Histone H4R3me2s Antibody

What is the H4R3me2s modification and what is its biological significance?

H4R3me2s refers to the symmetric dimethylation of arginine 3 on histone H4, a post-translational modification catalyzed primarily by protein arginine methyltransferases PRMT5 and PRMT7 . This epigenetic mark plays a crucial role in transcriptional repression and gene silencing mechanisms. Studies using human β-globin locus as a model have demonstrated that H4R3me2s serves as a direct binding target for DNA methyltransferase DNMT3A, establishing a mechanistic link between histone arginine methylation and DNA methylation . The interaction occurs through DNMT3A's ADD domain containing the PHD motif, creating a sequence of epigenetic events that leads to gene silencing . In primary erythroid progenitors from adult bone marrow, H4R3me2s has been shown to mark inactive methylated globin genes coincident with the localization of PRMT5, further supporting its role in gene repression . This modification represents an important regulatory mechanism controlling chromatin structure and accessibility, ultimately influencing cellular processes including differentiation and development.

What applications are Histone H4R3me2s antibodies validated for?

Histone H4R3me2s antibodies have been validated for multiple experimental applications essential to epigenetic research. Western blotting (WB) represents a primary application, with recommended dilutions typically ranging from 1:500 to 1:2000 depending on the specific antibody and experimental conditions . Immunohistochemistry on paraffin-embedded tissues (IHC-P) is another validated application, allowing researchers to examine the spatial distribution of H4R3me2s marks in tissue samples . Immunofluorescence (IF) can be used to visualize the nuclear localization and distribution patterns of this modification in cultured cells . Peptide array assays provide a powerful tool for confirming antibody specificity, with validated antibodies showing strong binding to H4R3me2s peptides but not to unmodified or asymmetrically methylated H4R3 peptides . Additionally, these antibodies have been validated for chromatin immunoprecipitation (ChIP) and ChIP-sequencing applications, enabling genome-wide mapping of H4R3me2s distribution and its correlation with transcriptional status . Researchers should verify the specific validation data for their antibody of choice, as performance may vary between manufacturers and applications.

How can I confirm the specificity of an H4R3me2s antibody?

Confirming antibody specificity is critical for reliable research outcomes when working with histone modifications. For H4R3me2s antibodies, peptide array assays represent the gold standard for specificity testing. In this approach, the antibody should be tested against peptides containing different modifications of histone H4, including unmodified, symmetrically dimethylated, and asymmetrically dimethylated R3 residues . A specific H4R3me2s antibody will show strong binding only to the symmetrically dimethylated peptide with minimal cross-reactivity. Western blot analysis using recombinant histone H4 proteins with defined modifications can provide additional confirmation of specificity . Researchers can also perform immunoprecipitation followed by mass spectrometry to identify the precise histone modifications being recognized by the antibody. Alternatively, a highly specific approach involves pre-incubating the antibody with competing peptides (both specific and non-specific) before immunoassays to demonstrate selective blocking of antibody binding . Loss of signal in samples treated with PRMT5 knockdown or inhibition (which reduces H4R3me2s levels) can serve as an additional functional validation of antibody specificity . Validation across multiple techniques strengthens confidence in antibody specificity and ensures reliable experimental results.

What are the optimal conditions for Western blot using H4R3me2s antibodies?

Successful Western blot detection of H4R3me2s requires careful optimization of several key parameters. Based on validated protocols, researchers should begin with sample preparation using a 4-12% Bis-tris gel under the MES buffer system, running at approximately 200V for 35 minutes . Transfer to a nitrocellulose membrane should be performed at lower voltage (around 30V) for an extended period (70 minutes) to ensure efficient transfer of the small histone proteins . Blocking should be conducted using 2% Bovine Serum Albumin rather than milk proteins, as milk contains bioactive compounds that can interfere with phospho-specific antibodies . For primary antibody incubation, optimal dilutions typically range from 1:500 to 1:2000, with incubation preferably performed overnight at 4°C to maximize specific binding . Secondary antibody selection should match the host species of the primary antibody, with anti-rabbit HRP conjugates being appropriate for most commercial H4R3me2s antibodies . When developing the blot, high-sensitivity ECL substrate kits are recommended due to the relatively low abundance of this specific modification . The expected molecular weight for histone H4 is approximately 11 kDa, though the observed band may appear at 14 kDa due to the effect of post-translational modifications on protein migration . Extended exposure times (up to 3 minutes) may be necessary for optimal visualization of bands, especially in samples with low levels of H4R3me2s modification .

How should I design ChIP experiments to analyze H4R3me2s distribution?

Chromatin immunoprecipitation (ChIP) experiments for H4R3me2s require careful consideration of fixation, sonication, antibody selection, and controls. Begin with standard formaldehyde crosslinking (1% for 10 minutes at room temperature) to preserve protein-DNA interactions . Sonication conditions should be optimized to generate DNA fragments between 200-500 bp, which is ideal for high-resolution mapping of histone modifications . When selecting antibodies, choose those specifically validated for ChIP applications with demonstrated specificity for the symmetric dimethylation (not asymmetric) . For immunoprecipitation, use 2-5 μg of antibody per ChIP reaction with chromatin from approximately 1-5 × 10^6 cells . Essential controls include: (1) input chromatin (non-immunoprecipitated) to normalize for differences in starting material, (2) immunoprecipitation with normal IgG to establish background signal, and (3) ChIP for a constitutive histone mark (such as H3) to normalize for nucleosome density . For site-specific analysis, design primers targeting regions of interest, including both putative target sites and known negative regions. For genome-wide studies, ChIP-seq libraries should be prepared with appropriate controls and sequenced to sufficient depth (typically 20-30 million uniquely mapped reads) . When analyzing data from H4R3me2s ChIP experiments, focus on correlation with transcriptional repression, DNA methylation patterns, and co-occupancy with PRMT5 or DNMT3A, as these relationships have been established in the literature .

What controls should be included when performing immunofluorescence with H4R3me2s antibodies?

Robust immunofluorescence experiments for H4R3me2s require multiple controls to ensure reliable interpretation of results. Primary antibody specificity controls should include: (1) peptide competition assays where pre-incubation of the antibody with the specific H4R3me2s peptide should abolish nuclear staining, while pre-incubation with unmodified or asymmetrically modified peptides should not affect signal , (2) PRMT5 knockdown or inhibitor-treated cells, which should show reduced H4R3me2s staining due to decreased enzymatic activity responsible for this modification . Technical controls must include: (1) secondary antibody-only controls to assess non-specific binding of the secondary antibody, (2) isotype control antibodies (usually normal rabbit IgG) to evaluate background staining due to non-specific binding of immunoglobulins . For co-localization studies, proper controls for each fluorophore channel should be included to account for potential bleed-through or cross-excitation. Fixed cell samples should be processed alongside experimental samples but without primary antibody addition. Positive control samples might include cell types known to have high levels of H4R3me2s, such as certain cancer cell lines, while negative controls could include embryonic stem cells which typically have lower levels of repressive histone marks . Image acquisition should be performed with consistent exposure settings across all samples and controls, with Z-stack imaging recommended to capture the three-dimensional distribution of this nuclear mark.

How can I extract and prepare histone samples for optimal H4R3me2s detection?

Optimal detection of H4R3me2s requires specialized histone extraction protocols that preserve this sensitive post-translational modification. For cultured cells, begin with harvesting approximately 1-5 × 10^6 cells by trypsinization followed by PBS washing . Cell lysis should be performed using a hypotonic lysis buffer (10 mM Tris-HCl pH 8.0, 1 mM KCl, 1.5 mM MgCl₂, 1 mM DTT) supplemented with protease inhibitors, phosphatase inhibitors, and importantly, methyltransferase inhibitors including 5 mM sodium butyrate and 60 μM DZNep to prevent artificial demethylation during processing . Following a 30-minute incubation on ice, nuclei should be pelleted by centrifugation at 10,000g for 10 minutes at 4°C. Acid extraction of histones is then performed by resuspending the nuclear pellet in 0.2 N HCl overnight at 4°C with gentle rotation . After centrifugation to remove nuclear debris, the supernatant containing acid-soluble histones should be neutralized with 1/10 volume of 2 M NaOH. Protein concentration should be determined using Bradford or BCA assays, with typical yields of 50-100 μg of histone proteins from 10^6 cells . For tissue samples, approximately 50-100 mg of tissue should be homogenized in the same hypotonic buffer before proceeding with nuclear isolation and acid extraction . Prior to loading on gels, histone samples should be mixed with Laemmli buffer and denatured at 95°C for 5 minutes. Loading 0.5-1 μg of histone preparation per lane is typically sufficient for detection with most commercial H4R3me2s antibodies .

How does H4R3me2s coordinate with DNA methylation and gene silencing mechanisms?

H4R3me2s establishes a sophisticated epigenetic crosstalk mechanism that coordinates histone modifications with DNA methylation to enforce gene silencing. Studies using the human β-globin locus have revealed that PRMT5-mediated symmetric dimethylation of H4R3 creates a direct binding platform for the DNA methyltransferase DNMT3A through its ADD domain containing the PHD motif . This interaction represents a critical mechanistic link between arginine methylation and DNA methylation, establishing a sequential epigenetic pathway leading to stable gene repression. Experimental evidence demonstrates that loss of H4R3me2s through PRMT5 knockdown results in reduced DNMT3A recruitment, subsequent loss of DNA methylation, and ultimately gene reactivation . This relationship appears to be unidirectional, as H4R3me2s deposition precedes and is required for DNA methylation, rather than the reverse. The repressive function of H4R3me2s extends beyond direct DNA methylation by facilitating the recruitment of additional repressive complexes, including histone deacetylases that remove activating acetyl marks and reinforce the repressed chromatin state . In developmental contexts, such as erythroid differentiation, H4R3me2s marks inactive methylated globin genes coincident with PRMT5 localization, suggesting a role in developmental gene regulation . Recent research has also implicated this modification in broader cellular processes, including adipocyte differentiation, where altered H4R3me2s levels influence the expression of key developmental regulators such as Dlk1 . Together, these findings establish H4R3me2s as a central coordinator in an epigenetic silencing pathway that connects histone arginine methylation, DNA methylation, and gene repression.

How do various PRMT inhibitors affect H4R3me2s levels and how can this be monitored?

PRMT inhibitors represent valuable tools for studying the functional significance of H4R3me2s in various biological contexts. Type II PRMT inhibitors targeting PRMT5 (the primary enzyme responsible for H4R3me2s) include EPZ015666 (GSK3235025), GSK3326595 (EPZ015938), and JNJ-64619178, each with distinct chemical properties and inhibitory mechanisms . When treating cells with these inhibitors, researchers should start with dose-response experiments ranging from 1-100 μM, typically with 24-72 hour treatment periods to allow for turnover of pre-existing modified histones. Monitoring H4R3me2s levels after inhibitor treatment can be accomplished through several complementary approaches. Western blotting provides a quantitative assessment of global H4R3me2s levels, with expected dose-dependent decreases following effective PRMT inhibition . ChIP-qPCR or ChIP-seq can reveal locus-specific changes in H4R3me2s distribution, particularly at known PRMT5 target genes, providing insights into the kinetics and specificity of inhibition . Immunofluorescence microscopy offers visualization of nuclear H4R3me2s patterns, which typically show reduced nuclear staining intensity with effective inhibition . For each monitoring approach, appropriate controls should include vehicle-treated cells and, ideally, PRMT5 knockdown cells as a positive control for H4R3me2s reduction. To comprehensively assess the specificity of inhibitor effects, researchers should simultaneously monitor other histone modifications, particularly H4R3me2a (asymmetric dimethylation), which is catalyzed by type I PRMTs and should be less affected by PRMT5-specific inhibitors . Functional readouts such as expression analysis of known H4R3me2s-repressed genes should be incorporated to connect the chromatin changes to transcriptional outcomes.

What is the relationship between H4R3me2s and other histone modifications in the histone code?

H4R3me2s operates within a complex network of histone modifications that collectively regulate chromatin structure and gene expression. This modification exhibits both cooperative and antagonistic relationships with other histone marks, creating a sophisticated regulatory system. H4R3me2s shows a strong negative correlation with active marks such as H3K4me3 and H3K27ac across the genome, consistent with its role in transcriptional repression . Mechanistically, H4R3me2s can inhibit the deposition of activating marks by preventing the recruitment of histone acetyltransferases and H3K4 methyltransferases to target loci. Conversely, H4R3me2s positively correlates with other repressive modifications including H3K9me3 and H3K27me3, suggesting cooperative functions in establishing heterochromatic regions . The temporal sequence of modification deposition is critical, with evidence suggesting that H4R3me2s often precedes DNA methylation and may facilitate the recruitment of repressive complexes that deposit H3K9me3 . Importantly, modifications on the same histone tail can directly influence H4R3me2s deposition, as acetylation of H4K5 and H4K8 has been shown to inhibit PRMT5 activity toward H4R3, creating a competitive relationship between these marks . Additionally, serine phosphorylation events on H4 can modulate the recognition of H4R3me2s by reader proteins, adding another layer of regulation. ChIP-seq studies examining the genome-wide co-occurrence of histone modifications have revealed that H4R3me2s is frequently found in large repressive domains rather than at specific regulatory elements, suggesting a role in establishing broader repressive environments rather than fine-tuning individual promoters . Understanding these complex relationships between H4R3me2s and other histone modifications is essential for deciphering the histone code and its role in gene regulation.

What are common problems in Western blotting for H4R3me2s and how can they be resolved?

Western blotting for H4R3me2s can present several technical challenges that may impact experimental outcomes. Weak or absent signals represent a common issue that may result from insufficient histone extraction, degradation of the modification during sample preparation, or inadequate antibody concentration . To address this, researchers should ensure complete extraction using acid-based histone isolation methods and include deacetylase and demethylase inhibitors (such as sodium butyrate and nicotinamide) in all buffers to preserve modifications . If signal remains weak, increasing antibody concentration or extending incubation time to overnight at 4°C may improve detection. High background signals often stem from insufficient blocking or excessive antibody concentration . This can be mitigated by extending the blocking step to 2 hours, using 5% BSA instead of 2%, and optimizing antibody dilutions through a titration experiment . Multiple bands or unexpected band sizes may indicate antibody cross-reactivity or histone degradation. Researchers should verify antibody specificity using peptide competition assays and ensure protease inhibitors are included during sample preparation . When H4R3me2s shows inconsistent levels between technical replicates, standardizing the sample preparation protocol and controlling for total histone loading using antibodies against unmodified histone H4 is essential . For quantitative Western blot applications, researchers should use recombinant histone standards with defined modifications to generate standard curves, ensuring signal linearity within the working range . If transfer problems are suspected, Ponceau S staining can confirm successful transfer of the small histone proteins, with adjusted transfer conditions (lower voltage, longer time) recommended for histones .

How can I optimize ChIP protocols specifically for H4R3me2s studies?

Optimizing ChIP protocols for H4R3me2s requires careful consideration of several key parameters to maximize signal-to-noise ratio and ensure reproducible results. Crosslinking conditions significantly impact H4R3me2s ChIP efficiency, with standard 1% formaldehyde fixation for 10 minutes typically being insufficient for optimal results . Researchers should test dual crosslinking approaches, first using protein-protein crosslinkers such as disuccinimidyl glutarate (DSG) at 2 mM for 30 minutes, followed by standard formaldehyde fixation, which can significantly improve recovery of protein-DNA complexes involving histone modifications . Chromatin fragmentation represents another critical variable, with excessive sonication potentially damaging epitopes while insufficient sonication results in poor resolution. For H4R3me2s, aim for fragments between 200-500 bp, optimizing sonication conditions by testing different cycle numbers and amplitudes . Antibody selection and concentration dramatically affect ChIP success - use antibodies specifically validated for ChIP applications at concentrations between 2-5 μg per reaction, with pre-clearing chromatin using protein A/G beads to reduce background . To optimize wash conditions, start with standard RIPA buffer washes but consider testing increased stringency (higher salt concentrations up to 500 mM NaCl) in later washes to reduce background . For challenging genomic regions or samples with low H4R3me2s levels, consider implementing carrier approaches (adding 10 μg of E. coli tRNA or 1-5 μg salmon sperm DNA to the IP reaction) to reduce non-specific binding and loss of material during handling . When analyzing results, normalize H4R3me2s signals to total H3 or H4 occupancy to account for nucleosome density variations, and include genomic regions known to be enriched for H4R3me2s (such as inactive globin genes in erythroid cells) as positive controls .

What factors affect H4R3me2s stability in experimental samples and how can they be controlled?

H4R3me2s stability in experimental samples can be compromised by multiple factors, requiring specific measures to preserve this modification throughout experimental procedures. Enzymatic demethylation by endogenous demethylases represents a primary concern during sample preparation . To mitigate this, all extraction buffers should include a cocktail of demethylase inhibitors including 5-10 mM sodium butyrate, 5 mM nicotinamide, and 1 μM MS-275, which inhibit various classes of histone-modifying enzymes . Temperature significantly impacts modification stability, with elevated temperatures accelerating enzymatic activity and chemical degradation. All sample processing should be performed at 4°C when possible, with samples kept on ice during brief room temperature steps . Repeated freeze-thaw cycles can also compromise H4R3me2s stability, so aliquoting samples after initial preparation is recommended . The chemical environment during sample processing influences stability, with extremes of pH potentially affecting arginine methylation. While acid extraction is commonly used for histones, exposure time should be minimized, and samples should be promptly neutralized after extraction . Buffer composition affects stability as well, with certain detergents (particularly ionic detergents at high concentrations) potentially interfering with antibody recognition of the modification. For long-term storage, histone samples should be kept at -80°C in the presence of both protease and demethylase inhibitors, preferably in small aliquots to avoid repeated thawing . When working with tissue samples, flash freezing in liquid nitrogen immediately after collection is essential, as post-mortem interval significantly affects histone modification patterns . For FFPE samples, overfixation can mask epitopes through protein crosslinking; therefore, antigen retrieval steps should be optimized for H4R3me2s detection in immunohistochemistry applications .

How is H4R3me2s involved in developmental processes and cell differentiation?

H4R3me2s plays critical roles in developmental processes and cell differentiation through its function in gene silencing and epigenetic programming. In hematopoietic development, particularly erythroid differentiation, H4R3me2s has been shown to mark inactive methylated globin genes coinciding with PRMT5 localization, suggesting its importance in the developmental switching of globin gene expression . This specific pattern of H4R3me2s deposition creates a repressive chromatin environment that silences fetal globin genes in adult erythroid cells, with dysregulation of this mechanism implicated in hemoglobinopathies. Recent studies have also revealed a significant role for H4R3me2s in adipocyte differentiation, where it contributes to the regulation of key developmental genes including Dlk1, a negative regulator of adipogenesis . In adipocyte precursors, the dynamic deposition and removal of H4R3me2s appears to coordinate the temporal expression of factors required for proper differentiation timing and adipocyte maturation . Embryonic stem cell differentiation represents another context where H4R3me2s functions prominently, as the transition from pluripotency to lineage commitment involves extensive epigenetic reprogramming with selective gene silencing mediated in part by H4R3me2s. Throughout neural development, studies have identified dynamic changes in H4R3me2s distribution that correlate with neuronal differentiation stages, particularly at genes involved in maintaining neural progenitor states . In all these developmental contexts, H4R3me2s does not function in isolation but rather coordinates with other epigenetic mechanisms including DNA methylation and repressive histone marks, creating a multi-layered system for precise developmental gene regulation . The enzymatic machinery responsible for H4R3me2s, particularly PRMT5, has proven essential for normal development, with knockout models demonstrating severe developmental defects and embryonic lethality in multiple organisms.

What role does H4R3me2s play in disease states and what are potential therapeutic implications?

H4R3me2s dysregulation has been implicated in various pathological conditions, highlighting its potential as both a biomarker and therapeutic target. In cancer biology, altered H4R3me2s patterns have been observed across multiple malignancies, with some studies reporting global increases in this modification correlating with disease progression and poor prognosis . Mechanistically, abnormal H4R3me2s distribution can silence tumor suppressor genes through recruitment of DNA methyltransferases, contributing to oncogenic transformation . Targeting the enzymatic machinery responsible for H4R3me2s, particularly PRMT5, has emerged as a promising therapeutic strategy, with several selective inhibitors currently in clinical trials for various cancers . In hematological disorders, particularly hemoglobinopathies like sickle cell disease and β-thalassemia, H4R3me2s contributes to the silencing of fetal hemoglobin genes in adult erythroid cells . Pharmaceutical approaches targeting PRMT5 to reduce H4R3me2s levels at these loci have shown promise in reactivating fetal hemoglobin expression, potentially ameliorating disease symptoms. Neurodegenerative conditions including Alzheimer's and Huntington's diseases exhibit altered histone methylation patterns, with evidence suggesting that aberrant H4R3me2s contributes to transcriptional dysregulation in affected neurons . Inflammatory disorders represent another area where H4R3me2s appears pathologically relevant, as this modification regulates genes involved in inflammatory responses and immune cell function . Importantly, the development of specific antibodies against H4R3me2s has enabled its evaluation as a diagnostic or prognostic biomarker, with immunohistochemical studies correlating H4R3me2s levels with clinical outcomes in several cancer types . As therapeutic approaches targeting histone modifications advance, the reversible nature of H4R3me2s makes it an attractive intervention point, with emerging evidence suggesting that pharmacological modulation of this mark can reprogram aberrant gene expression patterns in disease states .

What is known about the genome-wide distribution of H4R3me2s and its correlation with chromatin states?

Genome-wide mapping studies have revealed distinctive patterns in H4R3me2s distribution that provide insights into its functional roles in chromatin regulation. ChIP-sequencing analyses across various cell types have demonstrated that H4R3me2s is not uniformly distributed but shows preferential enrichment at specific genomic regions . This modification is predominantly found at silent gene promoters and enhancers, where it inversely correlates with active transcription markers such as RNA Polymerase II occupancy and H3K4me3 . Quantitatively, approximately 15-25% of gene promoters show significant H4R3me2s enrichment in differentiated cells, with lower percentages observed in pluripotent stem cells, suggesting developmental regulation of this modification . H4R3me2s typically forms broad domains rather than sharp peaks, often spanning several kilobases and extending from promoters into gene bodies of silenced genes . Intriguingly, comparative analyses across different cell types have revealed both constitutive and cell-type-specific patterns of H4R3me2s distribution. Constitutive H4R3me2s domains are frequently associated with permanently silenced genes and repetitive elements, while cell-type-specific patterns correlate with developmental gene regulation . Integration of H4R3me2s ChIP-seq data with DNA methylation profiles has confirmed the mechanistic link between these epigenetic marks, with approximately 70-80% of H4R3me2s-enriched promoters also showing DNA hypermethylation . When analyzed in the context of chromatin state maps, H4R3me2s predominantly associates with repressive chromatin states characterized by other repressive marks such as H3K9me3 and H3K27me3, though with distinct distribution patterns suggesting non-redundant functions . Time-course experiments during cellular differentiation have revealed dynamic changes in H4R3me2s distribution, with this mark often preceding DNA methylation at developmental genes transitioning to a silenced state . These genome-wide patterns underscore the role of H4R3me2s as a key component of the epigenetic machinery governing gene silencing and chromatin organization.

Comparative Analysis Table of Commercial H4R3me2s Antibodies

This comparative table demonstrates the range of commercial antibodies available for H4R3me2s detection, each with specific validated applications and working conditions. Researchers should select the appropriate antibody based on their experimental needs, considering factors such as validated applications, species reactivity, and documented performance in peer-reviewed literature .