Mono-methyl-HIST1H4A (R35) Antibody

Basic Research Considerations

• What is the biological significance of histone H4 arginine 35 methylation in chromatin regulation?

  Histone H4 arginine 35 (R35) methylation represents a critical modification that influences nucleosome structure and DNA accessibility. This residue is located at the lateral surface of the nucleosome where it interacts with DNA. According to studies of histone lateral surface modifications, R35 is positioned near the N-terminus of the H4 α1 helix with its side chain extended into the major groove of DNA . The methylation of R35 affects chromatin compaction through altered histone-DNA interactions.

  Unlike tail modifications, R35 methylation directly influences the structural properties of the nucleosome core by altering the water-mediated hydrogen bonds that normally exist between the unmodified arginine and the DNA phosphate backbone . This modification contributes to the "regulated nucleosome mobility" model, working alongside other modifications to determine chromatin states that impact transcriptional regulation, DNA repair, and replication .

• How does histone H4 arginine methylation differ functionally from lysine methylation?

  Arginine and lysine methylation on histone H4 serve distinct regulatory functions:

  Feature	Arginine Methylation (e.g., H4R35me1)	Lysine Methylation (e.g., H4K20me1)
  Chemical nature	Adds methyl groups to guanidino nitrogen	Adds methyl groups to ε-amino group
  Enzymatic writers	PRMTs (Protein Arginine Methyltransferases)	SET-domain containing HMTs and DOT1L
  Effect on charge	Preserves positive charge	Preserves positive charge
  Typical genomic locations	Often at active gene promoters (H4R3me1)	Varies (H4K20me1 often at silenced regions)
  Nuclear distribution	H4R35me1 shows uniform nuclear distribution	H4K20me1 often enriched at heterochromatin
  Functional outcomes	Often associated with transcriptional activation	Often associated with transcriptional silencing

  While both modifications can regulate chromatin states, arginine methylation generally functions in different molecular contexts than lysine methylation, with distinct reader proteins and downstream effects .

• What cellular processes are regulated by histone H4 arginine methylation?

  Histone H4 arginine methylation regulates multiple cellular processes:

  • Transcriptional activation: H4R3 methylation by PRMT1 is particularly associated with active transcription and facilitates subsequent histone acetylation .

  • Cell division regulation: Studies have shown that H4R35 methylation is notably enriched on mitotic chromosomes, suggesting a role in cell division processes .

  • Chromatin architecture: H4 arginine methylation contributes to higher-order chromatin structure by modulating nucleosome-nucleosome interactions .

  • DNA damage response: Arginine methylation can influence the recruitment of DNA repair factors to damaged chromatin sites.

  • Development: Changes in H4 arginine methylation patterns occur during cellular differentiation and development, as seen in parasite life cycles studied in T. gondii and P. falciparum .

Experimental Methodology

• What are the optimal conditions for using Mono-methyl-HIST1H4A (R35) Antibody in immunofluorescence experiments?

  For optimal immunofluorescence results with Mono-methyl-HIST1H4A (R35) Antibody:

  • Dilution ratio: 1:1-1:10 (as recommended for CSB-PA010429OA35me1HU)

  • Fixation protocol: 4% formaldehyde fixation is recommended (as demonstrated in successful experiments)

  • Permeabilization: 0.2% Triton X-100 ensures proper antibody access to nuclear epitopes

  • Blocking solution: 10% normal goat serum efficiently reduces background

  • Incubation conditions: Overnight incubation at 4°C provides optimal binding

  • Secondary antibody: Alexa Fluor 488-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L) works effectively with this primary antibody

  • Counterstaining: DAPI for nuclear visualization

  For reproducible results, it is critical to maintain consistent fixation times, antibody concentrations, and washing steps across experimental replicates.

• What controls should be included when using Mono-methyl-HIST1H4A (R35) Antibody?

  Rigorous controls are essential for reliable histone modification analysis:

  • Peptide competition assay: Pre-incubate antibody with synthetic H4R35me1 peptide to confirm specificity (similar to approaches used for other histone methylation antibodies)

  • Dot-blot specificity test: Test antibody against a panel of modified and unmodified histone peptides to ensure it doesn't cross-react with similar modifications (H4K31me1, H4K20me1, etc.)

  • Negative controls:

    • Secondary antibody-only control

    • Isotype control (rabbit IgG at equivalent concentration)

    • Unmodified histone H4 peptide control

  • Positive controls:

    • Cell lines known to express high levels of H4R35me1

    • Mitotic chromosomes (if analyzing mammalian cells)

  • Histone demethylase treatment: As a functional control, treating samples with appropriate demethylases should reduce antibody signal

• How can ChIP-seq be optimized for Mono-methyl-HIST1H4A (R35) Antibody?

  For successful ChIP-seq with Mono-methyl-HIST1H4A (R35) Antibody:

  1. Cross-linking optimization: 1% formaldehyde for 10 minutes at room temperature is typically effective for histone modifications

  2. Chromatin fragmentation: Aim for fragments of 150-300 bp using either sonication or enzymatic digestion

  3. Antibody amount: Start with 2-5 μg antibody per ChIP reaction and optimize based on preliminary results

  4. Immunoprecipitation conditions:

     • Dilution: 1:25 based on similar histone modification antibodies

     • Incubation: Overnight at 4°C with rotation

     • Washing: Stringent washing buffers to minimize background

  5. Library preparation: Use specialized library preparation kits designed for limited ChIP material

  6. Sequencing depth: Minimum 20 million uniquely mapped reads for sufficient coverage

  7. Bioinformatic analysis: Use specialized tools like MACS2 for peak calling, with appropriate input controls

  For genome-wide mapping, compare H4R35me1 distribution patterns with other histone marks (H3K4me3, H3K27ac) to understand its relationship to chromatin states and transcriptional activity.

Data Analysis and Interpretation

• How can quantitative analysis be performed with Mono-methyl-HIST1H4A (R35) Antibody staining?

  Quantitative analysis of H4R35me1 immunostaining involves several methodological approaches:

  1. Fluorescence intensity measurement:

     • Capture images using consistent exposure settings

     • Define nuclear regions (ROIs) using DAPI counterstain

     • Measure mean fluorescence intensity within nuclear ROIs

     • Subtract background from non-nuclear regions

  2. Nuclear pattern analysis:

     • Classify staining patterns (uniform, speckled, peripheral)

     • Quantify percentage of cells showing each pattern

     • Compare pattern distribution between experimental conditions

  3. Co-localization analysis:

     • Calculate Pearson's or Manders' coefficients for co-localization with other chromatin marks

     • Use specialized software (ImageJ with JACoP plugin, CellProfiler)

  4. Western blot quantification (for bulk analysis):

     • Use acid extraction protocols optimized for histones

     • Normalize H4R35me1 signal to total histone H4

     • Apply densitometric analysis using ImageJ or similar software

  5. Flow cytometry:

     • Fix and permeabilize cells appropriately

     • Use consistent antibody dilutions (1:100-1:200)

     • Gate populations based on cell cycle stages

     • Compare median fluorescence intensity between samples

• How do H4R35 methylation patterns correlate with gene expression data?

  The correlation between H4R35me1 and gene expression follows specific patterns:

  1. Genomic distribution analysis: H4R35me1 shows distinct genomic localization compared to other histone marks like H4K31ac and H4K31me1. While H4K31ac is typically enriched at promoters of active genes, methylation marks like H4R35me1 may have different distribution patterns .

  2. Correlation methodology:

     • Integrate ChIP-seq data for H4R35me1 with RNA-seq expression data

     • Calculate correlation coefficients between H4R35me1 enrichment at promoters/gene bodies and corresponding transcript levels

     • Perform gene set enrichment analysis (GSEA) to identify functional pathways associated with H4R35me1-marked genes

  3. Expression correlation patterns: Based on studies of similar modifications:

     • H4 methylation marks may have context-dependent correlations with gene expression

     • H4R35me1 might show inverse correlation with gene expression in some contexts, similar to H4K31me1 which is inversely correlated with gene expression in T. gondii

     • The correlation may differ between promoters and gene bodies

  4. Cell type specificity: The relationship between H4R35me1 and gene expression may vary between cell types, as observed with other histone modifications in studies comparing adult versus neonatal oligodendrocyte progenitors .

Advanced Research Applications

• What is the interplay between H4R35 methylation and other post-translational modifications on the nucleosome surface?

  The nucleosome lateral surface contains several modifiable residues that functionally interact:

  1. Modification crosstalk:

     • H4R35 methylation likely functions alongside H4K31 modifications, which can be either acetylated or methylated in a mutually exclusive manner

     • The proximity of R35 to K31 suggests their modifications may be interdependent or mutually influenced

  2. Structural implications:

     • Modifications at the protein-DNA interface, including H4R35me1 and H4K31ac/me1, collectively alter nucleosome stability and DNA accessibility

     • The "regulated nucleosome mobility" model suggests these modifications work together to control nucleosome dynamics

  3. Reader protein coordination:

     • Different reader domains recognize specific modifications: bromodomains for acetylation vs. tudor domains for methylation

     • The spatial arrangement of these modifications creates a complex "reading surface" for regulatory proteins

  4. Modification hierarchy:

     • Evidence from studies on H4K31 suggests that methylation and acetylation can counteract each other's effects

     • It remains to be determined whether H4R35 methylation precedes or follows other nearby modifications

  5. Condensability effects:

     • Recent research suggests that different histone modifications, including methylation of lateral surface residues, differentially affect chromatin condensability

     • Understanding H4R35me1's role in this context requires further investigation

• How does H4R35 methylation contribute to chromatin organization during cell division?

  H4R35 methylation has distinct roles during cell division:

  1. Mitotic enrichment pattern:

     • Similar to H4K31me1, H4R35me1 shows enrichment on mitotic chromosomes in mammalian cells

     • The signal on chromosome arms can be more pronounced than traditional mitotic markers like H3S10 phosphorylation

  2. Cell cycle dynamics:

     • H4R35me1 is often barely detectable in interphase nuclei of quiescent cells

     • The modification becomes prominent during mitosis, suggesting cell cycle-regulated deposition

  3. Chromosome condensation role:

     • The enrichment on mitotic chromosomes suggests a role in chromosome condensation

     • The positively charged methylated arginine may strengthen DNA binding, contributing to chromatin compaction

  4. Mitotic bookmarking potential:

     • H4R35me1 might serve as an epigenetic bookmark that preserves certain chromatin states through cell division

     • This could contribute to epigenetic memory mechanisms that maintain cell identity

  5. Cell type specificity:

     • The pattern of H4R35me1 during cell division may vary between different cell types and organisms

     • Apicomplexan parasites (like T. gondii) show different histone modification patterns compared to mammalian cells

• What are the enzymes responsible for H4R35 methylation and demethylation?

  The enzymatic regulation of H4R35 methylation involves specific writers and erasers:

  1. Methyltransferases (writers):

     • While the specific methyltransferase for H4R35 has not been definitively identified in the search results, protein arginine methyltransferases (PRMTs) are the likely candidates

     • PRMT1 is known to methylate H4R3 and may potentially target other arginine residues

     • The enzyme specificity may be influenced by the structural context of R35 within the nucleosome

  2. Demethylases (erasers):

     • Arginine demethylases are less well-characterized than lysine demethylases

     • JMJD family proteins or PADI4 (which converts arginine to citrulline) may be involved

     • The discovery of histone demethylases has established methylation as a reversible epigenetic marker

  3. Enzymatic assays:

     • In vitro assays for histone methyltransferases can be adapted to study H4R35 methylation

     • The HotSpot platform, originally developed for kinases, has been modified for histone methyltransferase assays

     • These assays can detect total methylation of substrates on both lysine and arginine residues

  4. Regulation of enzymatic activity:

     • The activity of these enzymes may be controlled by cellular signaling pathways

     • Inhibitors like histone deacetylase inhibitors (HDACi) can indirectly affect methylation patterns by altering the chromatin landscape

• How can H4R35 methylation patterns be manipulated experimentally to study its function?

  Experimental manipulation of H4R35 methylation can be achieved through several approaches:

  1. Enzymatic inhibition:

     • Small molecule inhibitors targeting PRMTs can reduce H4R35 methylation

     • Experimental designs similar to those using histone deacetylase inhibitors (like FR235222) could be adapted

  2. Genetic approaches:

     • CRISPR/Cas9-mediated mutation of H4R35 to a non-methylatable residue (R35K or R35A)

     • Knockdown/knockout of the responsible methyltransferase

     • Overexpression of candidate demethylases

  3. Peptide competition:

     • Cell-permeable H4R35me1 peptides can be used to compete with readers of this modification

     • This approach disrupts the function without altering the modification level

  4. In vitro nucleosome reconstitution:

     • Using synthesized histones with site-specific modifications or amino acid substitutions

     • Similar to approaches used for studying H4K31 modifications

     • These reconstituted nucleosomes can be used for structural studies and protein binding assays

  5. Experimental readouts:

     • Effects on gene expression can be measured by RNA-seq

     • Chromatin accessibility changes can be assessed by ATAC-seq

     • Changes in cell cycle progression can be monitored by flow cytometry

     • Effects on nuclear architecture can be visualized by super-resolution microscopy

Technical Troubleshooting

• How can specificity issues with Mono-methyl-HIST1H4A (R35) Antibody be resolved?

  Ensuring antibody specificity is critical for reliable results:

  1. Common cross-reactivity issues:

     • Cross-reactivity with other methylated arginines on histones (H3R2, H3R17, H4R3)

     • Non-specific binding to other histone modifications with similar chemical properties

  2. Specificity validation methods:

     • Peptide competition assays using H4R35me1 and other methylated peptides

     • Dot blot analysis against a panel of modified and unmodified histone peptides

     • Western blot analysis of acid-extracted histones from cells with genetically altered H4R35

  3. Optimization strategies:

     • Titrate antibody concentration to minimize background (starting with 1:50-1:200 dilution)

     • Increase blocking stringency (5% BSA or 10% normal serum)

     • Add competitive inhibitors for common cross-reactive epitopes

     • Optimize washing conditions (increase wash number, duration, or detergent concentration)

  4. Batch variability management:

     • Test each new antibody lot against a standard sample

     • Maintain reference images/data for comparison

     • Consider pooling multiple antibody lots for long-term studies

  5. Alternative confirmation approaches:

     • Use multiple antibodies targeting the same modification from different suppliers

     • Validate with orthogonal techniques (mass spectrometry)

• What are the challenges in detecting H4R35 methylation in different sample types?

  Different biological samples present unique challenges for H4R35me1 detection:

  1. Tissue sections:

     • Challenge: Epitope masking due to formaldehyde fixation and paraffin embedding

     • Solution: Optimize antigen retrieval (citrate buffer pH 6.0, 95-100°C for 20 minutes)

  2. Cell cultures:

     • Challenge: Variable expression levels across cell cycle

     • Solution: Synchronize cells or co-stain with cell cycle markers

  3. Mitotic chromosomes:

     • Challenge: Preserving chromosome structure while ensuring antibody accessibility

     • Solution: Use chromosome spreading techniques with gentle fixation

  4. Heterogeneous tissues:

     • Challenge: Cell type-specific variations in H4R35me1

     • Solution: Co-stain with cell type markers; consider single-cell approaches

  5. ChIP-seq samples:

     • Challenge: Low abundance of H4R35me1 and potential epitope masking during crosslinking

     • Solution: Increase starting material; optimize crosslinking conditions

  6. Western blot detection:

     • Challenge: Ensuring complete acid extraction of histones

     • Solution: Use specialized extraction protocols with HCl or H2SO4

     • Challenge: Low molecular weight (11-17 kDa) of histone H4

     • Solution: Use specialized SDS-PAGE systems for low molecular weight proteins

• How do storage and handling conditions affect Mono-methyl-HIST1H4A (R35) Antibody performance?

  Proper handling is essential for maintaining antibody activity:

  1. Storage recommendations:

     • Store at -20°C or -80°C for long-term storage

     • Avoid repeated freeze-thaw cycles by preparing small aliquots

     • Store in 50% glycerol buffer to prevent freezing damage

  2. Stability considerations:

     • Working dilutions should be prepared fresh

     • Most antibodies remain stable at 4°C for 1-2 weeks

     • Monitor for signs of deterioration (precipitation, declining signal)

  3. Buffer composition effects:

     • Preservatives like 0.03% Proclin 300 or 0.02% sodium azide help maintain antibody stability

     • Glycerol content (50%) prevents freeze-thaw damage

     • pH stability is typically maintained at 7.4 in PBS-based buffers

  4. Transportation considerations:

     • Shipping on ice or ice packs for short transits

     • Dry ice recommended for longer shipping times

  5. Performance monitoring:

     • Validate each aliquot before critical experiments

     • Maintain reference samples with known H4R35me1 levels for comparison

     • Record lot numbers and performance characteristics