Di-Methyl-Histone H3 (Lys27) Antibody

What is Di-Methyl-Histone H3 (Lys27) and what is its biological significance?

Di-methylation of histone H3 at lysine 27 (H3K27me2) is a critical epigenetic modification associated primarily with transcriptional repression. Histone H3 is one of the four core histones (H2A, H2B, H3, and H4) that make up the nucleosome core particle, which consists of 146 bp of DNA wrapped around an octamer of these core histones .

H3K27me2 is typically found at the promoters of polycomb-repressed genes and at inactive but transcriptionally poised genes. This modification plays a key role in the regulation of facultative heterochromatin, stem cell pluripotency, and cellular differentiation . Unlike activating histone marks, H3K27me2 modification usually results in a repressive chromatin state that prevents gene expression .

How is Di-Methyl-Histone H3 (Lys27) created and regulated in cells?

The di-methylation of H3K27 is catalyzed by the polycomb repressor complex 2 (PRC2), which contains either EZH1 or EZH2 methyltransferase proteins as its catalytic subunit . This enzyme complex is responsible for adding methyl groups to lysine 27 of histone H3, producing mono-, di-, and tri-methylated states.

The regulation of H3K27me2 levels involves a dynamic balance between methylation by PRC2 and demethylation by histone demethylases. Several demethylases, including those from the JMJD family, can remove methyl groups from H3K27, demonstrating that methylation is a reversible epigenetic marker . This reversibility is crucial for dynamic gene regulation during development and cellular responses to environmental stimuli.

What distinguishes antibodies targeting Di-Methyl-Histone H3 (Lys27) from other histone modification antibodies?

Di-Methyl-Histone H3 (Lys27) antibodies are specifically generated to recognize H3 when di-methylated at lysine 27. High-quality antibodies like the D18C8 XP® Rabbit mAb detect endogenous levels of H3K27me2 with minimal cross-reactivity to other methylation states .

These antibodies differ from other histone modification antibodies in their epitope specificity and application optimization. While some H3K27me2 antibodies may show cross-reactivity with mono-methylated Lys27, they typically do not cross-react with non-methylated or tri-methylated Lys27 . Furthermore, well-characterized antibodies do not cross-react with other methylated residues such as histone H3 Lys4, Lys9, Lys36, or histone H4 Lys20 .

What are the optimal conditions for using Di-Methyl-Histone H3 (Lys27) antibodies in ChIP experiments?

For optimal Chromatin Immunoprecipitation (ChIP) results with H3K27me2 antibodies, the following conditions are recommended:

For ChIP-qPCR analysis with H3K27me2 antibodies, primers should be designed for both positive controls (promoters of inactive genes like HBB and coding regions of inactive genes like MYOD1) and negative controls (promoters of active genes like GAPDH and EIF4A2) . This approach allows proper assessment of enrichment specificity, as H3K27me2 is preferentially present at silent genes .

How can I validate the specificity of a Di-Methyl-Histone H3 (Lys27) antibody?

Validating antibody specificity is crucial for reliable experimental results. A comprehensive validation approach includes:

• Peptide competition assays: Test antibody binding in the presence of competing peptides with different methylation states of H3K27 (non-methylated, mono-, di-, and tri-methylated).

• Western blot analysis: Perform western blots using recombinant histone H3 variants with defined methylation states and acid extracts from cells with known H3K27 methylation patterns .

• Cross-reactivity testing: Examine potential cross-reactivity with other methylated lysine residues in histone H3 and other histones. Quality antibodies should not cross-react with mono-methylated, di-methylated, or tri-methylated histone H3 Lys4, Lys9, Lys36, or histone H4 Lys20 .

• ChIP-qPCR at known genomic loci: Perform ChIP-qPCR at genomic regions known to be enriched or depleted for H3K27me2 to confirm antibody specificity in chromatin context .

What applications are supported by Di-Methyl-Histone H3 (Lys27) antibodies and what are the recommended dilutions?

Di-Methyl-Histone H3 (Lys27) antibodies support multiple research applications with specific optimal dilutions:

These dilutions represent starting points and may require optimization based on specific experimental conditions, antibody lot, and sample type . For applications like ChIP-seq, it's advisable to validate antibody performance with ChIP-qPCR before proceeding to sequencing.

How do I analyze ChIP-seq data for Di-Methyl-Histone H3 (Lys27) pattern identification?

Analysis of H3K27me2 ChIP-seq data requires a systematic approach:

• Quality control: Assess sequencing data quality, mapping rates, and library complexity to ensure reliable results.

• Peak calling: Use appropriate algorithms that account for the broad distribution pattern typical of H3K27me2, which differs from sharp transcription factor binding peaks.

• Genomic distribution analysis: Examine the distribution of H3K27me2 across genomic features (promoters, gene bodies, enhancers) to identify patterns of enrichment.

• Integration with gene expression data: Correlate H3K27me2 patterns with RNA-seq data to validate the expected inverse relationship between H3K27me2 enrichment and gene expression.

• Quantitative assessment: Calculate the recovery as a percentage of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis) . Higher recovery at inactive genes (like HBB and MYOD1) compared to active genes (GAPDH and EIF4A2) confirms the expected H3K27me2 distribution pattern .

What are the characteristic genomic distribution patterns of H3K27me2?

H3K27me2 exhibits specific genomic distribution patterns that differ from other histone modifications:

• Promoter regions: H3K27me2 levels are highest at the promoters of polycomb-repressed genes .

• Bivalent domains: H3K27me2 can be found at the promoters of inactive but transcriptionally poised genes that also contain the active tri-methyl-histone H3 Lys4 modification, forming "bivalent domains" .

• Silent genes: ChIP-qPCR analyses demonstrate significantly higher enrichment of H3K27me2 at promoters of inactive genes (like HBB) and coding regions of inactive genes (like MYOD1) compared to active genes .

• Facultative heterochromatin: H3K27me2 is associated with the formation and maintenance of facultative heterochromatin, contributing to developmental gene silencing .

Understanding these patterns is essential for proper interpretation of ChIP-seq results and for distinguishing between technical artifacts and biologically meaningful enrichment.

How can I address cross-reactivity issues with Di-Methyl-Histone H3 (Lys27) antibodies?

Cross-reactivity is a common challenge with histone modification antibodies. To address this issue:

• Select highly specific antibodies: Choose antibodies like D18C8 XP® Rabbit mAb that have been rigorously validated for specificity. Note that even well-characterized antibodies may show some cross-reactivity with mono-methylated Lys27 or histone H2B when di-methylated on Lys5 .

• Include peptide controls: Use modified and unmodified peptide controls in your experiments to assess and account for cross-reactivity.

• Perform validation experiments: Conduct specificity tests using recombinant histones with defined modifications or cell lines with genetically modified methyltransferases.

• Use antibody combinations: In some cases, using multiple antibodies targeting the same modification but raised against different epitopes can improve specificity through consensus detection.

• Consider alternative detection methods: For applications requiring absolute specificity, consider mass spectrometry-based approaches like the Mod Spec® Service, which can distinguish between closely related modifications without antibody limitations .

What methods are available for quantifying global H3K27me2 levels in cells?

Several methods are available for quantifying global H3K27me2 levels:

• High-Content Analysis (HCA): HCA assays enable primary high-throughput screening to identify, profile, and optimize cellular active small molecule inhibitors targeting histone methyltransferases that affect H3K27me2 levels .

• AlphaLISA assay: This chemiluminescent assay uses luminescent oxygen-channeling chemistry where the analyte is captured by a biotinylated antibody bound to streptavidin-coated donor beads and a second antibody conjugated to AlphaLISA acceptor beads .

• LanthaScreen assay: This technique employs terbium-based time-resolved fluorescence resonance energy transfer (TR-FRET) technology in conjunction with the Baculovirus-mediated gene transduction of mammalian cells (BacMam) gene delivery system .

• Western blotting: Using histone extracts and normalized loading, western blotting can provide semi-quantitative assessment of global H3K27me2 levels.

• Fluorescent polarization assays: The HiLite™ Histone H3 Methyl-Lys9/Lys27 Binding Assay allows measurement of the dissociation constant (Kd) of proteins binding to methyl-Lys27 of histone H3 through fluorescence polarization .

How do I design experiments to study the interplay between H3K27me2 and other histone modifications?

To investigate the relationship between H3K27me2 and other histone modifications:

• Sequential ChIP (Re-ChIP): Perform immunoprecipitation with an H3K27me2 antibody followed by a second immunoprecipitation with antibodies against other histone modifications to identify regions with co-occurrence of multiple marks.

• Integrated ChIP-seq analysis: Generate ChIP-seq data for multiple histone modifications in the same cell type and use computational approaches to identify patterns of co-occurrence or mutual exclusivity.

• Methylation state profiling: Use the Methyl-Histone H3 (Lys27) Antibody Sampler Kit, which contains antibodies specific to different methylation states (mono-, di-, and tri-) of H3K27, allowing comprehensive profiling of methylation patterns at this residue .

• Functional manipulation: Use inhibitors of specific histone-modifying enzymes or genetic approaches (CRISPR-Cas9) to alter one modification and examine the consequences for other modifications, including H3K27me2.

• Single-cell approaches: Consider new technologies for single-cell epigenomic profiling to examine the heterogeneity and correlation of different histone modifications at the single-cell level.

What are the latest methodological advances for studying H3K27me2 in limited cell numbers?

Recent technological developments have improved the ability to study H3K27me2 in samples with limited material:

• CUT&RUN and CUT&Tag: These techniques offer higher sensitivity than traditional ChIP, requiring fewer cells and less antibody. Several Di-Methyl-Histone H3 (Lys27) antibodies have been validated for CUT&RUN Assay and CUT&Tag approaches .

• Low-input ChIP-seq protocols: Modified ChIP-seq protocols have been developed specifically for H3K27me2 and other histone modifications that work with as few as 1,000-10,000 cells.

• Single-cell ChIP-seq: Although challenging, new approaches are being developed to assess H3K27me2 and other histone modifications at the single-cell level, providing insights into cellular heterogeneity.

• Carrier-based ChIP: Using exogenous carrier chromatin or proteins can improve chromatin immunoprecipitation efficiency for small cell numbers.

• Microfluidic approaches: Microfluidic devices can facilitate histone modification analysis from limited samples by reducing reaction volumes and increasing efficiency.

When working with limited samples, it is critical to use antibodies with documented performance in low-input applications and to include appropriate controls to distinguish genuine signal from technical noise.

How can computational approaches enhance the interpretation of H3K27me2 data?

Advanced computational methods can significantly improve H3K27me2 data analysis:

• Integrative analysis: Combining H3K27me2 data with other epigenetic marks, transcription factor binding, and gene expression data can reveal functional relationships and regulatory networks.

• Machine learning algorithms: These can identify complex patterns in H3K27me2 distribution and predict functional outcomes based on histone modification patterns.

• Comparative epigenomics: Cross-species analysis of H3K27me2 patterns can highlight evolutionarily conserved regulatory mechanisms and species-specific adaptations.

• Trajectory inference: For developmental studies, computational trajectory inference methods can map changes in H3K27me2 patterns during cell fate transitions.

• Three-dimensional chromatin structure integration: Correlating H3K27me2 patterns with Hi-C or other chromosome conformation capture data can reveal relationships between this histone modification and three-dimensional genome organization.

These computational approaches require careful experimental design, appropriate controls, and validation to ensure biological relevance of the identified patterns.