K79me3 abundance.

• Comparison to a standard curve allows absolute quantification.

This method offers high sensitivity and throughput for processing multiple samples simultaneously.

Mass Spectrometry-Based Approaches:
Mass spectrometry provides the most comprehensive and unbiased quantification of histone modifications:

• Histones are acid-extracted from nuclei and purified.

• Enzymatic digestion generates peptides containing the K79 residue.

• Liquid chromatography coupled to tandem mass spectrometry separates and identifies modified peptides.

• Heavy isotope-labeled internal standards allow absolute quantification.

This approach can simultaneously measure all methylation states (me1, me2, me3) at K79 and other modifications, providing a complete picture of the histone modification landscape.

Western Blot Semi-Quantification:
While less precise than the above methods, western blotting can provide relative quantification:

• Run acid-extracted histones on SDS-PAGE and transfer to membranes.

• Probe with the Tri-methyl-Histone H3(K79) antibody at 0.5 μg/mL .

• Detect using chemiluminescence or fluorescence.

• Normalize H3K79me3 signal to total H3 signal from the same samples.

For optimal results, researchers should combine complementary approaches and include appropriate controls to validate findings across different quantification platforms.

What experimental controls are essential when working with the Tri-methyl-Histone H3(K79) Monoclonal Antibody?

Robust experimental design when working with the Tri-methyl-Histone H3(K79) Monoclonal Antibody requires several critical controls to ensure data validity and reliability:

Antibody Specificity Controls:

• Peptide Competition: Pre-incubate the antibody with increasing concentrations of the H3K79me3 peptide before application in the experimental assay. This should progressively diminish signal, confirming specificity.

• Cross-Reactivity Testing: Include peptides with other methylation states (K79me1, K79me2) and unmodified H3K79 in dot blot or ELISA formats to verify the antibody only reacts with the trimethylated form .

• Knockout/Knockdown Validation: Use samples from cells where DOT1L (the enzyme responsible for H3K79 methylation) has been knocked out or knocked down, which should show reduced or absent H3K79me3 signal.

Procedural Controls:

• Loading Controls: For western blots, probe for total histone H3 to normalize for loading variations. Use commercially available recombinant histone H3.3 as a reference standard .

• Positive Controls: Include cell lines known to express high levels of H3K79me3, such as HeLa cells .

• Negative Controls: Use primary antibody isotype controls (rabbit IgG) at the same concentration as the experimental antibody.

• Input Controls: For ChIP experiments, reserve 5-10% of chromatin before immunoprecipitation as an input control to calculate percent enrichment.

Technique-Specific Controls:

• For ICC/IF: Include secondary-only controls to assess background fluorescence and include DAPI staining to confirm nuclear localization.

• For ChIP: Perform parallel IPs with antibodies against known active marks (H3K4me3) and repressive marks (H3K27me3) to verify the expected correlations with H3K79me3.

• For Quantification Assays: Include a standard curve using provided H3K79me3 standards and perform technical replicates to assess reproducibility.

Implementing these controls ensures that findings attributed to H3K79me3 detection are specific, reproducible, and biologically relevant.

How can researchers address inconsistent or contradictory H3K79me3 antibody results?

Inconsistent or contradictory results when working with H3K79me3 antibodies can arise from various sources. Here's a systematic approach to identify and resolve these issues:

Antibody-Related Factors:

• Antibody Batch Variation: Different lots of the same antibody may have variable specificity or sensitivity. Maintain detailed records of antibody lot numbers and validate new lots against previous results.

• Storage Conditions: Improper storage (repeated freeze-thaw cycles, storage at inappropriate temperatures) can compromise antibody performance. Store antibodies according to manufacturer recommendations, typically at -20°C in small aliquots to avoid repeated freezing and thawing .

• Working Concentration: Optimize antibody concentration for each application. For Western blot, a range of 0.2-1 μg/mL is recommended , but this may need adjustment based on sample type and detection method.

Sample Preparation Issues:

• Fixation Methods: Overfixation can mask epitopes. For ICC/IF or IHC, optimize fixation times (typically 10-15 minutes with 4% paraformaldehyde).

• Extraction Efficiency: Incomplete histone extraction can lead to inconsistent results. Use standardized acid extraction protocols for histones and verify extraction quality by Coomassie staining.

• Chromatin Fragmentation: For ChIP applications, inconsistent sonication can affect results. Verify fragment size distribution by agarose gel electrophoresis.

Biological Variables:

• Cell Cycle Effects: H3K79me3 levels may vary through the cell cycle. Synchronize cells or account for cell cycle distribution in experimental design.

• Cell Type Heterogeneity: Mixed cell populations may show variable H3K79me3 patterns. Use cell sorting or single-cell approaches when appropriate.

Data Analysis Approaches:

• Normalization Methods: For western blots, always normalize H3K79me3 signal to total H3 signal from the same samples to account for loading differences.

• Statistical Analysis: Employ appropriate statistical tests based on experimental design and data distribution. Include sufficient biological replicates (n≥3) to assess biological variability.

Creating a detailed troubleshooting table that tracks variables changed between experiments can help identify sources of inconsistency. When contradictory results persist, consider employing complementary techniques (e.g., mass spectrometry) to resolve discrepancies.

What are the key considerations when interpreting ChIP-seq data for H3K79me3?

Interpreting ChIP-seq data for H3K79me3 requires careful consideration of several factors that affect data quality and biological interpretation:

Data Quality Assessment:

• Sequencing Depth: Sufficient depth is crucial for detecting H3K79me3 peaks. Aim for at least 20 million uniquely mapped reads per sample for point-source peaks and higher coverage for broad domains.

• Signal-to-Noise Ratio: Calculate the fraction of reads in peaks (FRiP) score; values >1% generally indicate successful ChIP-seq experiments.

• Peak Reproducibility: Compare biological replicates to ensure peak calling consistency, using metrics such as the Irreproducible Discovery Rate (IDR).

• Control Normalization: Proper normalization against input controls is essential to account for biases in chromatin accessibility, mappability, and PCR amplification.

Genomic Distribution Analysis:

• Meta-Gene Profiles: H3K79me3 is typically enriched in the gene bodies of actively transcribed genes, with enrichment increasing from the 5' to 3' end, reflecting its association with transcriptional elongation.

• Integration with RNA-seq: Correlate H3K79me3 levels with gene expression data to verify the expected positive correlation.

• Comparison with Other Marks: Analyze co-occurrence with other active marks (H3K4me3, H3K36me3) and mutual exclusivity with repressive marks (H3K27me3, H3K9me3).

Bioinformatic Analysis Considerations:

• Peak Calling Algorithms: H3K79me3 forms broad domains rather than sharp peaks, so algorithms designed for broad marks (e.g., SICER or MACS2 with broad peak options) are more appropriate than those optimized for transcription factors.

• Differential Binding Analysis: When comparing H3K79me3 between conditions, use specialized tools like DiffBind that account for the characteristics of histone modification data.

• Genomic Feature Enrichment: Analyze enrichment of H3K79me3 relative to genomic features using tools like GREAT or HOMER to identify potential functional associations.

Biological Context Integration:

• Cell Type Specificity: H3K79me3 patterns vary between cell types, reflecting differences in transcriptional programs.

• Developmental Stage: Consider temporal dynamics of H3K79me3 during developmental processes.

• Disease Context: In disease states, particularly leukemias with MLL rearrangements, H3K79me3 may show altered distribution patterns that deviate from normal cells.

By accounting for these considerations, researchers can extract meaningful biological insights from H3K79me3 ChIP-seq data and place them in the appropriate context of gene regulation and cellular function.

How can researchers distinguish between direct and indirect effects when manipulating H3K79 methylation levels?

Distinguishing between direct and indirect effects of H3K79 methylation manipulation represents a significant challenge in epigenetic research. The following methodological approaches can help researchers make this critical distinction:

Temporal Resolution Approaches:

• Rapid Induction Systems: Employ systems that allow rapid and controlled induction of DOT1L activity or inhibition, such as:

  • Auxin-inducible degron (AID) system for rapid protein degradation

  • Chemical inhibitors of DOT1L with fast kinetics

  • Optogenetic control of DOT1L recruitment to specific loci

• Time-Course Analysis: Monitor changes in H3K79me3, transcription, and downstream effects at multiple time points after manipulation. Direct effects typically occur earlier than indirect ones. Create a temporal map that correlates:

  • Changes in H3K79me3 levels (measured by ChIP-qPCR or ChIP-seq)

  • Transcriptional changes (measured by RNA-seq or qRT-PCR)

  • Downstream protein changes (measured by proteomics or western blotting)

Genomic Targeting Approaches:

• Locus-Specific Manipulation: Use CRISPR-dCas9 fused to DOT1L or DOT1L inhibitory domains to alter H3K79me3 at specific genomic loci rather than globally. This allows observation of direct effects at targeted loci while minimizing system-wide perturbations.

• Genetic Complementation: In DOT1L knockout systems, reintroduce either wild-type DOT1L or catalytically inactive mutants. Phenotypes rescued only by the wild-type enzyme are likely direct consequences of H3K79 methylation.

Multi-Omics Integration:

• Integrated Analysis Framework: Combine multiple data types to distinguish direct from indirect effects:

  Data Type	Method	Direct Effect Evidence
  Histone modification	ChIP-seq	Altered H3K79me3 at specific loci
  Chromatin accessibility	ATAC-seq	Changes following H3K79me3 alteration
  Transcription	RNA-seq, GRO-seq	Rapid transcriptional changes at H3K79me3-marked genes
  Protein binding	CUT&RUN, ChIP-seq	Altered recruitment of readers/factors to H3K79me3 sites
  Chromatin interaction	Hi-C, 4C	Changes in chromatin looping at affected loci

• Causality Testing: Use computational approaches like Granger causality or dynamic Bayesian networks to infer causal relationships between H3K79me3 changes and downstream effects.

Biochemical Validation:

• Reader Protein Identification: Perform pulldown experiments with differentially methylated H3K79 peptides to identify proteins that specifically bind to H3K79me3. These represent potential mediators of direct effects.

• Mutational Analysis: Introduce point mutations at lysine 79 (e.g., K79R or K79A) to prevent methylation specifically at this residue while maintaining other histone functions.

By combining these approaches, researchers can build a comprehensive model that distinguishes between the direct consequences of H3K79 methylation and the cascade of indirect effects that follow from altered gene expression or other cellular processes.

What are the future directions for research using Tri-methyl-Histone H3(K79) Monoclonal Antibody?

Research utilizing the Tri-methyl-Histone H3(K79) Monoclonal Antibody is poised to advance in several promising directions as technology and understanding evolve. Future research will likely focus on:

• Single-Cell Epigenomics: Adapting H3K79me3 detection methods for single-cell analysis will reveal cell-to-cell variation in this modification and help understand its role in cellular heterogeneity and differentiation. The high specificity of the monoclonal antibody makes it particularly suitable for developing single-cell ChIP-seq or CUT&Tag protocols that require high signal-to-noise ratios .

• Dynamic Regulation: Investigating the temporal dynamics of H3K79me3 during cellular processes like differentiation, cell cycle progression, and response to environmental stimuli will provide insights into its regulatory mechanisms. This will require time-resolved experimental designs using synchronized cell populations or inducible systems.

• Therapeutic Applications: As DOT1L inhibitors enter clinical trials for MLL-rearranged leukemias, the antibody will be crucial for monitoring treatment efficacy through measurement of global and locus-specific H3K79me3 levels . Developing standardized protocols for H3K79me3 quantification in patient samples could lead to companion diagnostics.

• Cross-Talk with Other Epigenetic Mechanisms: Exploring the interplay between H3K79me3 and other epigenetic mechanisms such as DNA methylation, other histone modifications, and non-coding RNAs will provide a more comprehensive understanding of epigenetic regulation. Multi-omics approaches combining ChIP-seq, RNA-seq, and DNA methylation analysis will be particularly valuable.

• Structural Biology: Investigating the structural basis of H3K79me3 recognition by reader proteins and how this influences chromatin architecture could reveal novel therapeutic targets. Advanced imaging techniques like cryo-electron microscopy may help visualize H3K79me3-marked nucleosomes in complex with regulatory proteins.

• Development of Enhanced Detection Methods: Creating improved antibody formats or detection systems with greater sensitivity, specificity, or capability for multiplexing with other histone marks will expand research capabilities .

These future directions will contribute to a deeper understanding of H3K79 trimethylation's role in normal biology and disease, potentially leading to novel diagnostic and therapeutic approaches targeting this epigenetic modification.

How can researchers standardize H3K79me3 detection methods for cross-laboratory comparisons?

Standardization of H3K79me3 detection methods is essential for enabling reliable cross-laboratory comparisons and building a coherent body of knowledge. The following standardization framework addresses key aspects of experimental design, execution, and reporting:

Reagent Standardization:

• Reference Antibodies: Establish community-recognized reference antibodies with demonstrated specificity for H3K79me3, such as the RM157 clone, which has been extensively validated . Labs should report antibody clone, lot number, and source in publications.

• Standard Controls: Develop commercially available positive controls (synthetic H3K79me3 peptides, recombinant modified nucleosomes, or standardized cell extracts with known H3K79me3 levels) that can be used across laboratories for calibration purposes.

• Validation Requirements: Establish minimum validation criteria for antibodies, including demonstration of specificity against a panel of modified peptides, western blot analysis showing a single band of appropriate size, and reduced signal with DOT1L inhibition .

Methodological Standardization:

• Protocol Repositories: Create detailed, step-by-step protocols for common applications (ChIP, Western blot, immunofluorescence) in repositories like Protocols.io, including critical parameters and troubleshooting guidance.

• Normalization Approaches: Standardize normalization methods for different techniques:

  • For Western blot: Normalize to total H3 detected on the same membrane

  • For ChIP-seq: Use spike-in controls (e.g., Drosophila chromatin) for between-sample normalization

  • For quantitative assays: Include standard curves with defined H3K79me3 concentrations

• Reporting Templates: Develop structured reporting templates capturing key experimental variables like cell type, growth conditions, fixation method, antibody concentration, and detection method.

Data Analysis Standardization:

• Bioinformatic Pipelines: Establish standard computational workflows for processing H3K79me3 ChIP-seq data, including read mapping, quality control metrics, peak calling parameters, and visualization techniques.

• Quantification Metrics: Define standard metrics for reporting H3K79me3 levels, such as:

  • Relative enrichment over input

  • Signal-to-noise ratios

  • Absolute quantification using reference standards

  • Genomic distribution patterns

• Data Repositories: Encourage deposition of raw and processed data in public repositories with standardized metadata to facilitate reanalysis and meta-analyses.

Interlaboratory Validation:

• Round-Robin Studies: Conduct periodic interlaboratory studies where multiple labs analyze identical samples using their standard protocols, followed by comparison of results to identify sources of variation.

• Proficiency Testing: Develop proficiency testing programs where laboratories receive blinded samples with known H3K79me3 status to assess their detection accuracy.