Histone H3K36me3 Antibody

What is the biological significance of H3K36me3 in chromatin regulation?

H3K36me3 (trimethylation of lysine 36 on histone H3) serves as a critical epigenetic mark involved in numerous cellular processes. Most notably, it plays an essential role in DNA mismatch repair (MMR) by recruiting the mismatch recognition protein hMutSα (hMSH2-hMSH6) onto chromatin through direct interaction with the hMSH6 PWWP domain . Additionally, H3K36me3 is associated with transcriptional elongation by RNA polymerase II and serves as a marker of actively transcribed genes . The presence of this modification helps define chromatin states and influences gene expression patterns, making it a crucial regulatory mechanism in eukaryotic cells.

Which enzymes are responsible for establishing H3K36me3 marks, and how is this regulation maintained?

In humans, the primary enzyme responsible for H3K36 trimethylation is SETD2, a histone methyltransferase . This is distinct from yeast, where Set2 performs this function, or other mammalian systems where NSD1 can also contribute to this modification . Cells lacking SETD2 display microsatellite instability (MSI) and an elevated spontaneous mutation frequency, characteristics typical of MMR-deficient cells . The regulation of H3K36me3 is cell cycle-dependent, with maximum abundance occurring in early S phase, declining to very low levels by the end of S phase and G2/M, and beginning to increase again in G1 . This temporal pattern ensures that hMutSα is enriched on chromatin before and during DNA replication, potentially increasing the efficiency of MMR in actively replicating chromatin.

How does the distribution of H3K36me3 vary throughout the cell cycle?

H3K36me3 displays a dynamic pattern of abundance throughout the cell cycle. Research has demonstrated that H3K36me3 reaches maximum levels in early S phase, subsequently declining to very low levels by the end of S phase and G2/M phases, before beginning to increase again during G1 phase . This pattern is particularly significant because the abundance of H3K36me3 in G1 and early S phases ensures that hMutSα is enriched on chromatin before mispairs are introduced during DNA replication. Approximately 80% of hMSH6 foci colocalize with H3K36me3 in G1 phase, supporting this temporal relationship . This cell cycle-dependent distribution suggests a coordinated mechanism to prepare chromatin for efficient DNA repair prior to replication.

What criteria should be considered when selecting a H3K36me3 antibody for specific research applications?

When selecting a H3K36me3 antibody, researchers should consider several critical factors:

• Application compatibility: Verify that the antibody has been validated for your specific application (ChIP, ChIP-Seq, Western blot, immunofluorescence) .

• Host species and format: Consider whether you need a polyclonal, monoclonal, or recombinant antibody based on your experimental design .

• Cross-reactivity profile: Review validation data to ensure the antibody specifically recognizes H3K36me3 without cross-reacting with other histone modifications .

• Validation in comparable systems: Check if the antibody has been validated in cell types or organisms similar to your experimental system .

• Recommended working dilutions: Use manufacturer-specified concentrations for each application (e.g., 4 μg per ChIP for ChIP-Seq applications) .

These considerations are crucial as antibody performance can significantly impact experimental outcomes and data interpretation.

How can researchers validate the specificity of H3K36me3 antibodies to avoid cross-reactivity issues?

Thorough validation of H3K36me3 antibodies requires a multi-faceted approach:

• Peptide array testing: Screen antibodies against peptide arrays containing various histone modifications to identify potential cross-reactivity .

• Knockout/knockdown controls: Test antibody binding in cells lacking the modifying enzyme (e.g., SETD2 knockout cells) to confirm specificity .

• Competitive binding assays: Perform experiments using synthetic peptides containing the target modification to verify specific recognition .

• Recombinant histone comparison: Compare binding to native modified histones versus unmodified recombinant histones .

• Semi-synthetic nucleosome assays: Utilize DNA-barcoded mononucleosomes uniformly modified with H3K36me3 for direct specificity testing in IP conditions .

These validation steps are essential because some antibodies may cross-react with similar modifications (e.g., H3K27me3 antibodies cross-reacting with H3K4me3-marked histones) , potentially leading to misinterpretation of experimental results.

What are the key differences between polyclonal, monoclonal, and recombinant antibodies for H3K36me3 detection?

Each antibody type offers distinct advantages and limitations for H3K36me3 detection:

Recombinant antibodies like AbFlex contain additional features such as 6xHis Tags, Biotinylation Tags, and sortase recognition motifs (LPXTG) that allow for enzymatic biotin conjugation or attachment of various labels including fluorophores and enzymatic substrates , providing greater experimental flexibility.

What are the optimal conditions for using H3K36me3 antibodies in ChIP and ChIP-Seq experiments?

For optimal ChIP and ChIP-Seq experiments with H3K36me3 antibodies:

• Antibody amount: Use 4-10 μg of antibody per ChIP reaction, depending on the specific antibody and experimental system .

• Chromatin preparation: For many chromatin-bound proteins including histones with H3K36me3, use a High Salt/Sonication Protocol when preparing nuclear extracts as these proteins may not be soluble in low salt conditions .

• IP conditions: Consider both native and cross-linking IP conditions, as these can affect antibody performance. Some H3K36me3 antibodies perform better under native conditions .

• Controls: Include appropriate controls such as IgG negative controls and positive controls (e.g., HeLa nuclear extract) or semi-synthetic nucleosomes with defined modifications .

• qPCR validation: Use validated positive control primer sets (e.g., ACTB-1 for human samples) and negative control primer sets to validate ChIP experiments before proceeding to sequencing .

These methodological considerations ensure optimal enrichment of H3K36me3-modified regions and minimize background signal.

How should researchers optimize Western blot protocols for detecting H3K36me3?

For optimal Western blot detection of H3K36me3:

• Sample preparation: Use 0.5-2 μg/ml antibody dilution for Western blotting .

• Extraction method: Many chromatin-bound proteins including H3K36me3-modified histones are not soluble in low salt nuclear extracts and may fractionate to the pellet. Therefore, implement a High Salt/Sonication Protocol when preparing nuclear extracts .

• Controls: Include both positive controls (e.g., HeLa nuclear extract) and negative controls (recombinant unmodified histones) to confirm specificity .

• Blocking conditions: Optimize blocking conditions to reduce background while maintaining specific signal.

• Detection system: Choose a detection system with appropriate sensitivity for your experimental needs, considering that H3K36me3 migrates at approximately 17 kDa on SDS-PAGE .

These considerations help ensure specific detection of H3K36me3 while minimizing background and cross-reactivity issues.

What alternatives to antibodies exist for detecting H3K36me3, and what are their comparative advantages?

Alternative approaches for H3K36me3 detection include:

• Histone modification-specific interaction domains (HMIDs): Protein domains that naturally recognize specific histone modifications can be used as detection reagents . For H3K36me3, the PWWP domain of DNMT3A specifically binds to H3K36me2/3 and can be used in place of antibodies .

• Semi-synthetic nucleosome standards: DNA-barcoded mononucleosomes with defined modifications serve as internal standards for calibration (IceChIP) .

• Mass spectrometry: Provides quantitative analysis of histone modifications without antibody-related biases.

Comparative advantages:

These alternative approaches can complement antibody-based detection methods, particularly in cases where antibody specificity is a concern.

How can researchers address false positive/negative results when using H3K36me3 antibodies?

To address potential false results when using H3K36me3 antibodies:

• False positives due to cross-reactivity:

  • Verify antibody specificity using peptide arrays or competitive binding assays

  • Include knockout/knockdown controls (e.g., SETD2-depleted cells)

  • Confirm results with an alternative antibody from a different manufacturer or clone

• False negatives due to epitope masking:

  • Secondary modifications near H3K36 may prevent antibody binding despite the presence of H3K36me3

  • Try alternative antibodies that recognize different epitopes within the modified region

  • Consider native versus crosslinked ChIP conditions, as crosslinking can mask epitopes

• Validation approaches:

  • Compare results across multiple technical and biological replicates

  • Confirm findings using alternative methods (e.g., HMIDs as detection reagents)

  • Implement spike-in controls with semi-synthetic nucleosomes containing defined modifications

These strategies help ensure reliable detection of H3K36me3 and prevent misinterpretation of experimental results.

What genomic distribution patterns are typically observed for H3K36me3, and how should researchers interpret variations from these patterns?

H3K36me3 typically displays characteristic distribution patterns:

• Standard distribution:

  • Enriched in the bodies of actively transcribed genes, with increasing levels toward the 3' end

  • Associated with exons rather than introns, particularly in constitutively expressed genes

  • Generally depleted at promoters and transcription start sites

• Interpreting variations:

  • Cell type-specific variations may reflect tissue-specific transcriptional programs

  • Altered distribution in disease states may indicate dysregulated transcription

  • Redistribution following experimental interventions may suggest mechanistic connections to the manipulated pathways

• Analytical approaches:

  • Compare distribution to transcriptome data to correlate with gene expression levels

  • Analyze co-occurrence with other histone marks to understand combinatorial regulation

  • Examine changes across developmental stages or disease progression to identify dynamic regulation

Variations from typical patterns may indicate biological significance rather than technical artifacts, particularly when observed consistently across replicates and with validated antibodies.

How can conflicting ChIP-Seq data for H3K36me3 from different antibodies be reconciled?

When faced with conflicting ChIP-Seq data from different H3K36me3 antibodies:

• Assess antibody validation data:

  • Review specificity profiles of each antibody using peptide arrays or other validation methods

  • Check for known cross-reactivity with other histone modifications

• Compare experimental conditions:

  • Evaluate differences in ChIP protocols (native vs. crosslinked, sonication methods)

  • Consider chromatin preparation methods and cell cycle stage of samples

• Analytical approaches:

  • Calculate correlation coefficients between datasets to quantify similarities and differences

  • Identify consistently enriched regions across antibodies as high-confidence H3K36me3 sites

  • Implement spike-in controls with semi-synthetic nucleosomes to calibrate enrichment

• Validation strategies:

  • Confirm key findings with orthogonal methods (e.g., CUT&Tag, HMIDs)

  • Perform ChIP-qPCR on selected regions using both antibodies

  • Test antibodies in cells with genetic manipulation of SETD2

These approaches help distinguish genuine biological signal from antibody-specific artifacts, leading to more reliable interpretations of H3K36me3 distribution.

How does H3K36me3 coordinate with other histone modifications in regulation of cellular processes?

H3K36me3 functions within a complex network of histone modifications:

• Transcriptional regulation:

  • H3K36me3 positively correlates with H3K4me3 (a promoter-associated active mark) on actively transcribed genes

  • Anti-correlates with repressive marks like H3K27me3, with potential implications for bivalent domains

  • Works cooperatively with H3K79me2 during transcriptional elongation

• DNA repair mechanisms:

  • H3K36me3 recruits hMutSα through the hMSH6 PWWP domain to facilitate DNA mismatch repair

  • The histone mark's cell cycle-dependent abundance (maximum in early S phase) coordinates with DNA replication timing

  • May interact with other DNA damage response pathways through recruitment of additional repair factors

• Chromatin organization:

  • Influences nucleosome positioning and chromatin accessibility

  • May work with other modifications to define chromatin territories and boundaries

  • Potentially impacts higher-order chromatin structure through reader protein interactions

Understanding these complex interactions requires integrated analysis of multiple histone modifications across different cellular contexts and conditions.

What methodologies can be employed to study the dynamic changes of H3K36me3 throughout the cell cycle?

To study dynamic changes of H3K36me3 throughout the cell cycle:

• Cell synchronization techniques:

  • Use double thymidine block, nocodazole treatment, or serum starvation/release to obtain populations enriched at specific cell cycle stages

  • Employ Fluorescence Activated Cell Sorting (FACS) to isolate cells based on DNA content

• Time-resolved analyses:

  • Perform ChIP-Seq or CUT&Tag at defined time points after synchronization release

  • Combine with immunofluorescence microscopy to visualize H3K36me3 distribution changes

  • Implement pulse-chase experiments with labeled histones to track incorporation and modification

• Single-cell approaches:

  • Apply single-cell technologies to capture heterogeneity within populations

  • Combine with cell cycle markers to correlate H3K36me3 patterns with cell cycle stages

  • Implement live-cell imaging with fluorescently tagged readers of H3K36me3

• Quantitative assessments:

  • Measure global levels of H3K36me3 by quantitative Western blotting at different cell cycle stages

  • Employ mass spectrometry to quantify modification levels without antibody bias

These methodologies provide complementary information on the temporal dynamics of H3K36me3 regulation throughout the cell cycle.

How can researchers investigate the functional relationship between H3K36me3, SETD2, and DNA mismatch repair pathways?

To investigate the functional relationship between H3K36me3, SETD2, and DNA mismatch repair:

• Genetic manipulation approaches:

  • Generate SETD2 knockout/knockdown cells to eliminate H3K36me3

  • Create point mutations in the hMSH6 PWWP domain (e.g., W105A/W106A or Y103A) to disrupt interaction with H3K36me3

  • Perform rescue experiments with wild-type or catalytically inactive SETD2

• Protein-protein interaction studies:

  • Use co-immunoprecipitation to detect interactions between hMutSα and H3K36me3-modified nucleosomes

  • Perform pull-down assays with H3K36me3 peptides to identify additional interacting proteins

  • Apply proximity labeling techniques to identify proteins near H3K36me3 marks in vivo

• Functional assays:

  • Measure microsatellite instability (MSI) in SETD2-deficient cells

  • Determine mutation frequency at specific loci (e.g., HPRT) following manipulation of the H3K36me3-MMR pathway

  • Assess DNA damage response and repair kinetics following introduction of mismatches

• Genomic approaches:

  • Perform ChIP-Seq for H3K36me3 and MMR proteins to map their genomic co-occurrence

  • Analyze mutation spectra and frequencies in regions with varying H3K36me3 levels

  • Apply OK-Seq to correlate replication origins with H3K36me3 distribution and mutation rates

These integrated approaches provide mechanistic insights into how H3K36me3 coordinates with the DNA mismatch repair machinery to maintain genomic stability.

How might single-molecule and single-cell technologies advance our understanding of H3K36me3 function?

Single-molecule and single-cell technologies offer promising avenues to deepen our understanding of H3K36me3:

• Single-cell epigenomics:

  • Single-cell ChIP-Seq or CUT&Tag for H3K36me3 can reveal cell-to-cell variation in modification patterns

  • Correlation with single-cell transcriptomics can uncover relationships between H3K36me3 distribution and gene expression at unprecedented resolution

  • Integration with cell cycle markers can map dynamics without population synchronization artifacts

• Super-resolution microscopy:

  • Visualization of H3K36me3 distribution within nuclear territories at nanoscale resolution

  • Real-time tracking of H3K36me3 readers (e.g., hMSH6) to study kinetics and dynamics of interactions

  • Multi-color imaging to examine co-localization with DNA replication and repair machinery

• Single-molecule biochemistry:

  • Direct observation of SETD2 activity on individual nucleosomes

  • Real-time monitoring of reader protein binding to H3K36me3-modified chromatin

  • Force spectroscopy to measure binding strengths and kinetics of H3K36me3-protein interactions

These technologies promise to reveal heterogeneity, dynamics, and mechanistic details that are obscured in population-based analyses, potentially leading to novel insights into H3K36me3 function.

What therapeutic implications arise from understanding the role of H3K36me3 in DNA repair and genomic stability?

The functional connection between H3K36me3 and DNA repair suggests several therapeutic implications:

• Cancer therapeutics:

  • SETD2 mutations occur in various cancers, including clear cell renal cell carcinoma, suggesting potential vulnerabilities

  • H3K36me3-deficient tumors may display distinct DNA repair deficiencies that could be targeted synthetically lethal approaches

  • Combination therapies targeting both MMR pathways and H3K36me3-deficient cells could enhance therapeutic efficacy

• Biomarker development:

  • H3K36me3 levels could serve as biomarkers for DNA repair capacity in tumors

  • Alterations in H3K36me3 distribution patterns might predict response to certain chemotherapeutics

  • Combined assessment of SETD2 status and H3K36me3 levels could guide personalized treatment strategies

• Drug discovery opportunities:

  • Development of small molecules to modulate SETD2 activity or mimic H3K36me3 recognition

  • Targeting downstream effectors in H3K36me3-dependent pathways

  • Creating synthetic H3K36me3 mimetics to restore function in SETD2-deficient contexts