Histone H3R2me2s Antibody

What is Histone H3R2me2s and what is its biological significance?

H3R2me2s refers to symmetric dimethylation of arginine 2 on histone H3, a post-translational modification of the histone H3 N-terminal tail that is evolutionarily conserved from yeast to humans. This modification plays a crucial role in transcriptional regulation by influencing chromatin structure and accessibility. H3R2me2s is tightly correlated with H3K4me3 at active promoters throughout the genome and promotes global transcription at the 1-cell stage during zygotic genome activation . Unlike its asymmetric counterpart (H3R2me2a), H3R2me2s allows interaction with chromatin-associated proteins like WDR5, functioning as part of a binary switch that regulates gene expression . The presence of H3R2me2s creates a permissive environment for transcription by facilitating the recruitment of transcriptional machinery.

How does H3R2me2s differ from other histone H3 arginine methylation states?

H3R2me2s (symmetric dimethylation) differs structurally and functionally from H3R2me0 (unmethylated), H3R2me1 (monomethylated), and H3R2me2a (asymmetric dimethylation):

• Structural differences: In symmetric dimethylation, one methyl group is added to each of the two terminal nitrogen atoms of the guanidino group, while in asymmetric dimethylation, both methyl groups are added to the same nitrogen atom.

• Protein interactions: H3R2me2s allows interaction with WDR5 (a component of histone methyltransferase complexes), while H3R2me2a prevents this interaction . This creates a binary methylation switch that regulates downstream protein recruitment.

• Genomic distribution: H3R2me2s is found in euchromatic regions and associated with active transcription, while H3R2me2a is detected at low levels in centromeric regions (heterochromatin) .

• Functional outcomes: H3R2me2s promotes transcriptional activation and is compatible with H3K4 trimethylation, whereas H3R2me2a and H3K4me3 are generally mutually exclusive modifications .

What applications are most effective for H3R2me2s antibody use in epigenetic research?

H3R2me2s antibodies have been successfully employed in multiple applications:

For all applications, validation of antibody specificity is crucial to distinguish between symmetric and asymmetric dimethylation of H3R2.

How should researchers validate the specificity of an H3R2me2s antibody?

Thorough validation of H3R2me2s antibodies is essential to ensure reliable experimental results:

• Peptide dot blot analysis: Test antibody against various histone H3 peptides with different modifications (H3R2me0, H3R2me1, H3R2me2a, H3R2me2s, H3R2me2sK4me3). A specific α-pan-H3R2me2s antibody should show >25-fold preference for H3R2me2s over H3R2me2a .

• Peptide competition assays: Pre-incubate the antibody with specific peptides to demonstrate signal competition by the target peptide but not by unrelated peptides. This can be performed by Western blot of nuclear extracts with competing peptides .

• Western blot analysis: Confirm the antibody detects a band of the expected molecular weight (approximately 15-17 kDa) in nuclear extracts .

• Cross-reactivity testing: If working with non-human samples, verify species cross-reactivity. Most commercial H3R2me2s antibodies react with human, mouse, and rat samples due to high sequence conservation .

• Immunofluorescence patterns: Verify that the subcellular localization matches expected patterns (nuclear staining coincident with DNA/DAPI) .

How does the co-occurrence of H3R2me2s and H3K4me3 affect chromatin regulation?

The co-occurrence of H3R2me2s and H3K4me3 creates a unique chromatin environment with specific functional implications:

• Co-existence on the same histone tail: Specific antibodies (α-H3R2me2sK4me3) that recognize only the double modification confirm that H3R2me2s and H3K4me3 can exist simultaneously on the same histone H3 tail .

• Evolutionary conservation: The co-occurrence of these modifications is conserved throughout eukaryotic evolution, from yeast to humans, suggesting fundamental importance in chromatin regulation .

• Enrichment at antigen receptor loci: H3R2me2sK4me3 is highly enriched at antigen receptor gene segments poised for V(D)J recombination in developing lymphoid cells .

• Protein recruitment: The RAG2 PHD finger specifically binds to histone H3 tails carrying both modifications, facilitating V(D)J recombination. This demonstrates how the dual modification creates a specific binding platform for regulatory proteins .

• Dependency relationship: H3K4 is required for H3R2me2s deposition. Mutating H3K4 to alanine (H3K4A) completely abolishes H3R2me2s, indicating a hierarchical relationship between these modifications .

This co-occurrence pattern differs significantly from H3R2me2a and H3K4me3, which are mutually exclusive modifications, highlighting the distinct regulatory functions of symmetric versus asymmetric arginine dimethylation.

What is the binary arginine methylation switch and its functional significance?

The binary arginine methylation switch involving H3R2me2s and H3R2me2a serves as a sophisticated regulatory mechanism:

• Differential protein binding: H3R2me0 (unmethylated), H3R2me1, and H3R2me2s all have near equal affinity for WDR5, whereas H3R2me2a is not compatible with WDR5 binding .

• Structural basis: When WDR5 binds me2s-L-Arg, the guanidino moiety is rotated 180° about the Nδ-Cε bond compared to its orientation in me1-L-Arg complexes. This rotation allows water A to be retained in the WIN site while displacing water B, resulting in different hydrogen bonding patterns .

• Quantitative differences: H3R2me2s makes 39 van der Waals contacts with WDR5 and one with both water A and water B—nine more than compared to the me1-L-Arg structure. These structural differences explain the selective binding properties .

• Functional consequences: Since WDR5 is a component of histone methyltransferase complexes like MLL/SET1 that methylate H3K4, this switch regulates H3K4 methylation and downstream gene expression .

• Developmental implications: The binary switch may regulate critical developmental processes. For example, in zygotic genome activation, H3R2me2s promotes global transcription at the 1-cell stage .

This binary switch mechanism represents a sophisticated epigenetic regulatory system that influences chromatin structure, protein recruitment, and ultimately gene expression patterns.

How does H3R2me2s contribute to zygotic genome activation?

H3R2me2s plays a critical role in early embryonic development, particularly during zygotic genome activation (ZGA):

• Temporal presence: H3R2me2s is detected on chromosomes in MII oocytes and is consistently observed throughout the pronuclear (PN) stages of early embryo development .

• Transcriptional promotion: H3R2me2s promotes global transcription at the 1-cell stage, referred to as minor zygotic genome activation .

• Functional necessity: Experiments using histone mutants (H3.3R2A-EGFP) that cannot be methylated at R2 showed that prevention of H3R2me2s acquisition resulted in impairment of minor ZGA and 2-cell arrest in embryos .

• Chromatin localization: H3R2me2s is predominantly localized in euchromatic regions, consistent with its role in creating a transcriptionally permissive environment .

• Differential distribution: Unlike H3R2me2a, which is found in heterochromatic centromere regions, H3R2me2s is associated with transcriptionally active regions, supporting its role in genome activation .

This evidence suggests that H3R2me2s functions as a critical epigenetic mark that contributes to establishing a transcriptionally permissive chromatin state necessary for activating the embryonic genome after fertilization.

What are optimal ChIP-seq conditions for profiling genome-wide H3R2me2s distribution?

For successful ChIP-seq analysis of H3R2me2s distribution, researchers should consider the following protocol optimizations:

Data analysis should include correlation with H3K4me3 peaks, as these modifications are tightly correlated. Compare with H3R2me2a distribution to understand the binary switch dynamics in a genomic context. Focus analysis on promoters and enhancers to identify regulatory elements marked by H3R2me2s.

What factors influence the deposition and removal of H3R2me2s?

The regulation of H3R2me2s involves several key factors:

• Dependency on H3K4: H3K4 is absolutely required for H3R2me2s deposition. Mutating H3K4 to alanine (H3K4A) completely abolishes H3R2me2s, indicating that the presence of H3K4 (either unmethylated or methylated) is necessary for H3R2me2s deposition .

• Enzymatic writers: While the search results don't explicitly identify the specific enzymes responsible for H3R2me2s deposition, protein arginine methyltransferases (PRMTs) are the likely candidates. Type II PRMTs typically catalyze symmetric dimethylation of arginine residues.

• Coordinated regulation: The tight correlation between H3R2me2s and H3K4me3 suggests coordinated regulation of these modifications, potentially through protein complexes that recognize or deposit both marks .

• Developmental regulation: In early embryonic development, H3R2me2s levels appear to be developmentally regulated, as evidenced by their role in zygotic genome activation .

• Genomic context: The predominant localization of H3R2me2s in euchromatic regions suggests that chromatin accessibility and other histone modifications may influence its deposition and maintenance .

Understanding the complete enzymatic machinery and regulatory networks governing H3R2me2s deposition and removal requires further research, as the current literature doesn't fully elucidate all mechanisms involved.

What are the key differences in protocols when using H3R2me2s antibodies across different applications?

Different applications require specific protocol adjustments:

Each application requires careful optimization, and conditions may need adjustment based on sample type, fixation method, and antibody lot.

How does sample preparation affect H3R2me2s antibody performance?

Sample preparation significantly impacts H3R2me2s antibody performance across applications:

• Fixation effects: For immunohistochemistry and immunofluorescence, overfixation can mask epitopes. For H3R2me2s detection, standard fixation (4% paraformaldehyde for cells or formalin for tissues) followed by appropriate antigen retrieval is recommended .

• Nuclear extraction: For Western blot analysis, nuclear extraction is crucial for optimal detection of histone modifications. Cytoplasmic proteins can dilute the signal if whole-cell lysates are used .

• Cross-linking for ChIP: The standard 1% formaldehyde for 10 minutes at room temperature works well for most histone modifications including H3R2me2s. Over-crosslinking can reduce antibody accessibility .

• Storage conditions: Antibodies should be aliquoted and stored at -20°C to avoid repeated freeze/thaw cycles. For long-term storage of samples containing H3R2me2s, snap freezing and storage at -80°C is recommended to preserve modification integrity .

• Blocking reagents: For Western blots, 3% nonfat dry milk in TBST has been successfully used. For immunostaining applications, BSA-based blocking buffers may provide lower background .

Proper sample preparation is essential for maintaining epitope integrity and accessibility, particularly for methylation modifications that can be sensitive to preparation conditions.

How can researchers distinguish between H3R2me2s and H3R2me2a in experimental settings?

Distinguishing between these structurally similar modifications requires careful experimental design:

• Highly specific antibodies: Use antibodies with validated specificity. For example, α-pan-H3R2me2s antibodies should show >25-fold preference for H3R2me2s over H3R2me2a .

• Peptide competition assays: Perform Western blot or immunostaining with antibody pre-incubated with specific peptides (H3R2me2s vs. H3R2me2a) to demonstrate specific competition .

• Dot blot analysis: Test antibody specificity against a panel of synthetic peptides containing different H3R2 modifications to quantify specificity and cross-reactivity .

• Functional assays: Leverage the binary switch model where H3R2me2s allows interaction with WDR5 while H3R2me2a prevents it. Protein interaction assays (e.g., pulldown experiments with recombinant WDR5) can differentiate between these modifications .

• Immunofluorescence patterns: H3R2me2s is predominantly found in euchromatic regions, while H3R2me2a is detected at low levels in centromeric regions, providing a spatial distinction that can be visualized .

• Mass spectrometry: For definitive identification, liquid chromatography-tandem mass spectrometry (LC-MS/MS) can distinguish between symmetric and asymmetric dimethylation based on their different fragmentation patterns.

These approaches can be used individually or in combination to reliably distinguish between these two important regulatory modifications.