Histone H3R17me2a Antibody

What is Histone H3R17me2a and what role does it play in gene regulation?

Histone H3R17me2a refers to the asymmetric dimethylation of arginine 17 on histone H3, a post-translational modification catalyzed primarily by coactivator-associated arginine methyltransferase 1 (CARM1). This epigenetic mark plays a crucial role in transcriptional activation by creating binding sites for effector molecules that facilitate chromatin remodeling and gene expression . Specifically, H3R17me2a is associated with estrogen-responsive gene activation, where ChIP analysis has revealed elevated levels at promoters of estrogen-responsive genes such as pS2 . Unlike some histone modifications that remain stable, H3R17me2a exhibits dynamic cycling at approximately 40-minute intervals following estrogen stimulation, correlating with transcriptionally productive cycles .

How does H3R17me2a interact with other histone modifications in the epigenetic landscape?

H3R17me2a functions within a complex network of histone modifications. While H4R3me2a (catalyzed by PRMT1) establishes transcriptional competency and is required for subsequent histone acetylation, H3R17me2a appears in every transcriptionally productive cycle . This suggests a sequential relationship between different arginine methylation events. Interestingly, while H3R17me2a marks every transcriptionally productive cycle at 40-minute intervals, methylation of the R3 motif in H4 and H2A spans two cycles before being lost and re-established, indicating complex temporal regulation of these modifications . The H3R17me2a mark also influences subsequent modifications, as it recruits the PAF1 complex, which is involved in directing additional histone modifications including H3K4me3 and H2B ubiquitination .

What are the recommended protocols for using H3R17me2a antibodies in ChIP assays?

For chromatin immunoprecipitation (ChIP) assays using H3R17me2a antibodies, the following methodological approach is recommended:

• Sample Preparation: Culture cells (e.g., MCF7 or HEK293) and conduct appropriate treatments (for estrogen-responsive studies, treat with 20 nM 17β-estradiol after 3 days in phenol red-free medium with stripped FBS) .

• Cross-linking and Chromatin Preparation: Use 1% formaldehyde for cross-linking (typically 10 minutes at room temperature), followed by glycine quenching. Isolate nuclei and sonicate chromatin to fragments of approximately 200-500 bp.

• Immunoprecipitation:

  • Antibody dilution: Use H3R17me2a antibody at 1:50-1:200 dilution

  • Incubation: Overnight at 4°C with rotation

  • Capture: Protein A/G magnetic beads for 2 hours at 4°C

  • Washing: Use increasingly stringent buffers to reduce background

• Analysis: Perform qPCR targeting regions of interest (particularly estrogen-responsive gene promoters such as pS2) .

This protocol can be implemented using commercial ChIP kits with appropriate modifications for histone PTM-specific antibodies. For optimal results, include appropriate controls (IgG negative control and total H3 for normalization) .

How can Western blotting protocols be optimized for H3R17me2a detection?

Detecting H3R17me2a via Western blotting requires specific considerations to ensure sensitive and specific results:

• Sample Preparation:

  • Use a high salt/sonication protocol to extract histones, as many chromatin-bound proteins are not soluble in low salt nuclear extracts

  • Acid extraction methods (e.g., with 0.2N HCl) are particularly effective for enriching histones

  • Include phosphatase and deacetylase inhibitors to preserve post-translational modifications

• Gel Electrophoresis:

  • Use 15-18% gels for optimal resolution of histone proteins

  • Load 10-20 μg of histone-enriched extracts

• Transfer and Blocking:

  • PVDF membranes are recommended for histone transfer

  • Block with 5% non-fat dry milk or BSA in TBS-T

• Antibody Incubation:

  • Primary antibody dilution: 1:500-1:2,000

  • Incubate overnight at 4°C for optimal results

• Detection:

  • Use HRP-conjugated secondary antibodies and enhanced chemiluminescence

  • Expected molecular weight: ~17 kDa (observed) compared to calculated 15 kDa

• Controls:

  • Include unmodified H3 as a negative control

  • Include total H3 antibody for normalization

  • HeLa nuclear extract can serve as a positive control

What are the approaches for validating H3R17me2a antibody specificity?

Validating antibody specificity is crucial for histone modification research. For H3R17me2a antibodies, employ these approaches:

• Peptide Competition Assays: Pre-incubate the antibody with increasing concentrations of H3R17me2a peptide before applying to samples. Specific binding should be progressively reduced.

• Peptide Array Analysis: Test reactivity against a panel of differentially modified histone peptides to confirm specificity for H3R17me2a versus other arginine methylation states (H3R17me1, H3R17me2s) or modifications at other arginine residues (H3R2me2a, H3R26me2a).

• Enzyme Treatment Controls:

  • Use CARM1 knockdown or knockout cells to reduce H3R17me2a levels

  • Compare wild-type cells with CARM1 enzyme-deficient mutant cells

  • METTL23 knockdown models can also be used as control systems showing reduced H3R17me2a

• Biochemical Validation: Perform in vitro methylation assays using recombinant CARM1 or METTL23 and histone H3 substrates to generate control samples with defined modification states .

• Orthogonal Detection Methods: Validate findings using alternative detection methods such as mass spectrometry to confirm the presence and quantity of H3R17me2a in your samples.

How can H3R17me2a antibodies be used to investigate protein-protein interactions at specific chromatin regions?

H3R17me2a antibodies can be leveraged for studying protein-protein interactions through these advanced approaches:

• Sequential ChIP (Re-ChIP): This technique involves performing consecutive immunoprecipitations, first with H3R17me2a antibody followed by antibodies against suspected interaction partners (e.g., PAF1 complex components). This approach can confirm co-localization of H3R17me2a with specific proteins at the same genomic loci .

• Protein Pull-down Assays: Biotinylated histone H3 peptides containing asymmetrically dimethylated R17 can be used in pull-down assays to identify proteins that specifically recognize this modification. As demonstrated in research on PAF1c:

  • Immobilize biotinylated peptides (1 μg) on streptavidin-coated magnetic beads (200 μg)

  • Incubate with nuclear extracts or purified recombinant proteins for 2 hours at 4°C

  • Analyze bound proteins via Western blotting or mass spectrometry

• Chromatin Proteomics: Combining H3R17me2a ChIP with mass spectrometry (ChIP-MS) can identify the complete set of proteins associated with chromatin regions containing this modification.

• Proximity Ligation Assays (PLA): PLA can detect protein-protein interactions in situ by using H3R17me2a antibodies together with antibodies against potential binding partners (like PAF1c components or TDRD3), providing spatial resolution of these interactions.

• CRISPR-Based Approaches: Combining H3R17me2a antibodies with CUT&RUN or CUT&Tag protocols provides high-resolution mapping of this modification and its associated proteins with reduced background compared to traditional ChIP .

What is the relationship between H3R17me2a and RNA Polymerase II-associated factor 1 complex (PAF1c)?

The relationship between H3R17me2a and PAF1c represents a crucial mechanism in transcriptional regulation:

• Direct Binding Interaction: Through unbiased biochemical approaches, PAF1c has been identified as a specific binding partner for the H3R17me2a modification. Pull-down assays using biotinylated H3 tail peptides with or without R17me2a demonstrated that all five PAF1c components (hCtr9, hLeo1, hPaf1, hCdc73, and hSki8) specifically associate with H3R17me2a-modified tails .

• Recruitment Mechanism: H3R17me2a functions as a specific histone mark that recruits PAF1c to promoter regions. This recruitment is diminished when CARM1 (the enzyme responsible for H3R17me2a) is knocked down or when an enzyme-deficient CARM1 mutant is present, demonstrating the dependence of PAF1c localization on this specific modification .

• Functional Consequences:

  • PAF1c recruitment by H3R17me2a facilitates transcription elongation

  • PAF1c promotes additional histone modifications, including H3K4me3

  • PAF1c is involved in multiple steps of transcription, including transcription initiation, elongation, and pre-mRNA processing

• Regulatory Relationship:

  • H3R17me2a levels affect PAF1c occupancy at promoters

  • PAF1c knockdown does not affect H3R17me2a levels but reduces H3K4me3 marks

  • This suggests a unidirectional relationship where H3R17me2a acts upstream of PAF1c

This mechanism represents a novel pathway by which arginine methylation facilitates transcription activation through the specific recruitment of the transcription elongation complex.

How does METTL23 regulate H3R17me2a methylation in normal tissues versus disease states?

Recent research has revealed METTL23's role in regulating H3R17me2a methylation, particularly in retinal tissue:

• Normal Tissue Function: METTL23 catalyzes asymmetric dimethylation of arginine 17 in histone H3 (H3R17me2a) in murine retina and oocytes, serving as an alternative enzyme to CARM1 for this specific modification in certain tissues .

• Loss-of-Function Effects:

  • In METTL23 mutant mice (both Mettl23^G/G and Mettl23^-/-), H3R17me2a methylation activity is lost in retinal tissue compared to age-matched controls

  • This demonstrates METTL23's non-redundant role in establishing this modification in retinal cells

• Molecular Mechanisms:

  • METTL23 splicing products (resulting from mutations) show markedly reduced H3R17me2a methylation when expressed in cell models

  • In vitro methylation assays confirm the loss of methylation function by METTL23 splicing variants

• Disease Implications:

  • The high conservation between murine and human METTL23 exons suggests it likely catalyzes H3R17me2a in the human retina as well

  • Mutations affecting METTL23's ability to catalyze H3R17me2a may contribute to retinal pathologies

  • This establishes a potential epigenetic mechanism behind certain visual system disorders

This research highlights the tissue-specific regulation of H3R17me2a and suggests this epigenetic mark may have distinct functions and regulatory mechanisms in different cellular contexts.

What are common pitfalls when using H3R17me2a antibodies and how can they be addressed?

Researchers should be aware of these common challenges when working with H3R17me2a antibodies:

• Cross-Reactivity Issues:

  • Challenge: H3R17me2a antibodies may cross-react with other arginine methylation sites

  • Solution: Validate antibody specificity using peptide competition assays and controls like CARM1 knockout samples

• Detection Sensitivity:

  • Challenge: H3R17me2a may be present at low abundance in some cell types

  • Solution: Use enrichment techniques (acid extraction for histones) and optimize antibody concentration (1:500-1:2000 for WB, 1:50-1:200 for ChIP)

• Temporal Dynamics:

  • Challenge: H3R17me2a shows cyclic patterns (40-minute intervals) following stimulation

  • Solution: Perform time-course experiments with appropriate temporal resolution when studying inducible systems (e.g., estrogen response)

• Cell Type Variability:

  • Challenge: In some cell lines (e.g., 661W), detection may be challenging

  • Solution: Consider cell-type specific optimization or use more responsive cell lines (e.g., MCF7 for estrogen response studies)

• Storage Degradation:

  • Challenge: Antibody effectiveness may decrease with repeated freeze/thaw cycles

  • Solution: Store at -20°C in small aliquots; maintain in glycerol buffers (antibodies are typically supplied in buffers containing 30-50% glycerol)

How can researchers differentiate between asymmetric (me2a) and symmetric (me2s) dimethylation of H3R17?

Distinguishing between asymmetric and symmetric dimethylation of H3R17 requires specific approaches:

• Antibody Selection:

  • Use antibodies specifically validated for H3R17me2a versus H3R17me2s

  • Confirm the antibody's ability to distinguish these modifications using peptide arrays or competition assays

• Enzyme-Based Controls:

  • CARM1 catalyzes asymmetric dimethylation (me2a) while PRMTs like PRMT5 typically generate symmetric dimethylation (me2s)

  • Manipulating these enzymes (knockdown/overexpression) can help confirm the specific modification type

• Mass Spectrometry Approaches:

  • Tandem mass spectrometry can definitively distinguish between asymmetric and symmetric dimethylation

  • Diagnostic fragment ions differ between these isomeric modifications

• Functional Validation:

  • H3R17me2a specifically recruits proteins like PAF1c and TDRD3

  • Protein-binding assays can help confirm the specific methylation state

• Context-Specific Analysis:

  • Asymmetric dimethylation (me2a) is generally associated with transcriptional activation

  • Symmetric dimethylation (me2s) often correlates with transcriptional repression

  • Correlating modification detection with transcriptional states can provide supporting evidence

How should researchers interpret changes in H3R17me2a levels in different experimental conditions?

When analyzing changes in H3R17me2a levels across experimental conditions, consider these interpretation guidelines:

• Baseline Variations:

  • Cell type-specific differences in basal H3R17me2a levels reflect variable CARM1 or METTL23 activity

  • Always include appropriate controls matched for cell type, passage number, and culture conditions

• Temporal Dynamics:

  • H3R17me2a shows cyclic patterns (~40-minute intervals) following estrogen stimulation

  • Single timepoint measurements may miss important dynamics; consider time-course experiments

• Stimulation Responses:

  • In estrogen-responsive systems, elevated H3R17me2a at specific promoters indicates CARM1-mediated coactivation

  • Correlate changes with recruitment of PAF1c and transcriptional output of target genes

• Regulatory Mechanisms:

  • Decreased H3R17me2a may result from:
    a) Reduced CARM1/METTL23 expression or activity
    b) Increased demethylase activity
    c) Histone exchange

  • Distinguish between these mechanisms through additional experiments

• Interpreting ChIP Data:

  • H3R17me2a enrichment at promoters typically indicates transcriptional competence

  • Consider normalizing to total H3 occupancy to account for nucleosome density changes

  • Analyze in conjunction with other activation marks (H3K4me3) and elongation markers

What is the significance of H3R17me2a in estrogen-responsive gene regulation?

H3R17me2a plays a pivotal role in estrogen-responsive gene regulation through several mechanisms:

• Temporal Regulation:

  • CARM1 is recruited to estrogen-responsive promoters in a cyclic manner

  • H3R17me2a marks every transcriptionally productive cycle at 40-minute intervals following estrogen stimulation

  • This temporal pattern suggests H3R17me2a is a key signal in the transcriptional cycle of estrogen-responsive genes

• Coactivator Function:

  • H3R17me2a is deposited by CARM1, which functions as a coactivator for estrogen receptor (ER)

  • Estrogen treatment leads to increased H3R17me2a levels at responsive promoters (e.g., pS2)

  • This modification is important for transcriptional activation of estrogen-regulated genes

• Effector Recruitment:

  • H3R17me2a specifically recruits effector proteins that facilitate transcription:
    a) PAF1c: Promotes transcription elongation and directs additional histone modifications
    b) TDRD3: Contains a tudor domain that recognizes methylated arginines and functions as a coactivator

  • These recruitments form a mechanistic link between arginine methylation and transcriptional activation

• Sequential Histone Modifications:

  • In estrogen signaling, H4R3me2a (by PRMT1) occurs during the first non-productive cycle

  • H3R17me2a (by CARM1) then marks every productive transcription cycle

  • This sequential pattern suggests a histone modification code in estrogen-responsive transcription

• Clinical Relevance:

  • Altered H3R17me2a patterns may contribute to dysregulated estrogen signaling in hormone-responsive cancers

  • Understanding this mechanism provides potential epigenetic targets for therapeutic intervention in hormone-dependent diseases

How can H3R17me2a antibodies be utilized in single-cell epigenetic profiling?

Emerging approaches for single-cell epigenetic profiling using H3R17me2a antibodies include:

• Single-Cell CUT&RUN/CUT&Tag:

  • H3R17me2a antibodies can be adapted for these techniques that require fewer cells than ChIP

  • These methods offer improved signal-to-noise ratio and can profile rare cell populations

  • Recent commercial developments have enabled single-cell resolution for histone modifications

• Mass Cytometry (CyTOF):

  • Metal-conjugated H3R17me2a antibodies enable quantification across thousands of individual cells

  • Can be combined with other cellular markers to correlate H3R17me2a with cell states and identities

  • Provides quantitative measurement at the single-cell level

• Integrated Multi-Omics:

  • Combining single-cell H3R17me2a profiling with transcriptomics (scRNA-seq)

  • Correlating H3R17me2a patterns with gene expression in the same cells

  • Reveals cause-effect relationships between this epigenetic mark and transcriptional output

• Spatial Epigenomics:

  • Utilizing H3R17me2a antibodies for immunofluorescence (IF/ICC at 1:50-1:200 dilution)

  • Combining with spatial transcriptomics to correlate modification patterns with gene expression in tissue context

  • Reveals cell-type specific roles of H3R17me2a in complex tissues

• Technical Considerations:

  • Fixation protocols must be optimized to preserve epitope recognition in single cells

  • Antibody concentration should typically be higher than in bulk assays

  • Controls (CARM1/METTL23 knockdown cells) are essential for validating specificity in single-cell contexts

What are the emerging connections between H3R17me2a and disease mechanisms?

Recent research is uncovering important connections between H3R17me2a and various disease mechanisms:

• Retinal Disorders:

  • METTL23 mutations that disrupt H3R17me2a methylation in retinal tissue may contribute to visual system pathologies

  • Both Mettl23^G/G and Mettl23^-/- mouse models show loss of H3R17me2a methylation in retinas

• Cancer Biology:

  • Dysregulation of CARM1 and resulting alterations in H3R17me2a patterns are implicated in multiple cancer types

  • H3R17me2a can affect estrogen-responsive gene expression relevant to hormone-dependent cancers

  • The connection to PAF1c, which regulates multiple aspects of transcription, suggests broad implications for cancer progression

• Developmental Disorders:

  • CARM1 and METTL23 regulate H3R17me2a in different tissues, suggesting tissue-specific roles in development

  • Mutations affecting these enzymes and resulting H3R17me2a patterns may contribute to developmental abnormalities

• Transcriptional Dysregulation:

  • H3R17me2a recruits PAF1c, which influences RNA processing and transcription elongation

  • Disruption of this pathway could contribute to diseases characterized by transcriptional dysregulation

• Therapeutic Targeting:

  • Understanding the "readers" of H3R17me2a (PAF1c, TDRD3) offers potential therapeutic targets

  • Inhibiting specific reader-modification interactions could provide more precise control than targeting the modifying enzymes directly

  • Such approaches could potentially modulate specific gene expression patterns in disease contexts