Histone H4R3me2a Antibody

What is Histone H4R3me2a and what role does it play in epigenetic regulation?

Histone H4R3me2a refers to the asymmetric dimethylation of arginine 3 on histone H4, a core component of the nucleosome. This specific modification is associated with transcriptional activation by nuclear hormone receptors and plays a central role in regulating gene expression. As nucleosomes wrap and compact DNA into chromatin, they limit DNA accessibility to cellular machineries that require DNA as a template. Histone H4R3me2a represents one of many post-translational modifications that constitute the "histone code" which regulates DNA accessibility. This modification specifically facilitates subsequent acetylation of histone H4 by the acetyltransferase p300, creating a sequential modification pattern that influences chromatin structure and gene transcription. Understanding this modification provides insight into mechanisms of transcriptional regulation and epigenetic control of gene expression .

What are the key enzymes responsible for establishing the H4R3me2a mark?

The asymmetric dimethylation of histone H4 at arginine 3 (H4R3me2a) is catalyzed primarily by protein arginine methyltransferases (PRMTs), specifically PRMT1 and PRMT6. These enzymes transfer methyl groups to the arginine residue in an asymmetric configuration. This enzymatic activity is distinct from symmetric dimethylation, which is catalyzed by different PRMT family members. The selective activity of PRMT1 and PRMT6 on histone H4R3 contributes to specific gene expression patterns associated with this modification. Understanding the enzymes responsible for establishing H4R3me2a is crucial for interpreting the biological significance of this mark in different cellular contexts and for developing potential inhibitors for research or therapeutic applications .

How does H4R3me2a differ from symmetric dimethylation (H4R3me2s)?

The key distinction between H4R3me2a and H4R3me2s lies in their molecular configuration and biological functions. Asymmetric dimethylation (H4R3me2a) involves the addition of two methyl groups to the same terminal nitrogen atom of the arginine guanidino group, while symmetric dimethylation (H4R3me2s) distributes the methyl groups across both terminal nitrogen atoms. These structural differences result in distinct biological outcomes: H4R3me2a is generally associated with transcriptional activation, whereas H4R3me2s typically correlates with transcriptional repression. The two modifications are established by different enzyme families (PRMT1/PRMT6 for asymmetric; PRMT5/PRMT7 for symmetric) and interact with different protein complexes. Importantly, antibodies raised against H4R3me2a show high specificity for the asymmetric mark and do not cross-react with the symmetric modification, as demonstrated by dot-blot analysis using methylation peptides, making them valuable tools for distinguishing between these functionally distinct epigenetic marks .

What criteria should researchers use when selecting a Histone H4R3me2a antibody?

When selecting a Histone H4R3me2a antibody, researchers should consider several critical factors to ensure experimental success. First, evaluate the antibody's specificity through documented validation data, particularly dot-blot analyses showing discrimination between asymmetric and symmetric dimethylation and other histone modifications. The search results show that validated antibodies have been tested in dot-blot assays to confirm specific recognition of the H4R3me2a mark without cross-reactivity to other methylation states . Second, consider the validated applications—whether the antibody has been successfully used in Western blotting (WB), immunofluorescence (IF), immunoprecipitation (IP), or chromatin immunoprecipitation (ChIP). According to the search results, commercially available antibodies have been validated for multiple applications with recommended dilutions (e.g., 1:500-1:2000 for WB) . Third, verify species reactivity—many H4R3me2a antibodies react with human, mouse, and rat samples due to the high conservation of histone sequences . Finally, consider the antibody's format (polyclonal vs. monoclonal) and host species, which will influence secondary antibody selection and potential background in your experimental system.

How can researchers validate the specificity of H4R3me2a antibodies?

Validation of H4R3me2a antibody specificity requires a multi-faceted approach to ensure reliable experimental results. The primary method is peptide competition assays, where synthetic peptides containing H4R3me2a modifications are used to block antibody binding, demonstrating specificity for the target epitope. Dot-blot analysis using arrays of modified histone peptides (H4R3me2a, H4R3me2s, H4R3me1, unmodified H4, and other modified histones) provides visual confirmation of binding specificity, as shown in the validation data from commercial antibodies . Western blot analysis should show detection of the appropriate ~11 kDa band in histone extracts, and signal reduction following treatment with demethylase enzymes or PRMT inhibitors further confirms specificity. ChIP-qPCR at known H4R3me2a-enriched genomic regions, compared with IgG control, demonstrates functional specificity. Finally, immunofluorescence microscopy can reveal expected nuclear localization patterns. Researchers should also verify antibody performance across different lots and compare results with alternative antibody sources to ensure reproducibility and minimize bias from batch-specific variations.

What are the optimal conditions for using H4R3me2a antibodies in Western blotting?

For optimal Western blotting results with H4R3me2a antibodies, researchers should follow these methodological considerations. Sample preparation is critical—histone extraction should use acid extraction methods (such as 0.2N HCl) or commercial histone extraction kits to isolate histones efficiently. For whole cell extracts, a high salt/sonication protocol is recommended as many chromatin-bound proteins are not soluble in low salt nuclear extracts . Use 15-18% SDS-PAGE gels to achieve proper separation of the low molecular weight histone proteins (~11 kDa). Transfer to PVDF membranes (rather than nitrocellulose) at lower voltage (30V) overnight at 4°C to ensure efficient transfer of small proteins. For blocking, 5% non-fat dry milk or BSA in TBST is suitable, with BSA preferred when using phospho-specific antibodies. The recommended dilution range for H4R3me2a antibodies is 1:500 to 1:2000 , though this should be optimized for each specific antibody. Incubation should occur overnight at 4°C to maximize specific binding. Use appropriate HRP-conjugated secondary antibodies (typically anti-rabbit IgG) at 1:5000 to 1:10000 dilution. Enhanced chemiluminescence detection systems are suitable for visualization, with exposure times adjusted based on signal strength.

What protocol optimizations improve ChIP-seq results with H4R3me2a antibodies?

To optimize ChIP-seq experiments using H4R3me2a antibodies, several protocol refinements are crucial. First, crosslinking conditions should be carefully calibrated—standard 1% formaldehyde for 10 minutes at room temperature works for most histone modifications, but H4R3me2a may benefit from dual crosslinking with 1.5 mM EGS (ethylene glycol bis[succinimidylsuccinate]) prior to formaldehyde treatment to better preserve protein-protein interactions. Sonication parameters should be optimized to generate chromatin fragments between 200-300 bp, with fragment size verified by agarose gel electrophoresis. For immunoprecipitation, use 3-5 μg of validated H4R3me2a antibody per 25 μg of chromatin, and include IgG controls and input samples for normalization. Incorporate spike-in controls with exogenous chromatin (e.g., Drosophila) and species-specific antibodies to account for experimental variation. Pre-clearing chromatin with protein A/G beads reduces non-specific binding. For washing steps, use increasingly stringent buffers to reduce background while preserving specific interactions. During library preparation, minimize PCR cycles to reduce amplification bias. For bioinformatic analysis, compare H4R3me2a peaks with known transcriptionally active regions, as this modification is associated with transcriptional activation by nuclear hormone receptors . Integration with RNA-seq data can provide functional correlations between H4R3me2a enrichment and gene expression levels.

How should immunofluorescence experiments be designed for H4R3me2a detection?

Successful immunofluorescence detection of H4R3me2a requires careful experimental design and execution. Cells should be grown on glass coverslips and fixed with 4% paraformaldehyde for 10 minutes at room temperature. Permeabilization with 0.2% Triton X-100 for 10 minutes allows antibody access to nuclear epitopes, while antigen retrieval using 10 mM sodium citrate buffer (pH 6.0) may enhance signal detection by exposing masked epitopes. Blocking with 5% normal serum from the same species as the secondary antibody (typically goat) for 1 hour reduces non-specific binding. For primary antibody incubation, use H4R3me2a antibody at dilutions of approximately 1:200 to 1:500 (optimized for each specific antibody) overnight at 4°C in a humidified chamber. Secondary antibody incubation should use fluorophore-conjugated antibodies (e.g., Alexa Fluor 488 or 594) at 1:500 to 1:1000 for 1 hour at room temperature protected from light. Include DAPI (1 μg/mL) for nuclear counterstaining. Mount slides with anti-fade mounting medium to preserve fluorescence. Critical controls include primary antibody omission, peptide competition with H4R3me2a-modified peptides, and comparison with cells treated with PRMT1/6 inhibitors to validate signal specificity. Confocal microscopy with z-stack acquisition provides optimal visualization of nuclear distribution patterns of H4R3me2a .

How does H4R3me2a interact with other histone modifications in gene regulation?

H4R3me2a operates within a complex network of histone modifications that collectively regulate gene expression. This asymmetric dimethylation has been shown to facilitate the subsequent acetylation of histone H4 by the acetyltransferase p300, establishing a sequential modification pattern critical for transcriptional activation . The relationship between H4R3me2a and histone acetylation represents a classic example of histone modification crosstalk, where one modification influences the deposition or removal of another. H4R3me2a typically co-occurs with active chromatin marks such as H3K4me3 and various histone acetylation marks (H3K9ac, H3K27ac, H4K5ac, H4K8ac, H4K12ac, and H4K16ac) at transcriptionally active promoters and enhancers. Conversely, H4R3me2a appears to be mutually exclusive with repressive marks like H3K9me3 and H3K27me3 in many genomic contexts. The presence of H4R3me2a can also affect nucleosome stability and positioning, further influencing chromatin accessibility. ChIP-seq analyses have revealed that H4R3me2a patterns often overlap with binding sites for transcription factors associated with nuclear hormone receptor signaling pathways, consistent with its role in transcriptional activation by these receptors. Understanding these interactions is crucial for deciphering the histone code and its impact on gene regulation in normal development and disease states.

What are the emerging roles of H4R3me2a in disease pathogenesis?

Emerging research has implicated aberrant H4R3me2a patterns in various disease pathologies, particularly in cancer development and progression. Altered expression or activity of PRMT1 and PRMT6, the enzymes responsible for H4R3me2a deposition, has been observed in multiple cancer types, including rectal cancer as indicated by immunohistochemical studies . These changes can disrupt normal H4R3me2a distribution, affecting gene expression programs that control cell proliferation, differentiation, and apoptosis. In hormone-dependent cancers such as breast and prostate cancer, H4R3me2a may play a particularly significant role due to its association with nuclear hormone receptor-mediated transcription. Dysregulation of the interplay between H4R3me2a and subsequent histone acetylation by p300 can contribute to oncogenic transformation by altering the expression of key tumor suppressor genes and oncogenes. Beyond cancer, emerging evidence suggests potential roles for H4R3me2a dysregulation in inflammatory disorders, neurodegenerative diseases, and developmental disorders, though these connections require further investigation. The continued development of highly specific H4R3me2a antibodies enables more precise mapping of this modification in disease tissues, potentially leading to new biomarkers or therapeutic targets. Inhibitors targeting PRMT1 and PRMT6 are being explored as potential therapeutic agents, highlighting the clinical relevance of understanding H4R3me2a biology in disease contexts.

How can H4R3me2a analysis be integrated into multi-omics research approaches?

Integration of H4R3me2a analysis into multi-omics research frameworks provides comprehensive insights into epigenetic regulation of cellular processes. Researchers should first generate H4R3me2a ChIP-seq datasets and align them with other epigenomic data including additional histone modifications (H3K4me3, H3K27ac), transcription factor binding sites (especially nuclear hormone receptors), and chromatin accessibility profiles (ATAC-seq, DNase-seq). This integration reveals the chromatin context in which H4R3me2a functions. Correlation with transcriptomic data (RNA-seq) can establish functional relationships between H4R3me2a enrichment and gene expression patterns, particularly focusing on genes involved in hormone response pathways given H4R3me2a's association with nuclear hormone receptor-mediated transcription . Proteomic approaches such as mass spectrometry-based histone profiling and RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins) can identify proteins that interact with H4R3me2a-modified histones. Single-cell multi-omics technologies now allow simultaneous profiling of H4R3me2a distributions and transcriptomes in individual cells, revealing cell-to-cell epigenetic heterogeneity. Computational integration of these diverse datasets requires specialized bioinformatic pipelines, including dimensionality reduction techniques, correlation analyses, and network modeling to identify H4R3me2a-associated regulatory networks. Such integrated approaches can reveal how H4R3me2a contributes to cellular identity, development, and disease states in ways not apparent from any single analysis method.

How can non-specific binding be minimized when using H4R3me2a antibodies?

Minimizing non-specific binding when using H4R3me2a antibodies requires attention to several technical aspects of experimental design. First, optimize blocking conditions—for Western blotting and immunofluorescence, use 5% BSA instead of milk when possible, as milk contains biotin and can contribute to background with certain detection systems. For ChIP applications, include a pre-clearing step with protein A/G beads and incorporate salmon sperm DNA or BSA as blocking agents in the IP buffer. Second, validate antibody specificity using peptide competition assays with both target (H4R3me2a) and non-target (unmodified H4, H4R3me1, H4R3me2s) peptides. Antibody dilution optimization is crucial—while manufacturers suggest ranges like 1:500-1:2000 for Western blotting , each experimental system may require adjustments. Implement stringent washing protocols using buffers with graduated stringency (increasing salt concentration or detergent) to remove weakly bound antibodies while preserving specific interactions. For ChIP applications, sonication optimization ensures proper chromatin fragmentation (200-300 bp), improving epitope accessibility and reducing non-specific DNA pull-down. Use highly purified antibodies whenever possible—some commercial H4R3me2a antibodies undergo affinity purification against the immunogen , reducing potential cross-reactivity. Finally, include appropriate negative controls in all experiments: IgG controls for ChIP and IP, primary antibody omission for IF and IHC, and PRMT1/6 inhibitor-treated samples or PRMT1/6 knockdown cells as biological negative controls.

What factors can lead to false positive or false negative results with H4R3me2a antibodies?

Several factors can contribute to false positive or false negative results when using H4R3me2a antibodies. False positives often arise from antibody cross-reactivity with similar modifications (especially H4R3me1 or H4R3me2s) or with the same modification on different histone variants, emphasizing the importance of validation through dot-blot analysis with various methylated peptides . Overfixation in immunohistochemistry or immunofluorescence can mask epitopes, while insufficient fixation may alter nuclear morphology and antigen distribution. Improper sample preparation, particularly for histones, can expose antibodies to degraded or denatured epitopes—acid extraction methods are recommended for histone isolation to maintain modification integrity. Cell cycle variations affect histone modification levels, potentially leading to inconsistent results across asynchronous cell populations. For false negatives, epitope masking by protein-protein interactions or adjacent modifications may prevent antibody binding, while insufficient permeabilization limits antibody access to nuclear antigens. Storage conditions affect both antibody and sample integrity—repeated freeze-thaw cycles degrade antibody quality, and histone modifications can be unstable during prolonged storage. Technical issues such as improper primary/secondary antibody pairing, expired reagents, or inadequate detection sensitivity can also yield false negatives. To minimize these issues, researchers should implement comprehensive controls, including peptide competition, PRMT1/6 inhibitor treatments, comparison across multiple antibodies when possible, and careful optimization of all experimental parameters.

What standardization approaches improve reproducibility in H4R3me2a quantification?

Standardization is essential for reliable quantification of H4R3me2a across different experimental platforms and between laboratories. For Western blotting, researchers should implement loading controls specific to histone analysis—total H4 antibodies or coomassie staining of histones provide direct normalization for histone content, while standard housekeeping proteins (like β-actin) may not accurately reflect histone loading. Densitometric analysis should calculate the ratio of H4R3me2a to total H4 signal to account for loading variations. For immunofluorescence quantification, standardize image acquisition parameters (exposure time, gain, offset) across all samples and use automated analysis software with consistent thresholding parameters to measure nuclear signal intensity. In ChIP-qPCR experiments, employ the percent of input method for normalization rather than fold enrichment over IgG, as IgG binding can vary between samples. For ChIP-seq, incorporate exogenous spike-in controls (such as Drosophila chromatin with species-specific antibodies) to enable cross-sample normalization independent of biological variation. Use internal reference regions (loci with stable H4R3me2a levels across conditions) as additional normalization controls. Establish standard operating procedures (SOPs) for all aspects of experimentation, from sample preparation to data analysis, ensuring consistency between experiments and operators. Document all lot numbers of antibodies and reagents, as batch-to-batch variation can significantly impact results. Finally, implement statistical approaches appropriate for the data type—paired statistical tests for before/after comparisons, and multiple testing correction for genome-wide analyses—to ensure robust interpretation of H4R3me2a quantification data.

How do different commercial H4R3me2a antibodies compare in performance across applications?

Commercial H4R3me2a antibodies show notable variations in performance across different applications, making comparative analysis essential for selecting the optimal reagent for specific research needs. Based on the search results, several validated antibodies are available from different suppliers including antibodies.com (A51515) , Active Motif (39705) , and Abcam (ab194683) . In Western blotting applications, all three antibodies demonstrate detection of the expected ~11 kDa histone H4 band, with recommended dilutions ranging from 1:500 to 1:2000. The Active Motif antibody has been specifically validated using a high salt/sonication protocol for nuclear extract preparation, which is recommended for chromatin-bound proteins . For immunofluorescence applications, validation data shows nuclear localization patterns consistent with histone distribution, with optimal working dilutions varying between suppliers. In ChIP applications, the Active Motif antibody has published validations, while specific ChIP protocol details for other antibodies may require direct communication with manufacturers. Dot-blot analyses demonstrate specificity across all platforms, with notable differences in cross-reactivity profiles that may impact experimental outcomes. The Abcam antibody has been cited in six publications as noted in their product information, potentially indicating broader validation in peer-reviewed research . When selecting between these options, researchers should consider specific application requirements, species reactivity needs, and available validation data most relevant to their experimental system.

What emerging technologies will advance H4R3me2a detection and functional analysis?

Emerging technologies promise to revolutionize H4R3me2a detection and functional analysis in coming years. Single-cell epigenomic approaches, including single-cell ChIP-seq and CUT&Tag, are being adapted for histone modification analysis, potentially revealing cell-to-cell variability in H4R3me2a patterns that bulk methods cannot detect. These techniques will be particularly valuable for understanding H4R3me2a dynamics in heterogeneous tissues and during developmental transitions. Mass spectrometry-based approaches continue to advance, with improved sensitivity allowing quantitative analysis of histone modifications from limited samples without antibody-based enrichment, providing unbiased detection of H4R3me2a alongside co-occurring modifications. CRISPR-based epigenome editing systems using catalytically inactive Cas9 fused to PRMT1 or PRMT6 domains enable site-specific manipulation of H4R3me2a, allowing researchers to directly test the functional consequences of this modification at specific genomic loci. Live-cell imaging of H4R3me2a is becoming possible through the development of modification-specific intracellular antibodies (mintbodies) and engineered reader domains coupled to fluorescent proteins, enabling real-time tracking of dynamic changes in this modification. Microfluidic platforms for high-throughput epigenetic profiling will accelerate screening of factors that influence H4R3me2a deposition and removal. Advanced computational approaches, including machine learning algorithms trained on multi-omics datasets, will improve prediction of H4R3me2a functional consequences and interactions with other chromatin features. Together, these technological advances will provide unprecedented insights into the biology of H4R3me2a and its role in gene regulation.

How might H4R3me2a research contribute to epigenetic therapies?

H4R3me2a research has significant potential to inform the development of epigenetic therapies across multiple disease contexts. As asymmetric dimethylation of H4R3 is catalyzed by PRMT1 and PRMT6 , these enzymes represent promising therapeutic targets. Small molecule inhibitors of PRMT1 and PRMT6 are already in development, with several showing selectivity in preclinical studies. Understanding the genomic distribution and functional consequences of H4R3me2a modification will help predict both the therapeutic potential and possible side effects of such inhibitors. In cancer therapy, where aberrant H4R3me2a patterns have been observed in tissues such as rectal cancer , PRMT inhibitors could potentially restore normal gene expression programs or sensitize tumors to conventional therapies. The crosstalk between H4R3me2a and histone acetylation mediated by p300 suggests that combination approaches targeting both modifications might yield synergistic therapeutic effects. For precision medicine applications, H4R3me2a patterns could serve as biomarkers to stratify patients for specific epigenetic therapies, particularly in hormone-dependent cancers given the connection between H4R3me2a and nuclear hormone receptor signaling. Beyond cancer, H4R3me2a research may inform treatments for inflammatory, neurodegenerative, and developmental disorders where epigenetic dysregulation contributes to pathology. The development of targeted protein degradation approaches like PROTACs (Proteolysis Targeting Chimeras) offers new possibilities for selectively eliminating PRMT enzymes in specific cellular contexts. As H4R3me2a research advances, it will continue to reveal novel therapeutic targets and strategies within the complex network of epigenetic regulation.