Histone H3R8me2a Antibody

What is Histone H3R8me2a and what biological significance does it have?

Histone H3R8me2a refers to the asymmetric dimethylation of arginine 8 on histone H3. This post-translational modification plays significant roles in transcriptional regulation, particularly in:

• Oncogenic activation pathways

• Nuclear-receptor-mediated transcriptional activation

• Wnt/β-catenin signaling pathway regulation

Studies have shown that H3R8me2a is maintained by protein arginine methyltransferase 2 (PRMT2) . Genome-wide distribution analysis reveals that H3R8me2a is predominantly found at intragenic and intergenic regions, with approximately 13.71% located at promoters within ±3 kb from transcription start sites .

How do H3R8me2a antibodies function in epigenetic research applications?

H3R8me2a antibodies are designed to specifically recognize the asymmetrically dimethylated arginine 8 of histone H3. These antibodies have been validated for multiple applications:

These antibodies are typically raised against peptides containing asymmetrically dimethylated Arg8 of human histone H3 .

What enzymes are responsible for establishing H3R8me2a marks?

PRMT2 has been identified as the primary enzyme responsible for maintaining H3R8me2a levels in vivo. Experimental evidence supporting this includes:

• Knockdown of PRMT2 results in specific decrease of H3R8me2a levels as determined by Western blotting and immunofluorescence assays

• ChIP-seq analysis in PRMT2-depleted cells shows significantly decreased enrichment of H3R8me2a compared to control cells

• PRMT2 has been shown to exhibit H3R8me2a methylation activity in vitro using peptides or recombinant histones as substrates

While PRMT2 alone may not show strong activity in vitro, it is essential for maintaining H3R8me2a in cellular contexts .

What are the optimal protocols for ChIP-seq using H3R8me2a antibodies?

ChIP-seq for H3R8me2a requires optimized protocols due to the specific nature of arginine methylation. Based on published methodologies:

• Cross-linking and Sonication:

  • Standard formaldehyde cross-linking (1% for 10 minutes at room temperature)

  • Sonication to achieve fragments between 200-500 bp

• Immunoprecipitation:

  • Recommended antibody amount: 10 μl per ChIP reaction

  • Extended incubation period (overnight at 4°C) for optimal antibody binding

  • Include appropriate controls: IgG negative control and a positive control such as H3K4me3

• Washing and Elution:

  • Stringent washing conditions to reduce non-specific binding

  • Sequential elution to maximize recovery

• Library Preparation and Sequencing:

  • Standard ChIP-seq library preparation with attention to depth (>20 million reads)

  • Include input controls and spike-in normalization when possible

The enrichment patterns should be validated against published H3R8me2a distributions, which show approximately 13.71% at promoters and the majority at intragenic or intergenic regions .

How can specificity of H3R8me2a antibodies be validated?

Rigorous validation of H3R8me2a antibodies is critical due to potential cross-reactivity with similar histone modifications. Recommended validation approaches include:

• Peptide Competition Assays:

  • Pre-incubate antibody with H3R8me2a peptides before application

  • Include control peptides with other methylation states (H3R8me0, H3R8me1, H3R8me2s)

• Peptide Array Analysis:
  Evidence from peptide dot blot testing shows potential cross-reactivity that must be addressed:

  Peptide	Reactivity
  H3 unmodified	-
  H3R8me1	-
  H3R8me2s	+
  H3R8me2a	+++
  H3K9me2	+
  H3K4me3	-

  Some antibodies show cross-reactivity with H3K9me2 in peptide dot blot assays, requiring further optimization to minimize this in experimental applications .

• Genetic Validation:

  • Compare antibody signal in wild-type cells versus PRMT2 knockdown cells (which show decreased H3R8me2a levels)

• Mass Spectrometry Confirmation:

  • Validate immunoprecipitated material using mass spectrometry to confirm the presence of H3R8me2a

What experimental approaches can distinguish between H3R8me2a and H3R8me2s?

Distinguishing between asymmetric (H3R8me2a) and symmetric (H3R8me2s) dimethylation is crucial as they may have distinct biological functions:

• Antibody Selection:

  • Use antibodies specifically raised against H3R8me2a or H3R8me2s epitopes

  • Validate antibody specificity using peptide arrays containing both modifications

• Methyltransferase Assays:

  • PRMT type I enzymes catalyze asymmetric dimethylation

  • PRMT type II enzymes catalyze symmetric dimethylation

  • In vitro histone methyltransferase (HMT) assays using recombinant PRMTs can help distinguish the two forms

• Structural Binding Studies:

  • Different reader proteins show specificity for H3R8me2a versus H3R8me2s

  • For example, Spindlin1 specifically recognizes H3R8me2a in combination with H3K4me3

• Mass Spectrometry:

  • Fragmentation patterns in MS/MS can differentiate between asymmetric and symmetric dimethylation

How does H3R8me2a interact with other histone modifications?

H3R8me2a operates within the context of the histone code, interacting with other modifications:

• H3K4me3-H3R8me2a Dual Mark Recognition:

  • The Spin/Ssty repeat protein Spindlin1 specifically recognizes the dual cis-tail histone H3 methylation pattern involving H3K4me3 and H3R8me2a

  • Crystal structure analysis shows H3K4me3 is recognized by Spin/Ssty repeat 2, while H3R8me2a is recognized by repeat 1

  • Both marks are recognized using an "insertion cavity" recognition mode

• Functional Implications:

  • This dual mark contributes to Wnt/β-catenin signaling pathway regulation

  • PRMT2 and the MLL complex work together to generate the specific H3K4me3-R8me2a pattern

  • Mutagenesis of Spindlin1 reader pockets impairs activation of Wnt target genes

• Regulatory Cross-talk:

  • The presence of H3R8me2a may affect the deposition or removal of nearby modifications

  • The H3K4me3-R8me2a dual mark represents a methylation state-specific layer of regulation

What are the critical controls required for H3R8me2a ChIP experiments?

When designing ChIP experiments for H3R8me2a, the following controls are essential:

• Input Control:

  • Reserve 5-10% of chromatin before immunoprecipitation to normalize for DNA quantity and quality variations

• Antibody Controls:

  • IgG negative control to assess non-specific binding

  • Positive control antibody (e.g., H3K4me3) to verify ChIP success

• Genetic Controls:

  • PRMT2 knockdown or knockout cells to validate antibody specificity

  • Rescue experiments with wild-type versus catalytically dead PRMT2

• Peptide Controls:

  • Include peptide competition assays with H3R8me2a and other modified peptides

• Genomic Region Controls:

  • Include primers for regions known to be enriched or depleted for H3R8me2a

  • Validate findings using secondary methods (e.g., CUT&RUN or CUT&Tag)

How can recombinant H3R8me2a histones be generated for in vitro studies?

Generation of recombinant histones with specific modifications is valuable for controlled in vitro experiments:

• Expressed Protein Ligation (EPL) Technology:

  • This technology enables generation of methylated, acetylated, or phosphorylated histones

  • The histone globular domain is ligated to a peptide containing the N-terminal histone tail with the desired site-specific modification

  • The ligation reaction maintains native histone bonds, creating recombinant proteins that closely mimic natural histones

• Production Protocol:

  • Express truncated human Histone H3.2 in E. coli

  • Purify using FPLC

  • Ligate to an N-terminal histone tail peptide containing asymmetric dimethyl arginine 8

  • Repurify the full-length protein

  • Verify using high-resolution ESI-TOF mass spectrometry

• Applications:

  • These recombinant histones can be used as positive controls in analysis of histone post-translational modifications

  • They serve as substrates for histone modification enzymes

  • They can be used to generate chromatin in vitro

• Nucleosome Reconstitution:

  • Recombinant histones can be combined to form octamers

  • Sequential salt dialysis with DNA containing nucleosome positioning sequences allows assembly of nucleosomes

  • These can then be used in histone methyltransferase (HMT) assays

How do PRMT2 activity and H3R8me2a levels correlate in different cellular contexts?

The relationship between PRMT2 and H3R8me2a levels shows cellular context dependence:

• Cell Type Variations:

  • In U87 cells, PRMT2 knockdown leads to specific decrease in H3R8me2a levels

  • ChIP-seq analysis in these cells revealed 13,078 H3R8me2a-enriched regions

  • The enrichment levels of H3R8me2a are significantly decreased in PRMT2-depleted cells

• Enzymatic Activity:

  • While PRMT2 shows relatively weak activity alone in vitro, it is indispensable for H3R8me2a maintenance in vivo

  • This suggests potential co-factors or cellular conditions that enhance PRMT2 activity in cellular contexts

• Genomic Distribution:

  • H3R8me2a is distributed primarily at intragenic or intergenic regions

  • Approximately 13.71% of H3R8me2a marks are found at promoters (±3kb from TSS)

  • This genomic distribution pattern is dependent on PRMT2 activity

• Functional Implications:

  • PRMT2-mediated H3R8me2a is implicated in oncogenic activation

  • Together with H3K4me3, H3R8me2a forms a dual mark recognized by Spindlin1 to regulate Wnt/β-catenin signaling

How should contradictory H3R8me2a ChIP-seq results be interpreted?

When faced with contradictory H3R8me2a ChIP-seq results, consider these analytical approaches:

• Antibody Specificity Assessment:

  • Different antibodies may have varying specificities for H3R8me2a

  • Some antibodies show cross-reactivity with H3K9me2 in peptide dot blot assays

  • Validate antibody specificity using peptide competition assays

• Cellular Context Considerations:

  • H3R8me2a distribution may vary between cell types

  • PRMT2 activity may be differentially regulated across cellular contexts

  • Compare PRMT2 expression levels across the cell types being studied

• Technical Variability Analysis:

  • ChIP efficiency can vary between experiments

  • Normalize using appropriate controls (input, spike-in)

  • Consider using orthogonal approaches like CUT&RUN or CUT&Tag to validate findings

• Bioinformatic Reanalysis:

  • Standardize peak calling parameters across datasets

  • Employ multiple normalization methods to identify robust signals

  • Analyze H3R8me2a in conjunction with other histone marks or transcription factors

• Biological Validation:

  • Verify key findings using targeted ChIP-qPCR

  • Correlate with gene expression data

  • Perform genetic manipulation experiments (PRMT2 knockdown/knockout)

What factors affect the sensitivity and reproducibility of H3R8me2a detection?

Several factors can influence the detection of H3R8me2a:

• Antibody Quality and Batch Variation:

  • Different lots of antibodies may have varying specificities

  • Validate each new batch using peptide arrays or dot blots

  • Consider using monoclonal antibodies for increased reproducibility

• Fixation and Chromatin Preparation:

  • Over-fixation can mask epitopes

  • Insufficient fixation can result in poor recovery

  • Optimize cross-linking conditions for arginine methylation detection

  • Sonication efficiency affects chromatin fragment size and accessibility

• Cell Culture Conditions:

  • Cell density and growth phase can affect histone modification levels

  • Standardize culture conditions across experiments

  • Consider synchronizing cells when applicable

• Technical Parameters:

  • Buffer compositions may affect antibody binding

  • Wash stringency influences specificity

  • Incubation times and temperatures should be optimized

  • DNA purification efficiency affects yield

• Analysis Methods:

  • Peak calling algorithms may have different sensitivities

  • Sequencing depth affects detection of low-abundance marks

  • Background correction methods influence signal-to-noise ratios

How can H3R8me2a be studied in the context of histone methyltransferase assays?

Histone methyltransferase (HMT) assays for studying H3R8me2a should be designed with these considerations:

• Standard HMT Assay Protocol:

  • Use HMT buffer (50 mM Tris-HCl at pH 8.5, 5 mM MgCl₂, 4 mM DTT)

  • Include 3H-labeled S-adenosylmethionine (SAM) as methyl donor

  • Use recombinant oligonucleosomes as substrates

  • Include recombinant human PRMT2 or other candidate PRMTs

  • Incubate for 60 minutes at 30°C

• Detection Methods:

  • SDS-PAGE separation followed by:

    • Coomassie blue staining for protein visualization

    • Transfer to PVDF membranes

    • Autoradiography for detecting radioactive signals

  • Western blotting with H3R8me2a-specific antibodies

  • Mass spectrometry for precise identification of methylation sites

• Controls and Variations:

  • Include negative controls (no enzyme, catalytically dead enzyme)

  • Use histone H3 peptides with alanine substitutions at target residues

  • Compare wild-type and mutant enzymes

  • Titrate enzyme concentrations to determine linear range

  • Perform time-course experiments to assess kinetics

• Data Analysis:

  Parameter	Measurement Method	Expected Outcome
  Enzyme Activity	Quantification of 3H incorporation	Linear increase with enzyme concentration
  Substrate Specificity	Comparison of different histone substrates	Higher activity on preferred substrates
  Reaction Kinetics	Time-course analysis	Initial linear phase followed by plateau
  Inhibition	Addition of inhibitors	Dose-dependent decrease in activity

How does H3R8me2a contribute to the functional readout of the histone code?

H3R8me2a contributes to histone code functionality through several mechanisms:

• Dual Mark Recognition:

  • H3R8me2a works in conjunction with H3K4me3 to form a specific dual histone mark

  • This dual mark is recognized by Spindlin1 through its Spin/Ssty repeats

  • H3K4me3 is recognized by Spin/Ssty repeat 2, while H3R8me2a is recognized by repeat 1

  • Both modifications are recognized using an "insertion cavity" recognition mode

• Structural Basis of Recognition:

  • Asymmetrically dimethylated R8 is inserted into pocket 1 of Spindlin1

  • There is a snug fit between the asymmetric dimethyl-guanidino group and the aromatic residues (W62, W72, Y91, Y98, and F251) lining pocket 1

  • R8me2a binding is facilitated by salt bridge formation with E64 and water-mediated hydrogen-bonding with Y98

  • This specific recognition contributes approximately 216 Ų of buried surface area

• Signaling Pathway Integration:

  • The H3K4me3-R8me2a dual mark functions downstream of PRMT2 and the MLL complex

  • This epigenetic signature activates Wnt/β-catenin signaling

  • Mutagenesis of Spindlin1 reader pockets impairs activation of Wnt target genes

• Cross-regulation with Other Modifications:

  • The presence of H3R8me2a may affect the deposition or removal of adjacent modifications

  • This creates a complex regulatory network within the histone code

What methodological approaches can identify proteins that interact with H3R8me2a?

Identifying H3R8me2a-interacting proteins (readers) requires specialized approaches:

• Peptide Pull-down Assays:

  • Synthesize biotinylated histone H3 peptides with R8me2a modification

  • Include unmodified and differently modified peptides as controls

  • Incubate with nuclear extracts

  • Capture with streptavidin beads

  • Identify bound proteins by mass spectrometry or western blotting

• Structural Biology Approaches:

  • X-ray crystallography of candidate reader domains with H3R8me2a peptides

  • For example, the crystal structure of Spindlin1 with H3K4me3R8me2a peptide at 2.1 Å resolution

  • NMR studies to analyze binding dynamics

  • Isothermal titration calorimetry (ITC) to determine binding affinities

• CRISPR-Based Screening:

  • Create a library of potential reader proteins for knockout

  • Assess changes in H3R8me2a-dependent processes

  • Validate hits using biochemical approaches

• Proximity Labeling Methods:

  • Fusion of biotin ligase to modified nucleosomes containing H3R8me2a

  • Identification of proteins in close proximity to the modification

  • Comparison with control nucleosomes to identify specific interactors

How can temporal dynamics of H3R8me2a be studied during cellular processes?

Investigating the temporal dynamics of H3R8me2a requires specialized approaches:

• Time-Course ChIP-seq:

  • Collect samples at multiple time points during cellular processes (differentiation, cell cycle, etc.)

  • Perform ChIP-seq for H3R8me2a at each time point

  • Analyze changes in genomic distribution patterns

  • Correlate with transcriptional changes

• Live-Cell Imaging Approaches:

  • Develop specific reader domains fused to fluorescent proteins

  • Create systems for conditional expression or degradation of PRMT2

  • Monitor real-time changes in the localization of reader domains

  • Correlate with cellular events using additional markers

• Single-Cell Epigenomics:

  • Apply single-cell ChIP-seq or CUT&Tag methods

  • Capture heterogeneity in H3R8me2a patterns across cell populations

  • Identify cell state transitions associated with changes in H3R8me2a

• Mathematical Modeling:

  • Develop kinetic models of H3R8me2a deposition and removal

  • Integrate with data on PRMT2 activity and demethylase function

  • Predict temporal dynamics under different cellular conditions

  • Validate model predictions experimentally

These advanced methodological approaches provide researchers with comprehensive tools for investigating the complex roles of H3R8me2a in chromatin regulation and gene expression.