Histone H3K36me2 Antibody

What is the genomic distribution pattern of H3K36me2 compared to H3K36me3?

H3K36me2 and H3K36me3 exhibit distinct genomic localization patterns that reflect their different biological functions. H3K36me2 covers areas downstream of transcription start sites through the first intron, followed by a marked switch to H3K36me3 after the first splice junction. Additionally, H3K36me2 demarcates intergenic regions where H3K36me3 is absent . This distinct localization pattern suggests non-redundant functions between these methylation states.

Within genic regions of actively transcribed genes, the transition from H3K36me2 to H3K36me3 correlates with splicing boundaries, suggesting potential roles in RNA processing regulation. Quantitative profiling studies reveal that:

This distribution pattern indicates that H3K36me2 may function in regulating transcription initiation and early elongation, while H3K36me3 has been more closely associated with RNA splicing, prevention of intragenic transcription, and DNA damage repair processes .

Which enzymes are responsible for H3K36me2 deposition and removal?

The dynamic regulation of H3K36me2 involves specific enzymes for both deposition and removal of this mark. Based on current research findings:

H3K36me2 Writers (Methyltransferases):

• NSD1 (Nuclear receptor binding SET domain protein 1)

• NSD2 (WHSC1/MMSET)

• NSD3

• ASH1L (relatively H3K36M-insensitive)

H3K36me2 Erasers (Demethylases):

• KDM2A (Lysine Demethylase 2A)

The antagonistic activities of these enzymes maintain appropriate levels of H3K36me2 in different genomic contexts. For instance, NSD1 and NSD2 function as the primary H3K36me2 methyltransferases, while SETD2 is primarily responsible for H3K36me3 deposition . Notably, manipulation of NSD2 and KDM2A has been shown to regulate the epithelial-mesenchymal spectrum through their opposing catalytic activities on H3K36me2, with minimal effects on H3K36me3 levels .

How can researchers validate the specificity of H3K36me2 antibodies?

Antibody specificity is crucial for accurate H3K36me2 detection. Researchers should implement multiple validation approaches:

Peptide Competition Assays:

• Pre-incubate the H3K36me2 antibody with increasing concentrations of specific H3K36me2 peptides

• Include other methylated H3 peptides (H3K36me1, H3K36me3, H3K9me2) as negative controls

• Observe signal reduction only with the specific H3K36me2 peptide

Knockout/Knockdown Validation:

• Generate NSD1/NSD2 double-knockout cells (specifically depleted of H3K36me2)

• Compare antibody signal between wild-type and knockout cells by Western blot and ChIP

• A significant reduction in signal should be observed in knockout cells

Cross-reactivity Assessment:
Create a validation matrix testing antibody against multiple histone modifications:

Use of synthetic histone peptides or recombinant histones with defined modifications provides the gold standard for specificity validation. Western blot analysis should show a single band at approximately 17 kDa corresponding to histone H3 .

What are optimal applications and conditions for H3K36me2 antibody use?

H3K36me2 antibodies can be utilized across multiple experimental platforms with specific optimization requirements:

Western Blot:

• Recommended dilution: 1-2 μg/ml

• Sample preparation: Use high salt/sonication protocol for chromatin-bound proteins, as many are not soluble in low salt nuclear extracts

• Include appropriate loading controls (total H3 or H4)

Chromatin Immunoprecipitation (ChIP):

• Crosslinking: 1% formaldehyde for 10 minutes at room temperature

• Sonication: Optimize to achieve 200-500 bp fragments

• Antibody amount: 2-5 μg per ChIP reaction

• Include appropriate controls (IgG, input)

• Consider spike-in normalization for quantitative comparisons between conditions

Immunofluorescence:

• Fixation: 4% paraformaldehyde followed by permeabilization

• Blocking: Use BSA-based blocking buffer to reduce background

• Antibody concentration should be empirically determined

• Include peptide competition controls to verify specificity

Each application requires specific validation and optimization to ensure reliable and reproducible results.

How does H3K36me2 contribute to transcriptional regulation mechanisms?

H3K36me2 plays critical roles in transcriptional regulation through multiple mechanisms:

Recruitment of Histone Deacetylase Complexes:
H3K36me2 is sufficient to target the histone deacetylase complex Rpd3S both in vitro and in vivo . This recruitment maintains a hypoacetylated state at coding regions throughout the genome, which suppresses cryptic transcription initiation from within gene bodies . This function highlights how H3K36me2 contributes to transcriptional fidelity.

Enhancer Regulation:
H3K36me2 has been implicated in the regulation of enhancers associated with master regulators of epithelial-mesenchymal state . Modulation of global H3K36me2 levels through the opposing activities of NSD2 (writer) and KDM2A (eraser) can reprogram enhancer landscapes, affecting cellular plasticity and differentiation potential.

Interaction with DNA Methylation:
H3K36me2 is required for the localization of the DNA methyltransferase DNMT3A to maintain DNA methylation levels at intergenic regions . Consequently, loss of H3K36me2 through NSD1/2 knockout or H3K36M expression results in genome-wide intergenic DNA hypomethylation , demonstrating the interconnected nature of histone and DNA modification pathways.

What is the role of H3K36me2 in heterochromatin formation?

Recent research has revealed unexpected roles for H3K36me2 in heterochromatin formation:

H3K36me2 has been found enriched in pericentromeric heterochromatin in some mouse cell lines . The mechanism of this heterochromatin targeting involves NSD2 (a H3K36 methyltransferase) recruitment mediated through imitation switch (ISWI) chromatin remodeling complexes, specifically BAZ1B-SMARCA5 (WICH) .

The BAZ1B component of these complexes directly binds to AT-rich DNA via domains containing AT-hook-like motifs, facilitating the recruitment of NSD2 to heterochromatic regions . The abundance and stoichiometry of NSD2, SMARCA5, and BAZ1B determine the localization of H3K36me2 in different cell types.

Developmental studies have observed H3K36me2 heterochromatin localization in mouse embryos at the two- to four-cell stages, suggesting physiological relevance during early embryonic development . This unexpected enrichment in heterochromatin regions challenges previous assumptions about H3K36me2 being primarily associated with euchromatic regions.

How does H3K36me2 depletion contribute to cancer mechanisms?

H3K36me2 depletion has emerged as a critical mechanism in cancer development, particularly in contexts involving histone H3K36M mutations:

Epigenomic Reprogramming:
Depletion of H3K36me2, whether through H3K36M mutation or NSD1/2 knockout, leads to significant epigenomic reprogramming. This includes redistribution of H3K27 trimethylation (H3K27me3) and consequent changes in gene expression patterns . These alterations in the epigenetic landscape contribute to oncogenic transformation by affecting key cellular processes including differentiation and proliferation.

Differentiation Blockade:
Loss of H3K36me2 through knockout of NSD1/2 phenocopies the differentiation blockade induced by H3K36M mutation . This suggests that depletion of H3K36me2, rather than H3K36me3, is the primary mechanism by which H3K36M mutations block differentiation in certain cancer contexts.

Therapeutic Vulnerability:
Cells with depleted H3K36me2 (either through H3K36M mutation or NSD1/2 knockout) exhibit hypersensitivity to DNA-hypomethylating agents . This creates a potential therapeutic vulnerability that could be exploited in treating cancers characterized by H3K36M mutations or NSD1/2 dysfunction.

Metastatic Progression:
H3K36me2 has been identified as a key determinant in epithelial plasticity and metastatic progression . Mass spectrometry analysis revealed that H3K36me2 was the only histone post-translational modification to exhibit significant differences between epithelial and mesenchymal cell states across multiple cell lines . Treatment with TGF-β, a potent inducer of epithelial-to-mesenchymal transition, also resulted in global increases in H3K36me2 .

How do experimental approaches to manipulate H3K36me2 compare in research settings?

Researchers employ various experimental approaches to manipulate H3K36me2 levels, each with distinct advantages and considerations:

Genetic Knockout/Knockdown of Methyltransferases:

• Targeting NSD1 and NSD2 through double knockout specifically depletes H3K36me2 while largely preserving H3K36me3 levels

• Allows for distinguishing the specific roles of H3K36me2 from H3K36me3

• ChIP-seq profiling in these models reveals that drastic decreases in H3K36me2 are particularly pronounced at intergenic regions

Expression of H3K36M Mutant Histones:

• H3K36M oncohistone expression dominantly inhibits several H3K36-specific methyltransferases

• Results in decreases in all methylation states of H3K36, though to varying degrees

• H3K36me2 typically shows more complete depletion compared to H3K36me3

Modulation of Demethylases:

• Manipulation of KDM2A affects H3K36me2 levels with minimal impact on H3K36me3

• Can be used to examine the consequences of increased H3K36me2 levels

• Works antagonistically with NSD2 to regulate epithelial-mesenchymal identity

Comparative data from these approaches:

These distinct manipulation strategies enable researchers to dissect the specific contributions of different H3K36 methylation states to various biological processes .

What factors impact ChIP-seq quality with H3K36me2 antibodies?

ChIP-seq experiments with H3K36me2 antibodies require careful attention to several critical factors:

Antibody Quality and Specificity:
The choice of antibody is paramount for successful H3K36me2 ChIP-seq experiments. Polyclonal antibodies may provide higher sensitivity but can vary between lots, while monoclonal antibodies offer consistency but potentially lower affinity. Researchers should validate antibodies using peptide arrays to confirm specificity against related modifications (H3K36me1, H3K36me3) .

Chromatin Preparation:

• Fixation conditions significantly impact H3K36me2 ChIP-seq results

• Over-fixation can mask epitopes, while under-fixation may not preserve protein-DNA interactions

• Sonication parameters should be optimized to generate fragments of 200-500 bp

• Native ChIP (without crosslinking) may provide better resolution for histone modifications but requires careful nuclease digestion

Bioinformatic Analysis Considerations:

• H3K36me2 profiles should be normalized to input and total H3 ChIP to account for histone occupancy

• For differential analysis between conditions, spike-in normalization is recommended

• Peak calling algorithms optimized for broad marks rather than sharp peaks should be employed

• Integration with RNA-seq data can help correlate H3K36me2 patterns with transcriptional outcomes

Common Technical Artifacts:

• Fragment size bias can affect H3K36me2 distribution profiles

• PCR duplication during library preparation can create artificial enrichment

• GC content bias can affect mapping and lead to false positives/negatives

• Batch effects between experimental runs require appropriate normalization

Researchers should include appropriate controls, such as IgG ChIP, input DNA, and spike-in controls, to ensure robust and reproducible results.

How do different experimental systems affect H3K36me2 distribution patterns?

The distribution and function of H3K36me2 can vary significantly across different experimental systems:

Cell Type-Specific Patterns:
H3K36me2 distribution exhibits cell type-specific patterns, particularly in heterochromatin regions. For example, H3K36me2 enrichment in pericentromeric heterochromatin has been observed in some mouse cell lines but not others . These differences are influenced by the abundance and stoichiometry of NSD2, SMARCA5, and BAZ1B in different cell types .

Developmental Stage Variations:
In mouse embryos, H3K36me2 heterochromatin localization is observed specifically at the two- to four-cell stages , suggesting dynamic regulation during early development. Researchers studying H3K36me2 in developmental contexts should consider stage-specific patterns and regulation.

Species-Specific Considerations:
While H3K36me2 functions are broadly conserved, there are species-specific aspects to consider:

• In budding yeast, K36me2 is capable of directing histone deacetylation and repressing spurious transcripts

• In Arabidopsis thaliana, di- and tri-, but not mono-methylation on H3K36 marks actively transcribed genes

• In mammalian systems, H3K36me2 has broader distribution patterns including intergenic regions

Cancer vs. Normal Cells:
H3K36me2 patterns differ between cancer and normal cells, with H3K36M mutations in cancer cells leading to global depletion of H3K36me2 . Additionally, epithelial-mesenchymal transitions in cancer progression correlate with changes in H3K36me2 levels .

Researchers should carefully consider these system-specific variations when designing experiments and interpreting results related to H3K36me2 biology.